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Analysis of the effect of inorganic lead on calmodulin and calmodulin-dependent cell signaling pathways

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Analysis of the effect of inorganic lead on calmodulin and calmodulin-dependent cell signaling pathways
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Wisniewski, Michael
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English
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xi, 90 leaves : illustrations ; 28 cm

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Calmodulin ( lcsh )
Calcium-binding proteins ( lcsh )
Lead -- Physiological effect ( lcsh )
Calcium-binding proteins ( fast )
Calmodulin ( fast )
Lead -- Physiological effect ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 86-90).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Michael Wisniewski.

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|University of Colorado Denver
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ocm44105174
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Full Text
ANALYSIS OF THE EFFECT OF INORGANIC LEAD ON CALMODULIN
AND CALMODULIN-DEPENDENT CELL SIGNALING PATHWAYS
Michael Wisniewski
B.S., University of Florida, 1994
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment of the
I
requirements fcjr the degree of
Master of Arts
Biology
1909
by


1999 by Michael Wisniewski
All rights reserved.


This thesis for the Master of Biology
degree by
Michael Scott Wisniewski
has been approved
by
Gerald Audesirk
Terry Audesirk
Charles Ferguson

II
Date


Wisniewski, Michael (M.A., Biology)
ANALYSIS OF THE EFFECT OF INORGANIC LEAD ON CALMODULIN
AND CALMODULIN-DEPENDENT CELL SIGNALING PATHWAYS
Thesis directed by Gerald Audesirk, Ph D.
ABSTRACT
Calmodulin is a multipurpose calcium-binding protein involved in
the regulation of numerous calcium-mediated cell functions. Calmodulin
is a dumbbell-shaped protein with two globular domains connected by an
alpha helix. There are two calcium-binding sites on each globular
domain. When all four calcium-binding sites are occupied, a
conformational change occurs that exposes a hydrophobic region
capable of regulating more than twenty different proteins, including
phosphodiesterase and calmodulin-dependent protein kinase II.
Furthermore, calmodulin can be activated by inorganic lead (Pb2+) at the
calcium-binding sites. However, the free Pb2+ concentrations required for
cell activation have not been accurately determined. It is also possible
that Pb2+ and Ca2+ may be capable of activating calmodulin in an additive
fashion via the occupation of different sites by either cation on the same
IV


molecule. We attempted to determine the concentration of Pb2+ required
to activate calmodulin, and to determine if Pb2+ could act in an additive
fashion in the presence of Ca2+.
Phosphodiesterase (PDE1 isoform) activity is stimulated by the
calcium-activated conformation of calmodulin. We found that Pb2+-
activated calmodulin was capable of stimulating phosphodiesterase
activity in two independent assays. Using the EnzChek kit from
Molecular Probes, free Pb2+ concentrations as low as 200 pM were found
to stimulate phosphodiesterase activity, with maximum activity at free
Pb2+concentrations of 1 nM and higher, with an EC50 at about 500 pM
Pb2+. Similar activation was also observed with a second method using
the molybdate/malachite green reaction to measure PDE activation. Pb2+
and Ca2+ also functioned additively in both experiments to provide full
activation as determined by isobolographic analysis.
Calmodulin-dependent protein kinase II (CaMKII) is also
stimulated by calcium-activated calmodulin. Stimulation of CaMKII by
Pb2+-activated calmodulin was shown to occur with maximum activation
at 1 nM Pb2+, and an EC50 concentration of approximately 200 pM Pb2+.
However, full activity as compared to calcium-activated calmodulin was
never achieved at any concentration. At higher concentrations, it
appears that lead can bind to an inhibitory site directly on CaMKII,
v


functioning to inactivate the kinase. At lower concentrations, activation
appeared to occur in a positive cooperative fashion in conjunction with
the presence of Ca2+.
This abstract accurately represents the content of the candidates thesis.
I recommend its publication.
Signe
Gerald Audesirk
vi


ACKNOWLEDGMENT
I want to express my appreciation to Marcy Kern, Leigh Cabell-Kluch,
and Deana Luginbill. They showed me what to do on a daily basis, and
where to find it.
I want to thank Charlie Ferguson and Terry Audesirk for their advice,
support and encouragement with my research, teaching and learning.
I especially want to thank Gerry Audesirk. He was there from day one.
Finally, I want to thank my wife, Jennifer Wisniewski. Without her love
and support, I would not have had the courage to begin to realize my
potential.


CONTENTS
Figures..................................................x
CHAPTER
1. INTRODUCTION..........................................1
Calmodulin.........,................................2
Phosphodiesterase...................................11
Calmodulin-Dependent Protein Kinase II..............12
2. MATERIALS AND METHODS.................................19
Chemicals...........................................19
Cyclic Nucleotide Phosphodiesterase Assays..........20
Preparation of Stock Solutions................21
EnzChekPhosphate Assay.......................23
Promega Phosphate Assay.......................29
Calcium-Calmodulin Dependent Protein Kinase II......31
Preparation of Stock Solutions................32
CaMKII Experimental Protocol..................36
Graphical Analysis..................................48
viii


3. RESULTS.............|..............................50
Molecular Probes Phosphodiesterase Assay.........50
Promega Molybdate/Malachite Green PDE Assay......52
Calmodulin-Dependent Protein Kinase II Assays....53
4. DISCUSSION..........J..............................56
FIGURE LEGEND..........i..............................77
BIBLIOGRAPHY...........J..............................86
IX


FIGURES
Figure
1. Amino acid sequence of calmodulin..................................4
2. Depiction of EF-hand binding site..................................6
3. Model of feedback loop, synaptic activity, and CaM/CaMKII activity.8
4. Enzymes activated by calmodulin....................................9
5. Conformation changes in CaMKII.....................................14
6. CaMKII holoenzyme activity.......................................17
7. 5-Nucleotidase reaction.........................................20
8. Ca2+/PDE activation curve (Molecular Probes).......................63
9. Pb2+/PDE activation curve (Molecular Probes).....................64
10. 500 nM Ca2+/Pb2+/PDE activation curve............................65
11. Comparison of figures 9 & 10.......................................66
12. Ca2+/PDE activation curve (Promega)..............................67
13. Pb2+/PDE activation curve (Promega)..............................68
14. Ca2+/Pb2+/PDE activation comparison curve........................69
15. Isobolographic analysis..........................................70
16. Ca2+/truncated CaMKII activation.................................71
17. Ca2+/CaMKII activation........:..................................72
x


18. Pb2+/CaMKII activation...
19. 500 nM Ca2+ with and without vi
20. 1 mM Ca2+ with and without 100
21. 10 mM Ca2+ with and without
10D
.73
friable Pb2+ CaMKII activation...........74
nM Pb2+ CaMKII activation..............75
nM Pb2+ CaMKII activation.............76
XI


CHAPTER 1
INTRODUCTION
Lead exposure induces numerous neurological deficiencies. Low
levels of lead exposure are especially damaging in embryonic
development, and have been implicated as a cause of hyperactivity,
decreased I.Q., and seizures in children (David eta!., 1976; Lockitch, 1993;
McMichael et a/., 1988). Lead exposure remains a concern in many urban
areas. Lead paint and lead pipes are still found in many older buildings,
and there is lead contamination in the soil of many neighborhoods that are
built near highways. A clear cause and effect relationship between lead
exposure and impaired neural development has been established. The
next question to be answered is how. It is important to discover the
mechanism behind leads ability to affect neural development.
Inorganic lead may affect many different intracellular molecules in
different ways. Because of the similar valence and ionic radius between
Ca2+ and Pb2+ (Cheung, 1984), it is highly feasible that Pb2+could act as a
substitute for Ca2+ on many molecules. One likely target for Pb2+ activation
would be calmodulin.
l


No one knows what the intracellular Pb2+ concentrations are in
exposed cells, but it is likely that the intracellular free Pb2+ concentration
would be below 1 nM. Studies reported in 1983 indicated that inorganic
lead could activate calmodulin at pM total Pb2+ concentrations (Goldstein
and Ar, 1983; Habermann et al., 1983), which should theoretically activate
calmodulin-dependent pathways. However, these tests were performed
using assay buffers that included EGTA and sulfhydryl reagents that bind
free Pb2+ (Kern et al., 1999). The actual free Pb2+ concentrations present
were probably considerably lower than the total Pb2+ concentrations in the
buffers. This raises the question of whether or not calmodulin could be
activated by biologically feasible intracellular Pb2+ concentrations, and
whether Pb2+ and Ca2+ can operate cooperatively in the activation of
calmodulin. We performed experiments attempting to quantify inorganic
leads ability to activate calmodulin at biologically relevant concentrations
by measuring the activity of two different calcium/calmodulin-dependent
molecular pathways.
Calmodulin
The mechanisms responsible for modulating calcium-activated
intracellular signaling must be highly precise to properly perform the rapid
responses initiated by transient fluctuations in Ca2+ concentrations.
2


Calmodulin functions as one of the primary mediators of calcium-
dependent pathways. Through the cooperative occupation of all four of its
calcium-binding sites, calmodulin effectively provides an almost all-or-none
response for the activation of many different enzymes with very small
changes in Ca2+ concentrations (Nelson and Chazin, 1998).
Calmodulin is a multipurpose, intracellular calcium acceptor. It is a
ubiquitous protein that is highly conserved in all eukaryotic organisms.
Calmodulin is typically found in excess of 10 million molecules per cell and
can constitute as much as 1% of the total protein mass of the cell.
Calmodulin has a high affinity constant for Ca2+, with a Kg of about 1CT6 M'1
(Nelson and Chazin, 1998). Calmodulin is fairly specific for calcium in
order to avoid binding to other divalent cations found within the cytosol at
much higher concentrations, such as Mg2+(~10'3 M).
In its native conformation, calmodulin is a single polypeptide chain
made up of 148 amino acid residues. Calmodulin is a small, heat stable
protein with a molecular weight of around 17 kD. Due to the large number
of acidic amino acid residues found on its exterior, the isoelectric point is
typically around 4.2. The inactivated molecule has a dumbbell shape
composed of two similar globular domains connected by an extended 28
amino acid alpha helix.
3


N-ltrrrwuJ tobt
Figure 1: Amino acid sequence of calmodulin. The globular domains each
contain two EF-hand binding sites, and are separated by a 28 amino acid
alpha helix (Lafitte etal., 1995).
The exact shape of the alpha helix has been a point of some debate.
The long central helix structure was disputed based on evidence that the
4


two globular domains interact during protein binding (Nelson and Chazin,
1998) and that the structure of the alpha helix exposed to the solvent was
not thought to be entirely stable. It is now believed that the alpha helix
functions as a flexible tether, allowing the two globular domains to come
into close proximity during protein binding (Persechini and Kretsinger,
1988).
Calmodulins globular domains each contain two of the molecules
four calcium-binding sites. The binding sites are composed of a twelve
residue loop that is arranged in an EF-hand configuration
(Hinrichsen,1993). The EF-hand configuration of the binding site is a
highly conserved structure found throughout a wide variety of calcium-
binding proteins. The EF-hand binding site is composed of thirty amino
acids that form a characteristic helix-loop-helix structure (see figure #2).
5


X
EF-Hand Calcium-Binding
Domain
Calmodulin
Figure 2: Depiction of the EF-hand Ca2+ binding site that is highly conserved in
most calcium-binding proteins. The EF-hands in calmodulin are
interconnected on each globular domain, and the two globular domains can
interact through their connection via a 28 amino acid alpha helix (Nelson and
Chazin, 1998).
In the first alpha helix, the side chains of amino acids 2, 5, 6, and 9
face toward the inside of the helix and are hydrophobic. Likewise, the
second alpha helix is comprised of inward facing hydrophobic amino acids
at positions 22, 25, 26, and 29 (most commonly valine, leucine, isoleucine,
methionine, phenylalanine, or tyrosine). The 12 amino acid sequence that
forms the loop is composed of six amino acids with oxygen containing side
chains at positions 10, 12, 14, 16, 18, and 21. The oxygen containing side
chains function to coordinate the binding of calcium within the looped-
6


domain. The remaining amino acids are those with short side chains (like
glycine), presumably to allow the flexibility associated with the looped
domain (Kretsinger, 1985). The binding sites are arranged on each
globular domain so that their hydrophobic regions are facing one another
and the calcium-binding loops are connected with hydrogen bonds. These
bonds provide a means for the cooperative binding displayed in calmodulin
(Nelson and Chazin, 1998).
The four calcium-binding sites have been designated as I and II on
the amino terminal globular domain, and III and IV on the carboxyl terminal
globular domain. Sites III and IV have an affinity that is ten times higher for
calcium than that of sites I and II, and the suspected order of binding is III,
IV, I, and II. The occupation of sites I and III are thought to increase the
affinity for calcium at sites II and IV, but not the other way around
(Hinrichsen, 1993). Once all four calcium-binding sites are occupied, the
molecule undergoes a conformational change which exposes a
hydrophobic region that is responsible for binding to calmodulin-regulated
molecules (LaPorte, et al., 1980; Nelson and Chazin, 1998). The alpha-
helical region connecting the two globular domains functions to arrange the
globular domains in a position that allows them to work in concert.
Cleaving the alpha helical region will inactivate the molecule (Newton et al.,
1984).
7


Although calmodulin has no direct enzymatic activity, it functions by
binding to many different cellular proteins and altering their activity. In
doing so, calmodulin functions to mediate many different second
messenger systems within different cells (see figures 3 and 4).
Figure 3: Model of feedback loop, synaptic input, and CaM/CaMKII activity as
proposed as a possible mechanism for L.T.P. within a post-synaptic neuron.
Displays the complexities involved with trying to predict the cellular effect of
inorganic lead contamination (AC1/8 = adenylate cyclase, CaN = calcineurin,
PKA = protein kinase A, PP1 = protein phosphatase 1) (Bhalla and Iyengar,
1999).
8


Enzymes activated by calmodulin
Enzvme Source
Cyclic nucleotide PDE Protein kinases Ubiquitous
NAD Kinase Plants Invertebrates
Myosin light-chain kinase Muscle Kidney Platelets
Myosin heavy-chain kinase Fungi
Phosphorylase kinase Skeletal muscle Platlets Cardiac muscle Liver
CaMKI & II Brain
CaMKIII CaMKIV Pancreas
Myosin I heavy-chain kinase Acanthamoeba
IPs Mammals
Calcineurin Brain Skeletal muscle
Adenylate cyclase Mammals Invertebrates
Guanylate cyclase Ca2+-ATPase Protozoa
Dynein Ubiquitous
Plasma membrane Ubiquitous
Sarcoplasmic reticulum Cardiac muscle
NADPH oxidase Mammals
Phosphol ipidmethylase Fungi
Nitric oxide synthase Mammals
NMDA receptors Brain
Ca2+ dependent K+ channels Mammals
Figure 4: Examples of some enzymes activated by calmodulin (adapted from
Hinrichsen, 1993).
There are over 20 different enzymes that are activated by calmodulin in a
calcium-dependant manner (Hinrichsen, 1993). Atfree-Ca2+
9


concentrations as low as 50 to 100 nM, calmodulin appears to be capable
of at least partially activating several different enzymes, including the
plasma membrane Ca2+-ATPase, which functions to pump Ca2+ out of the
cell (Brandt and Vanaman, 1998). Activation of other molecules which
require significantly higher Ca2+ concentrations appear to occur at transient
hot spots for Ca2+ within the cell (Smith, et al., 1996; Naraghi and Neher,
1997). When the cell is stimulated, Ca2+ is released from intracellular
stores or allowed to diffuse into the cell through Ca2+-permeable channels
in the plasma membrane. This creates temporary localized hot spots
where the immediate free Ca2+ concentrations may reach several
micromolar. The activation of calmodulin and any calmodulin-mediated
proteins within the hot spot will be greatest at the source of the free Ca2+,
and will decline rapidly with distance from the source (Smith, et al., 1996;
Naraghi and Neher, 1997; Kern, et al., 1999). Furthermore, there is also
evidence indicating that some membrane-bound proteins may have an
affinity for inactivated calmodulin, which would essentially function to
sequester inactivated calmodulin in close proximity to the Ca2+ channels
(Liu and Storm, 1990).
10


Phosphodiesterase
The first calcium/calmodulin dependent pathway that we examined
was the calcium/calmodulin activated PDE1 isoform of phosphodiesterase.
Activated phosphodiesterase (PDE) normally functions to hydrolyze cyclic
adenosine monophosphate (cAMP) or cyclic guanine monophosphate
(cGMP) into 5 adenosine monophosphate (AMP) or 5 guanine
monophosphate (GMP), respectively. This decreases activity of cAMP and
cGMP mediated second messenger systems common in eukaryotic cells.
In the absence of Ca2+/calmodulin, PDE hydrolytic activity is maintained at
a low, but detectable, level. In the presence of Ca2+/calmodulin, activity is
four to seven times higher (Sonnenburg et al., 1998).
PDE is a multimeric macromolecule composed of two identical
subunits, each containing a catalytic domain and a regulatory domain. The
catalytic domain is located near the carboxyl terminus of the protein, and is
composed of a highly conserved 250 amino acid sequence that is found in
all PDE isoforms. The catalytic domain is spontaneously active in the
absence of a functional regulatory unit. The regulatory element possesses
the inhibitory domain, which inactivates the catalytic domain, and two
different CaM binding domains in close proximity. The two CaM-binding
domains are separated by approximately 90 amino acids. It is currently
believed that CaM only has to bind to one of the binding domains to
ll


remove the inhibitory effect of the regulatory domain (Sonnenburg et ai,
1998).
By using kits designed by Molecular Probes and Promega we were
able to quantify inorganic leads ability to activate calmodulin alone and in
the presence of calcium as compared to normal calcium-stimulated
calmodulin activation. We found that Pb2+ could activate calmodulin
independently, with full activation (as compared to activation by high Ca2+
concentrations) occurring at concentrations of 1 nM and higher. This
activation concentration for Pb2+ is approximately 1000 times less than
Ca2+-induced full activation. The EC50 for calmodulin activation by Pb2+
alone was approximately 500 pM. We also found that sub-saturating
concentrations of Pb2+ and Ca2+ together could achieve greater activation
levels than either could independently. Isobolographic analysis (Gessner,
1995) indicated that Ca2+ and Pb2+ were additive in their ability to activate
calmodulin.
Calcium-Calmodulin-Dependent Protein Kinase II
Calcium/calmodulin-dependent protein kinase II (CaMKII) was also
used in an attempt to quantify Pb2+s ability to activate calmodulin
independently and in conjunction with Ca2+. CaMKII is just one of many
different kinases known to be regulated by calcium/calmodulin activity
12


(Lukas et al, 1998). The common mechanistic step among all of the
calmodulin-dependent protein kinases is the selective recognition of Ca2+
and the initial decoding of the Ca2+ signal by CaM, followed by the
activation via relief of an autoinhibition mechanism. CaMKII is typically
found as a 10 to 12 unit holoenzyme, although each unit is capable of
activity independent of the activation status of the other units (Lisman,
1994). CaMKII is particularly abundant in neurons and is directly involved
in neurite initiation and elongation (Cabell and Audesirk, 1993). Activated
calmodulin binds to CaMKII and initiates the phosphorylation of over forty
different substrates (Schulman, 1988).
In the absence of Ca2+/calmodulin, CaMKII is inactive as a result of
an interaction between its inhibitory domain and the phosphorylation site.
When Ca2+ activates calmodulin, CaM undergoes a conformational change
exposing two hydrophobic regions within the globular domains that interact
with the inhibitory domain on CaMKII. Activated calmodulin literally wraps
around the inhibitory domain of CaMKII (Schulman, 1988) (see figure 5).
13


inhibitory domain
Figure 5: Conformational changes in an individual unit of CaMKII that are originally
initiated by Ca2+/calmodulin are able to be maintained even after the dissociation of the activating
Ca2+/CaM complex. This allows a CaMKII unit to maintain partial activity even after Ca2+
concentrations have decreased (Alberts et al., 1994).
The binding of calmodulin to the inhibitory domain interferes with the
interaction between the inhibitory domain and the catalytic domain. This
14


allows CaMKIIs catalytic domain to phosphorylate itself, other CaMKII
molecules within the same holoenzyme, and other proteins within the cell.
Phosphorylation of CaMKII causes calmodulin to remain bound to CaMKII
for a short period of time after Ca2+ levels have decreased (Lisman, 1994).
The phosphorylation also allows the enzyme to remain active even after
dissociation from calmodulin until it is dephosphorylated by a phosphatase.
Ca2+-independent phosphorylation within a CaMKII holoenzyme may allow
the holoenzyme to retain a degree of phosphorylation indefinitely and is the
basis for the hypothesis that CaMKII is directly involved in long-term
potentiation, the most probable model for synaptic formation and storage of
memory (Lisman, 1994).
Because long-term potentiation is partially regulated by activation of
CaMKII by Ca2+/calmodulin, it is believed to be an example of a cellular
pathway which may be affected by the presence of inorganic lead. The
process by which long-term potentiation is believed to function involves
both the long-term activation of CaMKII and the sequestration of the
holoenzyme near N-methyl-D-asparate (NMDA) channels (Lisman, 1994).
The post-synaptic terminal is excited by the binding of glutamate to the
NMDA channels. When glutamate is bound, the NMDA channel opens and
allows Ca2+ to enter the cell. As Ca2+ flows into the cell, it will activate
calmodulin and, in turn, CaMKII. As individual members of the holoenzyme
15


are relieved of autoinhibition, they will phosphorylate other proteins and,
more importantly, other members of the same holoenzyme. Furthermore,
the activity of the enzyme is now CaPVcalmodulin-independent.
It is believed that through Ca2+/calmodulin-independent activity,
CaMKII holoenzyme activity can be maintained for extended periods of
time. This is possible because, as a phosphatase removes the activating
phosphate from one member of the holoenzyme, dephosphorylated units
are reactivated by other members of the same holoenzyme (Lisman and
Goldring, 1989). As phosphorylated units are broken down during the
continual protein turnover within a cell, the new CaMKII units are simply
phosphorylated in their place. Thus, once a post-synaptic terminal has
been activated by glutamate, a residual level of CaMKII post-synaptic
activity can be maintained for a period of time after the Ca2+ concentration
has decreased see (figures 5 and 6):
16


Phosphatase
Ca2*-independent'
autophosphotytation
Ca2+-independent
autophosphotytation
Figure 6: Structure and function of the Ca27calmodulin-dependent protein kinase II holoenzyme
demonstrating the conversion of individual units between the phosphorylated (on) and non-
phosphorylated states (Lisman, 1994).
The maintenance of a temporarily increased level of activity allows
for the development of dendrite specific plasticity. After a holoenzyme has
been activated, it tends to be bound by a membrane protein that functions
to localize the active CaMKII holoenzymes in close proximity to the NMDA
channels (Lisman, 1994). This means that specific, active dendrites within
a pathway may maintain higher local concentrations of CaMKII
holoenzymes than inactive dendrites do. Thus, through activation by
Ca2+/calmodulin, CaMKII can maintain a level of autophosphorylation for a
prolonged period of time that could be the molecular basis for synaptic
storage of memory formation.
17


In order to quantify Pb2+ activation of the calmodulin/CaMKII
pathway, we used a colored peptide commercially produced by Pierce that
functions as a suitable phosphorylation substrate. We found that Pb2+,
while capable of initiating calmodulin-dependent CaMKII activity at low
concentrations (threshold activity was achieved at concentrations below
100 pM free Pb2+), never achieved full activation of CaMKII as compared
with Ca2+. Pb2+-stimulated activity peaked at 10 nM and sharply
decreased at higher concentrations. Pb2+ appeared to act cooperatively at
low concentrations in conjunction with in the activation of CaMKII.
However, higher free Pb2+ concentrations appeared to have an inhibitory
effect on CaMKII activity consistent with the presence of an inhibitory Pb2+-
binding site directly on the kinase. When massive quantities of Ca2+ were
used (1 mM, which should have excluded Pb2+ from occupying any of
calmodulins Ca2+-binding activation sites), free Pb2+ concentrations as low
as 100 nM still reduced CaMKII activity. Furthermore, Ca2+ concentrations
as high as 10 mM also decreased maximum CaMKII activity. Based on
these results, we have concluded that Pb2+, while still functioning to
activate calmodulin, is also reducing the activation of CaMKII by binding to
a different, inhibitory site directly on the CaMKII molecule.
18


CHAPTER 2
MATERIALS AND METHODS
Chemicals
Calmodulin was purchased from Calbiochem, Boehringer
Mannheim, or New England Biolabs. EGTA was purchased from Sigma or
Baker Chemical Company. Phosphate quantification was accomplished
using the EnzChek phosphate assay kit produced by Molecular Probes,
and the serine-threonine phosphatase assay kit produced by Promega.
The truncated version of CaMKII was purchased from New England
Biolabs. Whole CaMKII was purchased from Upstate Biotechnology
Incorporated (UBI). Glycogen synthase peptide was purchased from
Pierce, as were the affinity separation units and the binding and elution
buffers. All other materials were purchased from Sigma (Na2ATP, MgC^,
CaCI2, Pb(N03)2, Tris, and BSA ).
Cyclic Nucleotide Phosphodiesterase Assays
Phosphodiesterase (PDE) activity was measured by the release of
inorganic phosphate from cyclic AMP. Calmodulin-activated PDE
hydrolyzed cAMP to AMP, and AMP was then hydrolyzed to adenosine and
inorganic phosphate by 5nucleotidase. The inorganic phosphate was
19


measured by its reaction with 2-amino-6-mercapto-7-methylpurine
ribonucleoside (MESG) to form ribose-1-phosphate and 2-amino-6-
mercapto-7-methylpurine via catalyzation by purine nucleoside
phosphorylase (PNP) using the Molecular Probes EnzChek assay kit, or
the molybdate/malachite green reaction (Lanzetta et al., 1979).
PRINCIPLE S
5'-AMP + H20 5*-Mvslfpfclfoggo Adenosine + Pi
Abbreviations used:
5'-AMP *= Adenosine 5'-Monophosphate
Pi *= inorganic Phosphate
Figure7: Release of inorganic phosphate by 5-nucleotidase from 5-AMP permits the
quantification of PDE activity. Reaction of cAMP to 5-AMP is catalyzed by calmodulin activated
PDE1 isoform.
20


Preparation of Stock Solutions
1. 5-nucleotidase (Sigma N5880): 50 units (1 unit will hydrolyze 1.0 nmole
of inorganic phosphorus from adenosine 5 monophosphate per
minute at pH 9.0 at 37C) of lyophilized 5-nucleotidase and glycine
buffer(already included) were dissolved in 1 ml of deionized water to
create 0.05 units per pi solution. This was then aliquotted into 100
pi portions that were frozen in liquid nitrogen and stored at -70C.
One sample was thawed and used per day.
2. Phosphodiesterase (PDE): 0.25 units (1 unit will hydrolyze 1.0 pmole of
3:5-cAMP to 5-AMP per minute at pH7.5 at 30C) of PDE were
dissolved in 500 pi of 50% glycerol to create a 1 unit per 2 ml
dilution. 5 pi were used per assay for a final concentration of 0.0025
units per assay.
3. Calmodulin: The calmodulin was prepared as a 30 pM stock solution
dissolved in 50 mM Tris and 0.2% BSA. This was aliquotted into 25
pi portions, frozen in liquid nitrogen, and stored at -70C.
21


4. Cyclic AMP: 25 mg of cAMP was dissolved in 14.2 ml of deionized
water for a 5 mM solution.
5. Purine Nucleoside Phosphorylase (PNP): 500 pi of deionized water was
added to 50 units of the lyophilized enzyme (with one unit defined as
the amount of PNP needed to cause the reaction of 1.0 pmole of
inosine to hypoxanthine and ribose 1-phosphate per minute at pH
7.4 at 25C) to create a 100 U/mL stock solution. The PNP stock
solution was stored at 4C, as per the Molecular Probes instructions.
6. 2-Amino-6-Mercapto-7-Methyl-Purine Ribonucleoside (MESG): 1 mM
stock solution was prepared by adding 20 ml of deionized water to
6.3 mg of MESG. The solution was mixed extensively (over 10
minutes) and then aliquotted into 1 ml portions. The aliquots were
frozen in liquid nitrogen and stored at -20C.
7. Metal Buffers:
A. 200 mM KCI, 40 mM NaCI, 100 mM Tris, 20 mM EGTA, pH 7.3.
B. 200 mM KCI, 40 mM NaCI, 100 mM Tris, 20 mM EGTA, 20 mM
CaCI2, pH 7.3.
22


C. 200 mM KCI, 40 mM NaCI, 100 mM Tris, 20 mM EGTA, 20 mM
Pb(N03)2, pH7.3.
All solutions contain some Ca2+. However, according to the
Chelator program (Schoenmakers, 1992), even with allowing for Ca2+
contamination, the free calcium ion concentration in buffer A should be less
than 1 nM. The free Ca2+ in buffer B should be about 50 pM.
EnzChek Phosphate Assay
The reaction mixture consisted of:
1. 5 pi of 5-nucleotidase, which amounts to 0.25 units per assay.
2. 5 pi of PDE (0.0025 units, dissolved in a 50% glycerol solution).
3. 2 pi of 30 pM calmodulin (dissolved in 50 mM Tris and 0.2%
BSA), for a final concentration of 0.12 pM calmodulin per
assay.
4. 20 pi cAMP, for a final concentration of 0.2 mM cAMP per assay.
5. 100 pi of 2- amino-6-mercapto-7-methylpurine ribonucleoside
(MESG) solution, for a final concentration of 0.2 mM MESG
per assay.
6. 5 pi PNP (0.5 units) solution.
23


7. 5 to 8 pi of 100mM MgC^ for a final free Mg2+ concentration of
1 mM.
8. The metal/buffer solution concentrations as determined using the
Chelator program for free metal ion concentration (see pages
24 27).
9. 100 to 103 pi of deionized water to bring the final solution to a
volume of 500 pi.
The following mixture of the buffers was used to achieve the desired
free metal concentrations as determined by the Chelator program.
24


DEFINED FREE Ca2+ CONCENTRATIONS
Use mixtures of buffers A and B above to produce defined free Ca2+
concentrations in the assay media. Volumes below are for a total volume of
500 pi. The calculated free Ca2+ concentrations assume 10 mM EGTA, ionic
strength 0.16, 1 mM free Mg2+, pH 7.3 and temperature of 30C.
Free Ca2+ Total Ca pi of buffer A (Ca free) pi of buffer B (Ca/EGTA) pi of 100m MgCI2
<1 nM OpM 250 pi 0pl 7.9 pi
10 nM 0.540 mM 236 13.5 7.8
20 nM 1.02 mM 225 25.5 7.6
50 nM 2.22 mM 194 56 7.2
100 nM 3.63 mM 159 91 6.9
200 nM 5.33 mM 117 133 6.4
500 nM 7.40 mM 65 185 5.8
600 nM 7.74 mM 56 194 5.7
700 nM 8.00 mM 50 200 5.6
800 nM 8.20 mM 45 205 5.5
900 nM 8.37 mM 41 209 5.4
1 pM 8.51 mM 37 213 5.4
2 pM 9.20 mM 20 230 5.2
5 pM 9.67 mM 8 242 5.1
10 pM 9.84 mM 4 246 5.0
25


DEFINED FREE Pb2+ CONCENTRAtnONS
Free Pb2+ Total Pb pi of buffer A (Ca free) pi of buffer C (Pb/EGTA) pi of 100 mM MgCI2
100 fM 41 pM 249 pi 1.0 pi 7.9 pi
200 fM 81 pM 248 2.0 7.9
500 fM 201 pM 245 | 5.0 7.9
1 pM 394 pM 240 9.85 7.8
2 pM 759 pM 231 19 7.6
5 pM 1.70 mM 208 42.5 7.4
10 pM 2.91 mM 177 73 7.0
20 pM 4.51 mM 137 113 6.6
50 pM 6.72 mM 82 168 6.0
60 pM 7.11 mM 72 ! 178 5.8
70 pM 7.42 mM 64 I 186 5.8
80 pM 7.67 mM 58 192 5.6
90 pM 7.87 mM 53 197 5.6
100 pM 8.04 mM 49 201 5.6
200 pM 8.91 mM 27 i 223 5.3
500 pM 9.54 mM 12 238 5.1
1000 pM 9.76 mM 6 244 5.0
2000 pM 9.88 mM 3 247 5.0
26


VARIABLE FREE Ca2+ AND Pb2+ CONCENTRATIONS.
Use mixtures of buffers A, B, and C above to produce solutions
with defined free Ca2+ with variable free Pb2+ concentrations.
100 nM free Ca2+with variable free Pb2+ concentrations.
Free Pb2* plB(Pb) pi C (Ca) plA(EGTA) pi MgCI2 (100mM)
1 pM 6.1 85 159 6.8
5 pM 28 77 145 6.6
10 pM 50 70 130 6.5
20 pM 84 58 108 6.2
50 pM 139 38 73 5.8
100 pM 178 25 47 5.4
200 pM 208 14.5 28 5.3
350 pM 224 8.9 17 5.2
500 pM 232 6.4 12 5.2
1000 pM 240 3.4 6 5.1
2000 pM 245 1.7 3.3 5.1
27


200 nM free Ca2+ with variable free Pb2+ concentrations.
Free Pb2+ pi B (Pb) pi C (Ca) pi A (EGTA) pi MgCl2 (100 mM)
1 pM 4.6 126 119 6.3
5 pM 21 118 111 6.2
10 pM 39 108 103 6.2
20 pM 68 94 88 6.0
50 pM 120 67 63 5.7
100 pM 162 45 43 5.4
200 pM 197 27 26 5.3
500 pM 226 13 11 5.2
500 nM free Ca2+ with variable free Pb2+ concentrations.
Free Pb2+ pi B (Pb) pi C (Ca) pi A (EGTA) pi MgCl2 (100 mM)
1 pM 2.6 180 67 5.8
5 pM 12.4 173 65 5.7
10 pM 24 164 62 5.7
20 pM 43 150 57 5.6
50 pM 86 119 45 5.5
100 pM 128 89 33 5.4
200 pM 169 59 22 5.3
350pM 196 39 15 5.2
500 pM 210 29 11 5.1
1000 pM 228 16 6 5.1
2000 pM 238 8.3 3.7 5.1
28


The reaction mixture was prepared with all of the ingredients except
PDE and incubated at 30C for five minutes. The sample was placed in the
temperature controlled quartz cuvette within the spectrophotometer and the
reaction was initiated by the rapid addition of the PDE. The reaction was
allowed to proceed for 10 minutes in a temperature-controlled quartz
cuvette at 30C, and the absorbance was continuously measured at 360
nm in a Milton-Roy 1001 spectrophotometer, with readings taken every 60
seconds. PDE activity was determined on the basis of the presence of free
inorganic phosphates. Although the lower sensitivity of the EnzChek
assay precluded the detection of any calmodulin-independent PDE activity,
it allowed for the determination of the time course of the reaction. Maximal
activity was determined to occur between 300 and 480 seconds, and the
calculated activity was taken as an average of the 300s, 360s, 420s, and
480s readings. The data points were calculated as a percentage of the
maximum activity measured that day.
Promeaa Phosphate Assay
PDE activity was also measured by the molybdate/malachite green
reaction (Lanzetta et al., 1979), using the Promega serine-threonine
phosphatase assay kit. The molybdate/malachite green reaction is more
sensitive than the EnzChek assay, allowing for the determination of
29


calmodulin-independent activity, but it can not be used for the continuous
measurement of the reaction rate. The molybdate/malachite green assay
was performed using the same buffer/chelator reaction mixtures as the
EnzChek assay above. Predetermined free-metal ion concentrations
and the buffer/chelator solutions were combined with 212 pi of deionized
water, 6 to 8 pi of 100 mM MgCk, 2 pi calmodulin (120 pM), 20 pi of cAMP
(200 pM), and 5 pi (0.5 units) of 5-nucleotidase, vortexed, and pre-
incubated for 5 minutes at 30C. The reaction was initiated by the rapid
addition of 5 pi (0.0025 units) of PDE (creating a final reaction volume of
500 pi) and allowed to proceed for 20 minutes. The reaction was
terminated by the addition of an equal volume of the molybdate/malachite
green assay mixture and incubated at room temperature for another 30
minutes. Absorbance at 600 nm was then measured in a Milton-Roy 1001
spectrophotometer. Simultaneous assays were performed in metal-free
and/or calmodulin-free solutions to determine the extent of the calmodulin-
independent PDE activity. The calmodulin-dependent PDE activity was
then determined by subtracting the calmodulin-independent PDE activity
from the total PDE activity in the calmodulin/free metal containing assays.
Activity was then calculated as a percentage of maximum activity
measured that day.
30


Calmodulin-Dependent Protein Kinase II Assays
CaMKII activity was quantified by measuring the phosphorylation of
glycogen synthase peptide by activated CaMKII. Glycogen synthase
peptide is a synthetic analog of glycogen synthase, a protein kinase C
(PKC) substrate that also functions equally well as a CaMKII substrate. It
is a ten amino acid peptide (Pro1 Leu Ser Arg Thr Leu Ser Val
- Ala Ala10) that is phosphorylated at Ser 7 with a Km of 40.3 pM (House
et al., 1987). Attached to proline is a colored dye (lissamine Rhodamine B)
which absorbs light at 570 nm.
Phosphorylated glycogen synthase peptide was collected using
affinity separation units produced by Pierce. In an acid environment
(created by the binding buffer, pH 5.0), the affinity separation membrane
has a high affinity for the phosphorylated glycogen synthase peptide. The
rest of the ingredients, including nonphosphorylated peptide, wash through
the membrane. The phosphorylated glycogen synthase peptide can then
be collected with the application of a basic solution through the membrane.
The collected glycogen synthase peptide was then measured
colometrically at 570 nm in a quartz cuvette with a Milton-Roy 1001
spectrophotometer.
31


Preparation of Stock Solutions
1. Glycogen Synthase Peptide Substrate: The glycogen synthase
was prepared as per the instructions from Pierce. A
2.08 mg sample of the peptide was dissolved in 650 pi
of deionized water (Dl) to produce a 1,77mM solution.
The solution was aloquoted into 50 pi portions, frozen
in liquid nitrogen, and stored at -70 C. One tube was
thawed per trial and used that day.
2. Calmodulin-Dependent Protein Kinase II: Two different CaMKII
proteins were used, and each was prepared per the
manufacturers instructions. The first was a CaMKII
fragment (or truncated version) produced by New
England Biolabs. The CaMKII was supplied as a 10 pi
sample containing 5000 units (1 unit = amount of
CaMKII required to catalyze the transfer of 1 pmol of
phosphate to CaMKII peptide substrate in one minute
at 30C in CaMKII buffer in a 30 pi reaction volume) of
the CaMKII fragment suspended in a 50% glycerol
32


solution containing 20 mM Tris-HCI, 50 mM NaCI, 2.0
Na2EDTA, 1 mM DTT, and 0.02% Tween-20 (pH 7.5 at
25C). This was aliquotted into 1 pi (500 units)
portions, frozen in liquid nitrogen, and stored at -70 C.
One tube was thawed per experiment.
The truncated CaMKII molecule was composed of
individual alpha subunits isolated from Spodoptera
frugiperda cells infected with recombinant baculovirus
carrying the rat truncated CaMKII DNA (NEB catalog).
The truncated CaMKII molecule was previously
reported to be Ca2+/calmodulin dependent (Takeuchi-
Suzuki et al., 1992).
The Upstate Biotechnology CaMKII sample was
purified from rat forebrain. The sample was stored a
temperature of -20 C, eliminating the complications of
freeze/thaw cycles and the need for aliquotting into
separate samples. The CaMKII was supplied as a 2.5
microgram sample of the holoenzyme containing the
33


a, p, y, and 8 isoforms suspended in
HEPES/glycerol/ethylene, which does not freeze at
-20C
3. ATP: The ATP was prepared by mixing 55 mg of Na2ATP with 50
mM Tris in a total volume solution of 10 ml to make a
10 mM stock, with the pH adjusted to 7.5. The stock
was then aliquotted into 250 pi portions, frozen in liquid
nitrogen, and stored at -70C. One tube was thawed
per day, with any unused portion refrozen for reuse.
4. Calmodulin: The calmodulin was prepared as a 30 pM stock
solution dissolved in 50 mM Tris and 0.2% BSA (which
prevents loss of CaM due to sticking to the side of the
tubes). This was aliquotted into 25 pi portions, frozen
in liquid nitrogen, and stored at -70C.
5. CaCk solutions: CaCI2 stocks were prepared as 20 mM, 10 mM,
5 mM, 2 mM, and 1 mM solutions by diluting a 100 mM
34


solution prepared from Sigma CaCfe powder dissolved
in deionized water.
6. Pb(N03)2 solutions: Pb(N03)2 stocks were prepared as 20 mM,
10 mM, 5 mM, 2 mM, and 1 mM solutions by diluting a
100 mM solution prepared from Sigma Pb(N03)2
powder (which had been oven dried to remove any
excess water) dissolved in deionized water.
7. CaMKII Dilution Buffer: The CaMKII dilution buffer had 0.1% BSA
(1mg/ml) and 0.1% Tween 20 in a 50 mM Tris solution.
This solution was adjusted to pH 7.5 at 25C and
stored at 4C. The solution was kept ice cold using it
to diluting the CaMKII, and the diluted CaMKII was
then stored on ibe.
8. EGTA/Tris: The EGTA/Tris solution was prepared with 10 mM
EGTA and 20mM Tris, adjusted to pH 7.5 at 25C, and
stored at 4C.
35


9. 10x buffer: The 10x buffer was prepared with 200 mM Tris and
100 mM MgCb, adjusted to pH 7.5 at 25C, and stored
at 4C. This solution was used to prepare a 2x
EGTA/Tris/ATP buffer solution on the day of usage.
10. NTA/Tris: The nitrilotriacetic acid (NTA)/Tris solution was only
used for the high Ca2+ concentration solutions. The
stock was prepared with 10 mM NTA and 40 mM Tris,
adjusted to pH 7.5 at 25C, and stored at 4C.
CaMKII Experimental Protocol
1. Preliminary Setup: Before making any solutions, the water
bath was turned on and set to 30 C. The vacuum
manifold and 8-well affinity separation units (or the
centrifuge separation units) were set up, labeled, and
placed in a dark area to keep the affinity membranes
(which are light-sensitive) out of the light.
2. 2x Buffer Preparation: 2x buffer was prepared by mixing 40 jul
ATP (10 mM), 40 pi of EGTA (10 mM), 40 pi 10x buffer,
36


and 80 pi of deionized water. 12.5 jul of the 2x buffer was
used to create a final concentration of 36 mM Tris, 10 mM
MgCfe, 1 mM EGTA, 1 mM Tris, 1 mM ATP, at pH 7.5 at
25C. For 1 mM and 10 mM Ca2+ reactions, 40 pi of
NTA/Tris solution was also added and the amount of
deionized water was decreased to 40 ml in the 2x buffer.
3. CaMKII Dilution: The CaMKII was prepared by diluting 0.5 pi
of CaMKII with 2.0 pi of ice-cold BSA/Tween/Tris solution,
for a 2.5 pi total volume per sample (which was stored on
ice until used). This created a final concentration of 0.025
pg of CaMKII per 25 pi sample.
4. Glycogen Synthase Peptide'. One tube of glycogen synthase
peptide was thawed for use per eight samples daily and
kept on ice until used.
5. Assay Mixtures: All ingredients except glycogen synthase
peptide were mixed together and incubated at room
temperature for 5 minutes. The glycogen synthase
37


peptide was added and the final solution was incubated
for 1 hour at 30C.
For the CaMKII/Ca2+, CaMKII/Pb2+, and CaMKII/Ca2+/Pb2+ curves,
the following concentrations were prepared as calculated by the Chelator
program (Schoenmakers et al., 199?). The Chelator program determines
the concentration of metal ions that ^re actually free to participate in the
reaction by determining what proportion will be bound up by other
substances, including other substrates and chelators, within the reaction
media. A 0 CaMKII Blank and a 10 pM Ca2+Maximum was run with
each experiment to establish 0 and 100% parameters.
Blank:
12.5 pi of 2x (EGTA/ATP/10x) buffer
2.5 pi calmodulin
2.46 pi 10 mM CaCI2
2.54 pi deionized water
38


Maximum (positive control = 10 pM free Ca2+):
12.5 pi of 2x (EGTA/ATP/1
Ox) buffer
2.5 pi calmodulin
2.46 pi 10 mM CaCI2
0.04 pi deionized water
2.5 pi CaMKII
Zero Calmodulin Negative Control:
12.5 pi of 2x (EGTA/ATP/1
Ox) buffer
2.46 pi 10 mM CaCh
2.54 pi deionized water
2.5 pi CaMKII
Zero Ca2+/ Pb2+ Negative Control:
12.5 pi of 2x (EGTA/ATP/1 Ox) buffer
2.5 pi calmodulin
2.5 pi deionized water
2.5 pi CaMKII


100 nM free Ca2+:
12.5^1 of 2x (EGTA/ATP/
Ox) buffer
2.5 jLxl calmodulin
0.67 |xl 10 mM CaCI2
1.83 |j.l deionized water
2.5 n\ CaMKII
200 nM free Ca2+:
12.5 ^l of 2x (EGTA/ATP/10x) buffer
2.5 ^l calmodulin
1.05 fil 10 mM CaCI2
1.45 nl deionized water
2.5 nl CaMKII
500 nM free Ca2+:
12.5 nl of 2x (EGTA/ATP/10X) buffer
2.5 jlxI calmodulin
1.61 nl 10 mM CaCI2
0.89 jLtl deionized water
2.5 nl CaMKII


750 nM free Ca2+:
12.5 |^l of 2x (EGTA/ATP/1
Ox) buffer
2.5 jLtl calmodulin
1.83 i^l 10 mM CaCb
1.67 jul deionized water
2.5 nl CaMKII
1 free Ca2+:
12.5 nl of 2x (EGTA/ATP/1 Ox) buffer
2.5 fxl calmodulin
1.96 f^l 10 mM CaCb
0.54 jal deionized water
2.5 nl CaMKII
2 )liM free Ca2+:
12.5^1 of 2x (EGTA/ATP/1
Dx) buffer
2.5 ^l calmodulin
2.20 nl 10 mM CaCI2
0.30 jul deionized water
2.5 nl CaMKII
41


10 pM free Ca2+:
12.5 pi of 2x (EGTA/ATP/10x) buffer
2.5 jLit calmodulin
2.46 pi 10 mM CaCb
0.04 pi deionized water
2.5 pi CaMKII
10 pM free Pb2+:
12.5 pi of 2x (EGTA/ATP/10x) buffer
2.5 pi calmodulin
1.17 pi 5 mM Pb(N03)2
1.33 pi deionized water
2.5 pi CaMKII
50 pM free Pb2+:
12.5 pi of 2x (EGTA/ATP/10x) buffer
2.5 pi calmodulin
1.5 pi 10 mM Pb(N03)2
1.0 pi deionized water
2.5 pi CaMKII
42


100 pM free Pb2+:
12.5 pi of 2x (EGTA/ATP/lOx) buffer
2.5 pi calmodulin
1.8 pi 10 mM Pb(N03)2
0.70 pi deionized water
2.5 pi CaMKII
200 pM free Pb2+:
12.5 pi of 2x (EGTA/ATP/10x) buffer
2.5 pi calmodulin
2.10 pi 10 mM Pb(N03)2
0.40 pi deionized water
2.5 pi CaMKII
500 pM free Pb2+:
12.5 pi of 2x (EGTA/ATP/10x) buffer
2.5 pi calmodulin
2.32 pi 10 mM Pb(N03)2
0.18 pi deionized water
2.5 pi CaMKII
43


1 nM free Pb2+:
12.5 nl of 2x (EGTA/ATP/lOx) buffer
2.5 jtl calmodulin
2.41 |j.l 10 mM Pb(N03)2
0.09 |nl deionized water
2.5 jtl CaMKII
10 nM free Pb2+:
12.5 n\ of 2x (EGTA/ATP/lOx) buffer
2.5 ]x\ calmodulin
1.9 fxl 20 mM Pb(N03)2
0.6 M.I deionized water
2.5 jLtl CaMKII
50 nM free Pb2+:
12.5 nl of 2x (EGTA/ATP/10x) buffer
2.5 jul calmodulin
1.53 nl 30 mM Pb(N03)2
0.97 jtl deionized water
2.5 |il CaMKII
44


100 nM free Pb2+:
12.5 pi of 2x (EGTA/ATP|f 10x) buffer
2.5 pi calmodulin
1.60 |il 30 mM Pb(N03)2
0.90 pi deionized water
2.5 pi CaMKII
500 nM free Ca2+ plus 100 pM free Pb2+:
12.5 pi of 2x (EGTA/ATP/10X) buffer
2.5 pi calmodulin
0.86 pi 10 mM CaCI2
1.22 pi 10 mM Pb(N03)2
0.42 pi deionized water
2.5 pi CaMKII
45


500 nM free Ca2+ plus 1000 pM free Pb2+:
12.5 pi of 2x (EGTA/ATP/10x) buffer
2.5 pi calmodulin
0.64 pi 10 mM CaCI2
1.14 pi 20 mM Pb(N03)2
0.72 pi deionized water
2.5 pi CaMKII
1 mM free Ca2+ plus 100 nM free Pl)2+:
12.5 pi of 2x (EGTA/ATP/10X) buffer
2.5 pi calmodulin
0.86 pi 50 mM CaCI2
1.02 pi 30 mM Pb(N03)2
0.62 pi deionized water
2.5 pi CaMKII
46


1 mM free Ca2+:
12.5 pi of 2x (EGTA/ATPpOx) buffer
2.5 pi calmodulin
1.32 pi 50 mM CaCI2
1.18 pi deionized water
2.5 pi CaMKII ;
After a 5 minute, room-temperature incubation, 5 pi of glycogen synthase
peptide was added and the solution was vortexed briefly and incubated at
30C. for 1 hour. After the assay mixtures were incubated, the peptides
were recovered as per the instructions for the Pierce kit.
1. Place 20 pi from each sample directly in the center of the affinity
membrane.
2. Briefly apply a gentle vacUum (or centrifuge) so as to draw the
sample onto the membrane, then release the vacuum.
3. Add 250 pi of the binding puffer to each well and apply the
vacuum until all of thel solution has been drawn through.
47


Release the vacuum and repeat. Release the vacuum and
discard liquid.
4. Place the collection wells underneath the vacuum collection
tubes.
5. Add 250 jul of the elution buffer to each well and apply the vacuum
until all of the solution has been drawn through. Release the
vacuum and repeat. Release the vacuum.
The optical density of the recovered phosphorylated glycogen
synthase peptide was measured in a quartz cuvette using a Milton-Roy
1001 spectrophotometer at 570 nm; The final calculations of activity were
determined by subtracting the CaM| test sample activity, and presenting the activation as a percentage of the
maximal activation (as determined by 10p.M Ca2+) for that day.
Graphical Analysis
The activation curves for the CaMKII assay and the PDE assays
were plotted using Sigma Plot, andj when appropriate, a four parameter
logistic fit was calculated. Sigma Plot was also used to determine the
EC50, activation threshold, and maximum activity of the activation curves.
i
The ANOVA section of SigmaStat was used to determine the statistical
48


significance of the differences among treatment groups and for multiple
comparison procedures where appropriate.
49


CHAPTER 3
RESULTS
Molecular Probes Phosphodiesterase Assay
The Molecular Probes assay measured the enzymatic conversion
(via PNP) of MESG in the presence of inorganic phosphate to ribose di-
phosphate and 2-amino-6-mercapto-7-methylpurine. Enzymatic
conversion of MESG results in a spectrophotometric shift in maximum
absorbance from 330 nm for the substrate to 360 nm for the product
(Molecular Probes, 1999). The inorganic phosphate is produced by 5'
nucleotidase via the cleavage of the inorganic phosphate from adenosine
monophosphate (AMP). The AMP was produced by calcium/calmodulin
activated phosphodiesterase cleaving the cyclic bond of cyclic AMP. This
reaction is the most commonly used method of determining calcium
activation of calmodulin (Cheung, 1971).
The activation of calmodulin by Ca2+, Pb2+, or both was measured
over a ten-minute time frame. In all cases, maximum activation was
apparent by 300 seconds and continued through the 480 second reading,
after which activity decreased.
50


The calcium activation curve was established using several
concentrations of free Ca2+ as established by the Chelator program. The
activity at the lower concentrations was comparable to "blank" assays run
in the absence of calmodulin. The threshold for activation appears to be
about 1 pM Ca2+, with the EC50 of approximately 2.5 pM Ca2+, and
maximum activity was achieved at concentrations of 5 pM and greater (see
figure 8). Maximum activity was calculated as a percent of the activation
achieved at 10 pM Ca2+ for that day.
Free Pb2+ in the absence of Ca2+ was found to activate calmodulin at
significantly lower concentrations. The threshold for free Pb2+ activation
appears to occur at 200 pM Pb2+, with the EC50 at 500 pM Pb2+ (as
determined by a four parameter logistic fit using the Sigma Plot program)
and maximal activation occurring at concentrations of 1 nM and higher (see
figure 9). This concentration is 1000 times lower than the Ca2+
concentration required for full activation.
Activation of calmodulin with 500 nM free Ca2+ plus variable free
Pb2+concentrations was also determined (see figure 10). At 500 pM free
Pb2+ with 500 nM Ca2+, the activation was nearly 30% higher than 500 pM
Pb2+achieved alone (see figure 11), and nearly 70% higher than 500 nM
free Ca2+ alone. However, maximum activity is still the same and still
occurs at about 1 nM free Pb2+.
51


Promeqa Molvbdate/Malachite Green PDE Assay
The Promega PDE assay functioned by measuring calmodulin
stimulated activity in the same reaction as the Molecular Probes assay.
While the Promega assay was not able to measure the time course of the
reaction, it was more sensitive and allowed for the determination of
calmodulin-independent activity. The threshold concentration for activation
with calcium was 200 nM Ca2+, with an EC50 of approximately 1200 nM
Ca2+. Maximum activity was achieved with Ca2+ concentrations of 10 ^M
and higher (see figure 12).
The free Pb2+ activation of calmodulin showed a threshold
activation concentration of approximately 200 pM and an EC50 of 360 pM
Pb2+. Maximum activation occurred at concentrations of 1 nM and higher
(see figure 13). Both Ca2+ and Pb2+ showed similar maximal stimulation of
PDE as measured by the moles of cAMP hydrolyzed per minute per unit
PDE. Maximal Ca2+stimulated activity was 215 +/-14 nmoles cAMP
hydrolyzed/minute/unit PDE, and maximal Pb2+stimulated activity was 220
+/- 8 nmoles cAMP hydrolyzed/minute/unit PDE.
Activation of PDE was also determined with Pb2+ and Ca2+ together
at subsaturating concentrations. When Ca2+ and Pb2+concentrations were
combined, activity was achieved at concentrations which neither cation
would have induced alone. At 100 nM Ca2+, threshold activation occurred
52


at a free Pb2+ concentration between 50 and 100 pM Pb2+ The EC50 was
approximately 330 pM Pb2+. At 1000 nM Ca2+, Pb2+ concentrations
between 20 and 50 pM Pb2+ induced additional activation of calmodulin
(see figure 14). Isobolographic analysis of this activation showed that the
calmodulin-stimulated PDE activity was approximately additive (see figure
15). When the EC50 data points for Pb2+ alone and Ca2+ alone were
plotted, the EC50 data points for different combinations of Pb2+ with Ca2+ all
fell very near to the line connecting the Pb2+alone and Ca2+ alone points.
This supports the conclusion that Pb2+ and Ca2+ are functioning in an
additive fashion for the activation of PDE (via calmodulin).
Calmodulin-Dependent Protein Kinase II Assays
The calmodulin-dependent protein kinase II (CaMKII) assays were
originally performed using a truncated, single polypeptide unit version of
CaMKII produced by New England Biolabs. The Ca2+ activation curve that
was observed was similar to that produced in the PDE assays. The
activation threshold was observed to be about 300 nM Ca2+ with the EC50
occurring at approximately 1250 nM Ca2+ (see figure 16). Maximal activity
was observed at concentrations of 10 pM and higher. However, when a
Pb2+ activation curve was attempted, no significant activity was achieved at
53


any concentration. In fact, Pb2+ was found to inhibit activation of CaMKII
despite the presence of stimulatory concentrations of Ca2+
Purified rat forebrain CaMKII was obtained from Upstate
Biotechnologies. A Ca2+activation curve was established. Maximal activity
occurred at concentrations of 10 pM and greater, with an activation
threshold at 500 nM and an EC50 at 1250 nM Ca2+ (see figure 17).
Activation by Pb2+ was also observed. Maximal activation occurred at 10
nM, with the activation threshold occurring below 50 pM Pb2+ and an EC50
of approximately 200 pM Pb2+ (because of the suspected inhibition of
CaMKII, the calculated EC50 may be inaccurate for the actual 50%
activation of calmodulin by Pb2+). However, maximum activation as
compared with 10 pM Ca2+ was not observed at any Pb2+ concentration,
and at higher concentrations activation of CaMKII actually decreased (see
figure 18).
In the presence of Ca2+, Pb2+ was found to have a cooperative effect
for activating CaMKII at low concentrations, but was inhibitory at higher
concentrations (see figure 19). 500 nM Ca2+ with 100 pM Pb2+ was found
to induce over 60% activation, which was more than twice the activation
achieved by either cation alone. When 500 nM Ca2+ was combined with
1000 pM Pb2+, activation did not continue to increase, and was not found to
be significantly different from that achieved by 1000 pM Pb2+ alone.
54


At even higher concentrations, Pb2+ continued to have an inhibitory
effect on CaMKII activation. 100 nM Pb2+was found to decrease the
activation achieved by 1 mM Ca2+ ffom 112% of maximum to 68% (see
figure 20). These results are consistent with the presence of an inhibitory
binding site for Pb2+ that prevents the activation of CaMKII by activated
calmodulin. Extremely high concentrations of Ca2+ also decreased the
activation of CaMKII. 10 mM Ca2+ and 10 mM Ca2+ with 100 nM Pb2+ both
only activated CaMKII to slightly abbve 50% (see figure 21).
55


CHAPTER 4
DISCUSSION
Inorganic lead has the ability to inappropriately initiate, intensify,
modify, or inhibit a wide range of cellular processes that are normally
regulated by calcium. Furthermore, inorganic lead can potentially influence
molecules at non-calcium-binding sites as well. Because Pb2+ has the
ability to activate calmodulin, it would be easy to want to generalize the
effect of Pb2+ exposure as limited to the activation of Ca2+-binding sites.
However, the actual effect that Pb2+-exposure has within a cell is much
more complicated.
Ca2+ concentrations are tightly regulated within a cell. Calmodulin is
normally only activated in discrete locations where Ca2+ concentrations are
temporarily elevated, such as in close proximity to calcium channels within
the cell membrane. There are data indicating that inactivated calmodulin
may even be sequestered by plasma membrane bound proteins near the
calcium channels (Liu and Storm, 1990). As calcium diffuses away from
the source and is bound up by other calcium-binding proteins, calmodulin
activity ceases. This allows for the localization of a hot-spot or a
microdomain of calcium/calmodulin regulated activity (Smith et a/., 1995;
56


Naraghi and Neher, 1997; Kern, et al., 1999). If the cell is contaminated
with even small levels of free Pb2+, this may have the effect of amplifying
the activation of calmodulin over a much larger area within the cell, and the
level of activity within the hot-spot may also be elevated beyond the norm.
If Pb2+ concentrations are high enough, Pb2+ could potentially initiate
activation of calmodulin independent of the presence of any free calcium.
There is evidence that Pb2+ is internalized into cells through calcium
channels to begin with (Simons and Pocock, 1987). This would suggest
that the potentially highest concentration of Pb2+contamination within the
cell could be at the very same spots that would have the highest potential
for altering calcium/calmodulin-regulated systems.
In the PDE assays, we found that Pb2+ could act as a full agonist in
the activation of calcium/calmodulin-activated phosphodiesterase, as
previously shown (Goldstein and Ar, 1983; Habermann et al., 1983).
However, we found that Pb2+ could activate calmodulin at much lower
concentrations than previously reported. This is likely due to the use of
EGTA and other metal chelators that were previously unaccounted for
(Kern et al., 1999). Our results indicate that Pb2+ can function to activate
calmodulin independently of Ca2+, and at concentrations one thousand
times lower than calcium. Pb2+ also appears to be capable of functioning in
an additive fashion in the presence of Ca2+.
57


Despite the ability of inorganic lead to activate calmodulin, the
generalization that inorganic lead will modify cell-signaling systems by
acting as a Ca2+ substitute on calmodulin cannot be made. Fluorescent
studies have shown that activation of calmodulin by Pb2+ appears to cause
a slightly different conformational change than that which is induced by
Ca2+. Analysis with hydrophobic probes such as N-phenyl-1-
naphthylamine (NPN) or 8-anilino-1-naphthalenesulfonate (ANS)
determined that the hydrophobic region within the globular domains that is
normally exposed during calcium activation is also exposed by Pb2+
activation. However, when activated by Pb2+, the calmodulin molecule
induces a greater increase fluorescence than that initiated by Ca2+ (Kern et
ai, 1999). This could possibly indicate that, when activated by lead, the
calmodulin molecule undergoes a larger conformational change, exposing
a greater area of hydrophobic residues.
It is difficult to predict the effect that the difference in the
conformational change initiated by inorganic lead on calmodulin will have
on target molecules. Consideration must be given to whether or not the
binding site of the target molecule is specific enough to be affected by the
difference in the degree of hydrophobic exposure. Activation of calmodulin
by inorganic lead does not appear to change the activation of PDE. The
maximum activity initiated by Ca2+, Pb2+, and Ca2+ with Pb2+ in the
58


calmodulin/PDE assays (both the molybdate/malachite green reaction and
the EnzChek assay) were roughly the same. However, given the high
degree of specificity displayed by most enzymes, it is unlikely that all of the
different classes of calmodulin-activated molecules will be unaffected.
Whether the effect would be an increase or decrease in activity must also
be determined on a case-by-case basis.
The EF-hand Ca2+-binding sites on the globular domains of
calmodulin display cooperative binding, with the affinity for calcium at each
site increasing with the binding of each Ca2+ It is currently not known what
effect the presence of inorganic lead would have on the cooperative
binding of Ca2+. The determination that Pb2+ and Ca2+ act in an additive
fashion for PDE activation suggests that it is possible that Pb2+ and Ca2+
are capable of occupying different binding sites on the same molecule.
However, this raises several questions as to the interactions between the
binding sites on calmodulin. Since the EF-hand molecules have a higher
affinity for Pb2+ than Ca2+, does Pb2+ bind in the same order as Ca2+
would? If the order of binding site occupation is altered by the presence of
inorganic lead, how does this affect the kink formation within the alpha
helix that is required for the interactions between the globular domains?
Does the binding of Pb2+ at one site on the globular domain affect the
affinity for ion binding at the neighboring site? If different sites are
59


occupied by different ions on the same molecule, what is the effect on the
exposure of the hydrophobic domains?
Further consideration must be given as to what effect the binding of
calmodulin to the target molecule has on the EF-hand site affinity for Pb2+.
Some target molecules, such as Ca2+/ATPase, initiate a conformational
change within calmodulin that greatly increases the affinity for Ca2+ within
the EF-hand binding sites. This allows calmodulin to remain active for
extended periods of time despite a low Ca2+ concentration (Alberts et al.,
1994). It cannot be accurately predicted what effect inorganic lead would
have on this type of system.
Attempts to generalize the effects of inorganic lead exposure are
also complicated by the fact that Pb2+ has different, calmodulin-
independent effects on other proteins. When we first attempted to analyze
Pb2+ activation of CaMKII using a truncated version of the protein
(produced by New England Biolabs), we assumed that the inhibitory effect
that Pb2+ had on CaMKII was a caused by the monomeric property of the
synthetic protein. Indeed, we did see a decrease in the magnitude of the
inhibition of whole CaMKII. However, activity initiated by Pb2+ never
achieved full activation as it did in the PDE assays. Even activity
stimulated by massive quantities of calcium was still inhibited from ever
reaching full activation by small quantities of free Pb2+. At Ca2+
60


concentrations of 1mM and higher, the competition by 100 nM free Pb2+ for
the calmodulin binding sites should have been minimal. This led to the
conclusion that the Pb2+ inhibitory effect must be caused by a Pb2+
interaction at another site on the kinase. Pb2+ is likely providing full
activation of calmodulin just as it did in the PDE assays. But, once it
reaches a particular concentration (possibly as low as 100 pM free Pb2+),
Pb2+ begins to bind to the inhibitory site directly on CaMKII, decreasing the
activation of CaMKII by calmodulin in a non-competitive manner.
Extremely high quantities of Ca2+ (10 mM) had a similar inhibitory effect.
Pb2+-inhibition similar to what we measured in CaMKII has been
observed in other molecules as well. Preliminary data (unpublished)
suggests that protein kinase A may also be inhibited by Pb2+ at higher
concentrations. Effects similar to this have also been observed with
calcineurin (a calcium/calmodulin-dependent phosphatase) and protein
kinase C. Low concentrations of Pb2+ increased the activation of
calcineurin, but higher concentrations reduced activity in a manner very
similar to our CaMKII results (Kern and Audesirk, 1999). Activation of
calcineurin via Pb2+-activated calmodulin was reported to occur at Pb2+
concentrations as low as 100 pM Pb2+. As the Pb2+ concentration was
increased, activation of calcineurin decreased. At low concentrations, Pb2+
was reported to be a partial agonist in the activation of protein kinase C
61


(PKC). However, at higher concentrations it appears that Pb2+ can reduce
the stimulatory effect that Ca2+ has on the activation of PKC (Long et al.,
1994; Tomsig and Suszkiw, 1995).
One possible common mechanism for the inhibition of CaMKII, PKA,
and PKC could be that inorganic lead interferes with of Mg2+/phosphate
interactions at higher concentrations. Mg2+ is required for stabilization of
the phosphate bonds in most triphosphate cleavage mechanisms. It is not
unlikely that high enough concentrations of Pb2+ could interfere with
Mg2+/phosphate associations. This could affect the addition of a phosphate
by kinases, or the removal of phosphates by phosphatases.
In conclusion, Pb2+ causes complete activation of calmodulin
independently, and in the presence of Ca2+, at biologically relevant
concentrations. Since calmodulin is capable of regulating the activation of
over twenty different proteins and is found in high quantities in virtually
every eukaryotic cell, the importance of understanding inorganic leads
ability to alter the activating mechanisms of calmodulin cannot be over-
estimated. Furthermore, since inorganic lead has also shown that it is
capable of inhibiting other calmodulin-dependent proteins, the effects that
Pb2+ has on different cell systems need to be assessed one at a time.
62


PDE Activity
(Percent of Maximum)
Effect of Variable Free Ca2+ Concentrations
on Calmodulin-Stimulated
Phosphodiesterase Activity
100.00
75.00
50.00
25.00
0.00
7^
J---tV-*-----------------1--------------L~
0 100 1000 10000
Free Ca2+ Concentration (nM)
Figure 8
63


PDE Activity
(Percentage of Maximum)
Effect of Variable Free Pb2+ Concentrations on Calmodulin
Stimulated Phosphodiesterase Activity
Pb2+ Concentration (pM)
Figure 9
64


Phosphodiesterase Activity
(Percent of Maximum)
Effect of 500 nM Ca2+ with Variable Pb2+
Concentrations on PDE Activity
100.00
75.00
50.00
25.00
0.00
0 10 100 1000 10000
Pb2+ Concentration (pM)
7^
t-V/*i-------r
Figure 10
65


Phosphodiesterase Activity
(Percent of Maximum)
Effect of 500 nM Ca2+ with Variable Pb2+
Concentrations on PDE Activity
100.00
75.00
50.00
25.00
0.00
0 10 100 1000 10000
Pb2+ Concentration (pM)

T-V^-T
Figure 11
66


PDE normalized Ca2+ curve Malachite Green
Phosphodiesterase Assay
9+
free Ca concentration (nM)
Figure 12
67


Malachite Green PDE Assay with
Variable Pb2+ Concentrations
CL
s
(/>
O
0
CL
9+
free Pb concentration (pM)
Figure 13
68


2+
free Pb concentration (pM)
o 1000 nM free Ca2+ with variable Pb2+
100 nM free Ca2+ with variable Pb2+
A Zero free Ca2+ with variable free Pb2+
Figure 14
69


Isobolographic Analysis of PDE Activity
Figure 15
70


Truncated CaM Kll Activation (percent of maximum)
Effect of Variable Free Ca2+ on Calmodulin-Stimulated
Truncated CaM Kinase II
Calcuim Concentration (nM)
Figure 16
71


CaM Kll Activity
(Percent of Maximum)
Effect of Variable Concentrations of
Free Ca2+ on Calmodulin-Stimulated
CaM Kinase II Activity
Free Ca2+ Concentration (nM)
Figure 17
72


CaM Kll Activity
(Percent of Maximum)
Effect of Variable Concentrations of Free Pb2+
on Calmodulin-Stimulated
CaM Kinase II Activity
100.00
75.00
50.00
25.00
0.00

Activation Threshold ~ 50 pM
EC50 200 pM
Maximum Activation ~ 10 nM
i
i ~//~i I I I r
0 101 102 103 104 105
Pb2+ Concentration (pM)
Figure 18
73


CaMKII Activity (Percent of Maximum)
Comparison of Activation of CaMKII
by 500 nM Ca2+ with Variable Pb2+
vs. Pb2+ Alone
100.00
75.00
50.00
25.00
0.00
l I Pb2+ with 500 nM Ca2+
0 100 1000
Lead Concentration (in pM)
Figure 19
74


CamKII Activity (Percent of Maximum)
Comparison of Activation of CaMKII
with 0 Ca2+, 1 mM Ca
2+
1mM Ca2* with 100 nM Pb
2+
and 100 nM Pb
2+
Figure 20
75


Effect of 10mM Free Ca2+ w/ & w/o 100nM Free Pb2+
On Calmodulin-Stimulated Cam Kinase II
150
125 -
§ 100
k-
><
£ 75 -|
o
<
ro
O
0 100
Free Lead Concentration (nM)
Figure 21
76


FIGURE LEGENDS
Figure 1: Amino acid sequence of calmodulin. The globular domains each
contain two EF-hand binding sites, and are separated by a 28 amino
acid alpha helix (Lafitte, 1995).
Figure 2: Depiction of the EF-hand Ca2+ binding site that is highly
conserved in most calcium-binding proteins. The EF-hands in
calmodulin are interconnected on each globular domain, and the two
globular domains can interact through their connection via a 28
amino acid alpha helix (Nelson and Chazin, 1998).
Figure 3: Model of feedback loop, synaptic input, and CaM/CaMKII activity
as proposed as a possible mechanism for L.T.P. within a post-
synaptic neuron. Displays the complexities involved with trying to
predict the cellular effect of inorganic lead contamination (AC 1/8 =
adenylate cyclase, CaN = calcineurin, PKA = protein kinase A)
(Bhalla and Iyengar, 1999).
77


Figure 4: Examples of enzymes activated by calmodulin (adapted from
Hinrichsen, 1993).
Figure 5: Conformational changes in an individual unit of CaMKII that are
originally initiated by Ca2+/calmodulin are able to be maintained
even after the dissociation of the activating Ca2+/CaM complex. This
allows a CaMKII unit to maintain partial activity even after Ca2+
concentrations have decreased (Alberts et ai, 1994).
Figure 6: Structure and function of the Ca2+/calmodulin-dependent protein
kinase II holoenzyme demonstrating the conversion of individual
units between the phosphorylated (on) and non-phosphorylated
states (Usman, 1994).
Figure 7: Release of inorganic phosphate by 5-nucleotidase from 5-AMP
permits the quantification of PDE activity. Reaction of cAMP to 5-
AMP is catalyzed by calmodulin activated PDE1 isoform.
Figure 8: Activation of phosphodiesterase (via calmodulin) vs.
concentration of free Ca2+. Data points are means +/- the
s.e.m.(minimum n=4 collected in four different experiments). The
78


curve shows a four-parameter logistic fit, as calculated using Sigma
Plot. The activation threshold was 1000 nM Ca2+. The EC50 was
2600 nM, and maximum activation occurred at concentrations equal
to or greater than 5000 nM Ca2+. Activation was measured using
the Molecular Probes EnzChek as described in the methods
section. The 2000 nM Ca2+ point was significantly different from
either the 1000 nM Ca2+ or the 5000 nM Ca2+. The data were
analyzed by ANOVA using the Student-Newman-Keuls method
(where appropriate) for multiple comparisons.
Figure 9: Activation of phosphodiesterase (via calmodulin) vs.
concentration of free Pb2+. Sample sizes ranged from four to seven
collected in four separate experiments. The activation threshold
was 200 pM Pb2+. The EC50 was 500 pM Pb2+, and maximum
activation occurred at concentrations equal to or greater than 1000
pM Pb2+. The 350 pM and 500 pM data points were significantly
different from one another and from the 200 pM and 1000 pM Pb2+
points. The data were analyzed by ANOVA using the Student-
Newman-Keuls method (where appropriate) for multiple
comparisons. Conventions as in figure 8.
79


Figure 10: Activation of phosphodiesterase (via calmodulin) vs.
concentration of free Pb2+ with 500 nM free Ca2+. Sample sizes
ranged from four to seven, collected on four separate experiments.
The activation threshold was 100 pM Pb2+. The EC50 was 290 pM
Pb2+, and maximum activation occurred at concentrations equal to
or greater than 1000 pM Pb2+. Conventions as in figure 8.
Figure 11: This graph is an overlay of figure 9 and figure 10 for the purpose
of comparing Pb2+activation of phosphodiesterase (via calmodulin)
alone and in conjunction with 500 nM Ca2+ (which is below the
activation threshold by itself). Comparisons of these two curves
show a significant increase in activation at 350 pM and 500 pM Pb2+
when used in conjunction with 500 nM free Ca2+. The data were
analyzed with the statistical t-test. Conventions as in figure 8.
Figure 12: Activation of phosphodiesterase (via calmodulin) vs. free Ca2+
concentration. Data points are means +/- s.e.m (minimum n =13
collected in 5 different experiments). Curve shows a four-parameter
logistic fit, as calculated by Sigma Plot. The activation threshold
was 200 nM Ca2+. The EC50 was 1200 nM Ca2+, and the maximum
80


activation occurred at 10 pM free Ca2+. Activation was determined
using the Promega phosphate kit, as described in the materials and
methods section.
Figure 13: Activation of phosphodiesterase (via calmodulin) vs.
concentration of free Pb2+. Minimum sample size was 8 collected in
5 separate experiments. The activation threshold was 200 pM Pb2+.
The EC50 was 360 pM Pb2+, and maximum activation occurred at
concentrations equal to or greater than 1000 pM Pb2+. Conventions
as in figure 12.
Figure 14: Activation of phosphodiesterase (via calmodulin) vs.
concentration of free Pb2+ with 0 Ca2+ (triangles), 100 nM free Ca2+
(filled circles), and 1000 nM free Ca2+ (open circles), overlaid for
comparison. The threshold for enhancement of activation by Pb2+
with 1000 nM free Ca2+ was between 20 and 50 pM Pb2+, as
compared with an activation threshold of 200 pM Pb2+ in conjunction
with 100 nM Ca2+, and an activation threshold of 200 pM Pb2+
without Ca2+. The maximum activity is approximately the same with
all combinations of Pb2+ and Ca2+. Data points are means +/-
s.e.m., with a minimum sample size of 7 collected in 5 separate
81


experiments. The data were analyzed by ANOVA using the Student-
Newman-Keuls method (where appropriate) for multiple
comparisons. Conventions as in figure 12.
Figure 15: Isobolographic analysis of phosphodiesterase activity as
activated by free Pb2+ and free Ca2+, alone and in combined
concentrations. All data points are concentrations of Ca2+, Pb2+, or
both that initiate 50% activation of phosphodiesterase (via
calmodulin). The line connects the 0 Ca2+ EC50 on the Y axis, and
the 0 Pb2+ EC50 on the X axis. The other points are EC50 values
achieved with different combinations of Ca2+ and Pb2+
concentrations.
Figure 16: Activation of truncated CaMKII (via calmodulin) vs.
concentration of free Ca2+. Data points are means +/- the s.e.m. (n
= 5 to 20 collected in a minimum of 5 different experiments). The
curve shows a four-parameter logistic fit, as calculated using Sigma
Plot. The activation threshold was 500 nM Ca2+. The EC50 was
1250 nM, and maximum activation occurred at concentrations equal
to or greater than 10 pM Ca2+.
82


Figure 17: Activation of CaMKII (via calmodulin) vs. concentration of free
Ca2+. Data points are means +/- the s.e.m. (n = 5 to 20 collected in
a minimum of 5 different experiments). The curve shows a four-
parameter logistic fit, as calculated using Sigma Plot. The activation
threshold was 500 nM Ca2+. The EC50 was 1250 nM, and maximum
activation occurred at concentrations equal to or greater than 10 pM
Ca2+. Conventions as in figure 16.
Figure 18: Activation of CaMKII (via calmodulin) vs. concentration of free
Pb2+. Sample sizes ranged from 4 to 18, collected in a minimum of
4 different experiments. The activation threshold was 50 pM Pb2+.
The EC50 was calculated to be 200 pM Pb2+, however it should be
noted that this calculation is likely skewed by the inhibition of
CaMKII at higher concentrations. Maximum activation occurred at
10000 pM Pb2+. Conventions as in figure 16.
Figure 19: Comparison of Pb2+ initiation of CaMKII activation in the
presence of absence of 500 nM Ca2+ (which by itself is below the
activation threshold). Data points are means +/- s.e.m, with sample
sizes ranged from 4 to 14, collected in a minimum of 3 different
experiments. Pb2+ in conjunction with 500 nM Ca2+ shows a
83


significant increase in activation at 100 pM Pb2+ as compared with
Pb2+ alone, but the activation level is not significantly different from
either 1000 pM Pb2+with 500 nM Ca2+ or 1000 pM Pb2+ alone. This
is consistent with the possible activation of a Pb2+ inhibitory site at
higher concentrations on the CaMKII molecule. The 1000 pM Pb2+
with and without 500 nM Ca2+ data points were not significantly
different from each other. The data were analyzed by ANOVA using
the Student-Newman-Keuls method (where appropriate) for multiple
comparisons.
Figure 20: Comparison of Pb2+ initiation of CaMKII activation in the
presence of absence of 1 mM Ca2+ (which is a high enough
concentration to theoretically eliminate most Pb2+ activation of
Ca2+/calmodulin binding sites). Data points are means +/- s.e.m,
with sample sizes ranged from 5 to 17, collected in a minimum of 5
different experiments. 1 mM Ca2+ without Pb2+ activates CaMKII
completely. When 100 nM Pb2+ is used in conjunction with 1 mM
Ca2+, activation drops to below 70% activation. This is again
consistent with the possible activation of a Pb2+ inhibitory site at
higher concentrations on the CaMKII molecule. The data were
analyzed by ANOVA using the Student-Newman-Keuls method
84


(where appropriate) for multiple comparisons. Activity with 1 mM
i
Ca2+ was significantly greater than the activity with 1 mM Ca2+ and
100 nM Pb2+.
Figure 21: Comparison of 10 mM Ce2+ (which is a high enough
concentration to theoretically eliminate most Pb2+ activation of
Ca2+/calmodulin binding site^) activation with and without 100 nM
Pb2+. The data were examined for significant variance using the t-
test and determined not to b^ significantly different from each other.
85


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