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Detection and quantification of xenopus laevis phospholipase d

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Title:
Detection and quantification of xenopus laevis phospholipase d
Creator:
Savi, Kai Alina
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Language:
English
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x, 57 leaves : illustrations ; 28 cm

Thesis/Dissertation Information

Degree:
Master's ( Master of arts)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
Department of Integrative Biology, CU Denver
Degree Disciplines:
Biology

Subjects

Subjects / Keywords:
Phospholipases ( lcsh )
Xenopus laevis ( lcsh )
Cell interaction ( lcsh )
Cell interaction ( fast )
Phospholipases ( fast )
Xenopus laevis ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 56-57).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Kai Alina Savi.

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University of Colorado Denver
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Auraria Library
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
47120981 ( OCLC )
ocm47120981
Classification:
LD1190.L45 2000m .S38 ( lcc )

Full Text
DETECTION AND QUANTIFICATION OF
XENOPUS LAEVIS PHOSPHOLIPASE D
by
Kai Alina Savi
B.S., University of Arizona, 1991
B.S., Metropolitan State College of Denver, 1998
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
2000


This thesis for the Master of Arts
degree by
Kai Alina Savi
has been approved
by
limberly Roane


Savi, Kai Alina (M.A., Biology)
Determination of Molecular Weight of Xenopus Laevis Phospholipase D
Thesis directed by Associate Professor Bradley J. Stith.
ABSTRACT
Phospholipase D (PLD) is an enzyme found in Xenopus eggs and oocytes. When
activated, PLD catalyzes the cleavage of phosphatidylcholine (PC) to phoshpatidic
acid (PA) and choline. Phosphatidic acid can then either be converted to
diacylglycerol (DAG) or lysophosphatidic acid through binding to phospholipase C- y.
Phosphatidic acid is thought to cause an increase in intracellular Ca 2+ levels subsequent
to fertilization. As an alternative pathway, phosphatidic acid can be converted to
DAG by phosphatidate phosphohydrolase. It is thought that DAG could then
activate Protein Kinase C to induce fertilization events.
PLD exists in a variety of isoforms and many are not characterized. However, several
have been purified and their molecular weights determined. Comparisons with PLD
from other sources indicates molecular weights in the range of 100 to 120 kDa. For
example, human membrane-associated PLD1 has been found to be 124kDa. A human
iii


cytosolic PLD is suggested to have a molecular weight of 1 lOkDa.
We sought to identify PLD from Xenopus oocytes and eggs and to determine the
molecular weight of the enzyme. Using a variety antibodies to mammalian forms of
PLD we performed a series of immunoprecipitations to isolate and Western blots to
identify the size of the protein. We found bands at 65kDa and lOOkDa.
This abstract accurately represents the content of the candidates thesis. I recommend
its publication.
rv


ACKNOWLEDGEMENTS
I would like to thank Dr. Bradley Stith for allowing me to work in his laboratory. He
has been an excellent teacher both in the classroom and in the laboratory. Thank you.
I would like to thank Dr. Ellen J. Levy for her support and instruction. I will never
cease to be amazed by her teaching abilities.
1 would like to thank Dr. Timberly Roane for her encouragement and assistance in all
things academic.
Finally, I would like to thank Patricia Medina for all her help and patience. Leslie
Waggoner for sharing her experience and her friendship. Douglas Petcoff for his
advice and expertise. William Holland for his assistance. Thanks to all of you.


DEDICATION
This thesis is dedicated to my friends and family for their patience during my
research, coursework and writing of this thesis. To my husband Robert for his love,
tireless support and confidence in my abilities. To my son Remy for bringing such
joy to my life. To my parents for their never-ending encouragement. To my mother
and father-in-law for their concern and interest.
-vi-


CONTENTS
Figures........................................................ ix
CHAPTER
1. INTRODUCTION................................................. 1
Background ............................................... I
Phospholipid Signaling ....................................4
Phospholipase C in Fertilization ..........................4
Phospholipase Products.....................................7
Phospholipase D in Fertilization ..........................8
Measurement of Phospholipase D Activity.................. 12
Role of Phosphatidic Acid in the Cell.................... 13
Phospholipase D and the Cell Cycle ...................... 13
Other Mechanisms of Phospholipase D Activation........... 16
Phospholipase D Isoforms................................. 16
Structure of Phospholipase D............................. 17
Molecular Weight of Phospholipase D.......................21
Xenopus laevis............................................21
2. MATERIALS, METHODS AND RESULTS...............................24
Xenopus laevis Oocytes and Eggs...........................24
Deielly Xenopus laevis Eggs...............................25
Xenopus laevis Sperm......................................26
Control Fertilization.....................................26
Initial Immunoprecipitation with Anti-PLD/KLH Antibody...27
Comparison of Human and Mouse Anti-PLD Antibodies
to Anti-PLD/KLH .......................................32
vii


Verification of Human and Mouse Anti-PLD Antibodies
and Anti-PLD/KLH Using Xenopus Oocytes .............35
Incubation of Oocytes in Progesterone to Stimulate Meiosis and
Identify a Cell-Cycle Related Change in PLD Levels.38
Fertilization Induced Change in PLD Levels
at 15 minutes, 30 minutes and 1 hour ...............42
3. DISCUSSION ..............................45
Molecular Weight of Xenopus Phospholipase D............45
Densitometry Readings of PLD 1 and PLD2 ...............49
Future Experiments ....................................49
APPENDIX
A. CHEMICALS, SOLUTIONS AND EQUIPMENT...................51
Chemicals..............................................51
Solutions .............................................52
Equipment .............................................55
REFERENCES .........................................................56
vm


FIGURES
FIGURE
1 .A Signal amplification by a membrane-bound receptor ............3
l.B Phospholipase C mediated breakdown of PIP2 ...................5
1 .C Phospholipid signaling by Phospholipase C ....................6
1 .D Diacylglycerol levels after cell stimulation..................7
1 .E Dephosphorylation of phosphotidic acid
to sn 1,2-diacylglycerol ..................................8
1 .F Phospholipase D mediated breakdown of phosphatidylcholine .... 9
1 .G Interactions of PLC and PLD signaling paths ................ 11
l.H Transphosphatidylationreaction ............................. 12
1.1 The cell cycle and corresponding PC levels ................. 15
1 .J. Structural domains of phospholipase D....................... 18
1. K Mechanism of PLD activity....................................20
2. A Initial immunoprecipitation with anti-PLD/KLH antibody......30
2.B Plot of molecular weight markers and determination of
sample molecular weight...................................31
2.C Lane loading of gel..........................................34
2.D Compare human and mouse anti-PLD antibodies to
anti-PLD/KLH using dejellied eggs.........................36
2.E Verification of human and mouse anti-PLD antibodies and
anti-PLD/KLH using oocytes................................37
2.F Incubation of oocytes in progesterone to stimulate meiosis
and identify a cell cycle related change in PLD levels...40
2.G Incubation of oocytes in progesterone........................41
IX


FIGURE
2.H Fertilization induced change in PLD levels at 15 minutes,
30 minutes and 1 hour..........................................44
2.1 Change in PLD levels at Fertilization .................................45
x


CHAPTER 1
INTRODUCTION
Background
Intercellular communication is an important component for survival in most any
multicellular animal. Individual cells must coordinate their activity with other cells in
an organism if the organism is to survive and function. Individual cells need to be
able to sense both the general status of the organism, as well as the functional status
of other cells within the organism (Morgan, 1989).
Cellular communication can occur in a variety of ways, including; direct contact via
gap junctions, membrane channels, or membrane-bound receptors. Mechanisms
involving membrane-bound receptors are initiated by the binding of a ligand to a
specific cellular receptor. The binding of the ligand to the receptor then triggers an
intracellular molecular switch (Alberts et. al.,1994).
In such communication systems, an extracellular soluble molecule that binds initially
to the cell via a membrane bound receptor is known as the first messenger. This is
often a hormone or neurotransmitter, but it can also include the binding of a sperm
molecule to an egg. The term first messenger is due to the fact that this initial
1


binding is the first link in a series of events resulting in a cellular response. This
contact of the first messenger initiates the next chemical signal. Receptors the act
through the activation of G-proteins, ion channels or kinase activity. The receptor
itself could be a kinase. The receptor could also activate a protein that is a kinase.
The intracellular signal that is generated in amplified amounts by the binding of the
first messenger is known as the second messenger. This signal begins the chain of
intracellular responses to the first messenger (Morgan, 1989).
A hypothesis proposed by Earl W. Sutherland in 1959 introduced the principle of the
second messenger (Robison, 1971). To be a second messenger, a molecule or ion
must satisfy the following criteria: The concentration of the second messenger in the
cytoplasm must rise and fall in response to the binding and release of the hormone
and receptor. The cellular metabolism must be affected in the way that the first
messenger is known to work. There must be a mechanism for removing the second
messenger to terminate the signal. There must be a demonstrated relationship
between the binding of the hormone, the change in second messenger concentration
and a physiological response.
It is with the second messenger that the signal begins to be amplified. The binding of
one hormone or extracellular signaling molecule propagates the message by causing
an increase in concentration of the second messenger (Figure 1 .A). These second
2


messengers then act to activate the next step in the pathway. Amplification (i.e.,
production of a large number of second messengers) is often accompanied by
hydrolysis of molecules already present in a cell to produce many second messengers.
Activation of a kinase that can act on many substrates. Movement of molecules via
an ion channel is another example of amplification.
Figure 1 .A
Signal amplification by a membrane-bound receptor
The binding of a hormone to a receptor is the first messenger. Upon activation of the
receptor, a second messenger is initiated which results in amplification of the signal.
3


Phospholipid Signaling
Phospholipid breakdown is an important step in signaling pathways stimulated by
fertilization and hormones. Phospholipid breakdown plays a role in the control of
such cellular responses as regulation of proliferation, secretion, platelet aggregation,
macrophage function and fertilization (Camero and Lacal, 1993).
Phospholipase C in Fertilization
The hydrolysis of one phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) by
phospholipase C (PLC) is known to produce two signaling molecules: inositol
trisphosphate (IP3) which act to release intracellular Ca2+ and diacylglycerol (DAG)
(Figure 1 .B) which activate protein kinase C (Figure 1 .C) (Exton et. al., 1991). Two
isoforms of phospholipase C have been very well studied. Phospholipase CP (PLC P)
is known to be activated by G-protein linked receptors. Phospholipase Cy (PLCy) is
known to be activated by tyrosine kinase receptors.
4


H3-CH2(n)-C0CH2
OH OH
Phosphatidylinositol 4,5-bisphosphate
(PIP2)
sn 1,2-diacylglycerol Inositol 1,4,5-trisphosphate
IDAG) (IP3)
Figure l.B
Phospholipase C mediated breakdown ofphosphaditylinositol 4,5-bisphosphate
PLC acts to cleave PIP2 into DAG and IP3.
5


Membrane
Biological
Response
(transcription,
translation,
mitogenesis,
cell
growth)
Figure l.C
Phospholipid signaling by phospholipase C
The hydrolysis of one phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) by
phospholipase C (PLC) is known to produce two signaling molecules: inositol
trisphosphate (IP3) which act to release intracellular Ca2+ and sn 1,2-diacylglycerol
(DAG) which activate protein kinase C.
6


Phospholipase Products
Previous research in our lab has shown that IP3 increases only 150 fmol at
fertilization (Stith, 1993). Yet, the DAG increase is 300-500 times greater. This
would indicate that DAG mostly comes from a non-PLC source. One such source is
phospholipase D (PLD).
It has also been shown that DAG levels respond to stimulation by a hormone with a
biphasic increase (Figure 1 .D); an initial rapid, transient peak is followed by a slowly
developing, prolonged accumulation (Exton, 1994). It has been suggested that the
initial DAG spike is due to the action of PLC-stimulated hydrolysis of PIP2, while the
second, larger increase may be due to the activity of PLD mediated breakdown of
phosphatidylcholine (PC) into choline and phosphatidic acid (PA) and the subsequent
conversion of PA into DAG (Figure 1 .E).
Figure l.D
Diacylglycerol levels after cell stimulation
1


Phospholipase D in Fertilization
When activated, PLD catalyzes the cleavage of phosphatidylcholine (PC) to
phoshpatidic acid (PA) and choline (Figure 1 .F). PA is thought to cause an increase
in intracellular Ca2+ levels subsequent to fertilization by binding PLCy and initiating
the PLC pathway. As an alternative, PA can be converted to DAG by phosphatidate
phosphohydrolase (Figure 1 .E). It is thought that DAG could then activate Protein
Kinase C to induce fertilization events.
OH
op=o
Ctj j CH 2 CH 2
0 0
phosphotidate phosphohydrolase
phosphatidic acid
OH
sn 1,2-diacylglycerol
(DAG)
Figure l.E
Dephosphorylation of phosphotidic acid to sn 1,2-diacylglycerol
8


Phosphatidic Acid
Figure l.F
Phospholipase D mediated breakdown of phosphatidylcholine
9


Previous research in our lab has shown that another PLD product, choline, mass in
Xenopus eggs at fertilization peaks before DAG (Stith et. al., 1997). This would
provide support for a suggestion that the dephosphorylation of PA to DAG occurs
(Figure 1 .G). The PA produced by PLD would then be converted to DAG by
phosphatidate phosphohydrolase (PAP) (Exton, 1994). DAG derived from the
hydrolysis of PC is thought to be capable of activating Ca2+-independent protein
kinase C (PKC) (Bocckino et. al., 1996). It has also been shown that the choline
derived from the PLD mediated breakdown of PC is converted into phosphorylcholine
(Pcho) by choline kinase.
10


Figure 1 .G
Interactions of PLC and PLD signaling paths
11


Measurement of Phospholipase D Activity
A unique feature of PLD is its ability to use either water or primary alcohol in its
enzymatic activity. Transphosphatidylation is the transfer of the phosphatidate of
phosphocholine to water or a primary alcohol (Figure l.H) (Meier and Gibbs, 1999).
The binding pocket of mammalian PLD has been shown to be specific for primary
alcohols. Ella et. al. (1997 ) performed competition studies that showed that
secondary and tertiary alcohols cannot fit the binding pocket of PLD. Research in our
lab attempted to use 1-butanol to inhibit PLD and 2-butanol as a control. If 1-butanol
could fit into the binding pocket, it could block the binding of PLD to PC. However,
we were unable to measure production of phosphatidylbutanol.
Phosphatide Acid Phosphatidyl ethanol
Figure l.H
Transphosphotidylation reaction
12


Role of Phosphatidic Acid in the Cell
PA, ARF and Rho are involved in vesicular formation and trafficking. PA has also
been shown to mediate transport of proteins from the ER to Golgi apparatus and the
formation of coated vesicles. Possibly through PLC or stimulation of other enzymes,
PA is thought to be involved in mitogenesis (including cell proliferation and actin
assembly), inflammation, tissue damage management, respiratory burst, protein
secretion and the synthesis of DNA. It has also been suggested that PA can be an
activator of tyrosine-kinase signaling molecules, GTPase activating protein (GAP),
PI-4 kinase and RAF. PA is also thought to be involved in Ca2+ signaling beyond
that stimulated by IP3 (Gomez-Cambronero and Keire, 1998).
Phospholipase D and the Cell Cycle
The main substrate for PLD is PC. Since, PC levels rise and fall through the cell
cycle, PLD activity must oscillate as well. PC degradation increases as G1 phase is
reached and decreases at S-phase (Figure 1.1) (Gomez-Cambronero and Keire, 1998).
A tight correlation between PC levels, PLD activity and the cell cycle can be seen in a
study by Camero and Lacal, in 1995 where they looked at the ability of
phospholipases to activate specific signaling kinases. PLD in Xenopus oocytes was
found to effectively activate maturation-promoting factor (MPF), mitogen-activated
protein (MAP) kinase and S6 kinase. MPF is known to peak in the middle of M-
phase of the cell cycle (Alberts et. al.,1994).
13


G2
G1
Figure 1.1
The cell cycle and corresponding PC levels
14


As mentioned above, PC degradation increases as the cell enters G1 which is
immediately after M-phase. If PLD is most active during M-phase, it is hydrolyzing
PC at a high rate. After MPF breaks down at the start of G1 PLD becomes less active
allowing for a reduced degradation rate of PC during S-phase.
It has also been suggested that PLD activity is involved in oncogenic transformation
by ras (or unregulated cell division). The ras proteins are low molecular-weight G-
proteins (approximately 21kDa) (Morgan, 1989). When these proteins become
transformed or mutated, they lose their ability to hydrolyze GTP and remain
constitutively active. The ras proteins are routinely involved in cell proliferation and
differentiation. They are also responsible for germinal vesicle breakdown in Xenopus
oocytes (Camera and Lacal, 1994).
It has been shown that high levels of phospholipid metabolites are related to the
functions of ras proteins. A correlation between elevated levels of DAG and ras
function has been reported. Cells that have been ras-transformed have also shown a
sustained activation of PLD, whereas mitogen activated cells show transient PLD
activity. Choline kinase, the enzyme responsible for creating PCho from the choline
product of PLD mediated PC breakdown has been shown to be constitutively active in
ras-transformed cells (Camera and Lacal, 1994).
15


Other Mechanisms of Phospholipase D Activation
Two forms of Protein Kinase C (PKC) are known to activate PLD. Both a and P-
PKC. The mechanism of action in-vivo is not yet known. However, in purified
preparations, a and P-PKC have been shown to have a stimulatory effect on PLD.
Other isozymes of PKC, specifically PKCy, have not been shown to activate PLD.
This would suggest the presence of a unique domain in the a and P isozymes that
interacts with PLD (Exton, 1998). Prior PLC activation may lead to PLD activation.
Activation of PLC results in the hydrolysis of PIP2 to IP3 and DAG resulting in an
increase in Ca2+ (Figure 1 .B). This increase in DAG and Ca2+ causes the
translocation of PKC to the plasma membrane (in some cases, other cellular
membranes). It is near the plasma membrane that PLD is activated.
Phoshpholipase D Isoforms
Two mammalian isoforms of PLD have been identified, PLD1 and PLD2. Meier and
Gibbs (1999) noted that PLD1 is known to be activated by PIP2 and PKC. Gomez-
Cambronero and Keire (1998) noted that PLD1 is insensitive to PKCa. However,
they also specified that PKC could not activate an enzyme whose activity is already at
peak. PLD1 is maintained at very low basal levels until it is activated by a signaling
event. PLD1 has been found in the Golgi, endoplasmic reticulum, late endosomes
and the nuclear membrane (Gomez-Cambronero and Keire, 1998).
16


PLD2 is also activated by PIP2 (Meier and Gibbs, 1999). PLD2 is constitutively
active. Since Camera and Lacal (1993) showed that PLD is independent of PKC in
Xenopus oocytes, it is most likely that they were working with PLD2. Mammalian
studies have shown that PLD2 is localized near the plasma membrane when inactive,
but upon activation becomes associated with endocytic vesicles (Exton, 1998).
However, certain isoforms can be found in other locations within the cell.
Both PLD isoforms are also known to be activated by growth factors, hormones,
cytokines and agonists that bind to high molecular-weight G-protein linked receptors
that also activate PLC (Meier and Gibbs, 1999). Mammalian PLD1 and PLD2 appear
to have about 50% amino acid sequence homology (Gibbs and Meier, 2000).
Mammalian PLD1 is thought to be composed of two differentially spliced proteins.
PLDla is 38 amino acids longer than PLDlb. However, the activities of PLDla and b
are the same so the splicing is apparently not from the catalytic domain (Hammond et.
al. 1997). It may be found true at some point that these differentially spliced forms
may account for the previously described low basal levels of PLD 1. Perhaps, the
missing amino acids provide for a more specific activation mechanism than PLD2.
Structure of Phospholipase D
PLD has a weak pleckstrin homology (PH) domain (Figure 1 .J). This domain is most
likely responsible for recruitment of the enzyme to the inner surface of the plasma
17


membrane. It also appears that PLD does not possess any common binding motifs
like SH2, SH3 or PTB. This lack of expected motifs would suggest that PLD is
activated by a unique mechanism yet to be identified (Exton, 1998).
Two HKD motifs (histidine -x- lysine -xxxx- aspartic acid) have been found in all
mammalian PLD isoforms. Experiments have shown that these two motifs are
required for the catalytic activity of PLD. It has been suggested that this activity may
involve a phosphohistidine intermediate (Figure l.K) (Exton, 1998).
Menbrane Association?
Subcellular Targeting?
Catalysis
I
Catalysis
I HKD
HKD
PLD1 N
PX PH -CHH> loop
Figure l.J
Structural domains of phospholipase D
The PH domain is thought to be responsible for membrane association. The two
HKD domains are essential for catalytic activity.
18


At the start of the reaction, the N-terminal HKD histidine is protonated (the C-
terminal HKD is not). A PA-PLD intermediate is formed by liberating a proton from
the N-terminal HKD histidine causing a release of choline from PC, covalently
linking PA to HIS486. Then, a proton is transferred from water to the N-terminal
HKD histidine. This frees up a hydroxyl group to hydrolyze the intermediate and
release PA (Frohman, 1999).
19


Figure l.K
Mechanism of PLD activity
20


Molecular Weight of Phospholipase D
As has already been described, PLD exists in a variety of isoforms and many are not
yet characterized. However, several examples have been purified and their molecular
weights determined. Human membrane-associated PLD1 has been found to be
124kDa. Another human form of PLD 1 that has been found to be dependent on ARF
and P1P2 has been described as 95kDa. A human cytosolic PLD is suggested to have
a molecular weight of 11 OkDa. PLD2 purified from pig lung has been found to be
PIP2 insensitive and identified at 190kDa. Plant PLD has been characterized at
85kDa. Yeast (Saccharomyces cerevisiae) is more than twice as large at 195kDa.
(Gomez-Cambronero and Keire 1998). Gibbs and Meier (2000) found what they
believed to be a band at lOOkDa to be PLD1 and a band at 120kDa to be PLD2 in PC-
3 and LNCaP cells.
Although quite a bit of research has already been done on the activity of PLD in
Xenopus, the enzyme itself is yet to be purified from a cell. We sought to identify
PLD1 and PLD2 from Xenopus oocytes and eggs and to determine the molecular
weight of the enzyme. Using a variety antibodies to mammalian forms of PLD we
performed a series of immunoprecipitations and Western blots.
Xenopus laevis
The frog Xenopus laevis is also called the African clawed frog. Xenopus is commonly
21


used in research laboratories because it is a relatively long lived and easy to maintain
in captivity. They are have large eggs and oocytes. They can be induced to produce
oocytes at any time with injection of pregnant mare serum gonadotropin and, for eggs,
human chorionic gonadotropin. Meiotic cell division begins quickly after addition of
progesterone or insulin to oocytes. An egg is then produced. The eggs (Stith, web),
are arrested at metaphase II of meiosis. Addition of sperm to the egg results in
activation of the egg. This and other similarities to human eggs may make
fertilization in Xenopus analogous to fertilization in humans.
The stages of Xenopus oocytes are very easy to determine. They possess a dark
animal pole and a light vegetal pole. Oocytes can be harvested in stages ranging from
I to VI. Stage I oocytes are very small (50-300 pm in diameter), translucent cells.
Stage VI oocytes have clearly defined animal and vegetal poles, they are very large
1200/im in diameter (Seidman and Soreq, 1997). Stage VI oocytes were most often
used in our laboratory experiments because they respond to hormone (Stage VI or less
do not). Stage VI oocytes are arrested at the G2/M gap of meiosis I. The oocytes can
be forced to re-enter the cell cycle by insulin, progesterone or ras proteins (Camera
and Lacal, 1994).
During the induction of meiosis (and thus, production of the egg from the oocyte) the
nucleus moves near the surface of the animal pole. The dark pigment of the animal
22


pole is pushed aside by the nucleus. The formation of this white spot in the egg is
known as germinal vesicle breakdown (GVBD) (Stith et. al, 1991). Progesterone- or
insulin-induced GVBD is an easy indicator that the cell has activated the biochemical
pathways for cell division.
23


CHAPTER 2
MATERIALS, METHODS AND RESULTS
Xenopus laevis Oocytes and Eggs
Xenopus laevis females were obtained from Xenopus One (Ann Arbor, MI) or
Xenopus Express (Homosassa, FL) and maintained on a diet of 2.0 grams of ground
beef heart three times per week. Female frogs were injected in the lymph sac 3-4 days
prior to use with a solution of 50IU of pregnant mares serum gonadotropin (PMSG)
(Calbiochem, La Jolla, CA) in O.lmg/ml bovine serum albumin (BSA) (Sigma, St.
Louis, MO) with deionized water (dH20)for an injection volume of 0.3cc.
Frogs that were to be used to obtain eggs were given another injection approximately
12-14 hours before eggs were required. These frogs were injected in the lymph sac
with 850IU of human chorionic gonadotropin (HCG) (Calbiochem, LaJolla, CA) in a
solution of O.lmg/ml BSA and deionized water for an injection volume of 0.3cc.
To obtain oocytes, the frogs were placed under ice for 15-20 minutes and sacrificed
with a guillotine. The ovaries were immediately removed and placed in room
temperature oocyte Ringers solution (OR2: 83mM NaCl, 0.5mM CaCl2, ImM MgCl2,
lOmM HEPES, pH adjusted to 7.9 if necessary). The oocytes were then manually
24


defolliculated, separated into sample groups and held in OR2.
When used, eggs were harvested by gently rubbing the belly and back of the female
frog while holding the posterior over a dish containing 100% Modified Barths
Solution (MBS: 440mM NaCl, 5mM Kcl, 50mM HEPES, 4.1mM Magnesium
Sulfate Hydrate, 1.65mM Calcium Nitrate tetrahydrate, 2.05mM Calcium Chloride
dihydrate, 0.202g/l NaHC03). Care was taken not to drip water from the frog into the
dish of MBS so as not to change the osmolarity of the solution. The eggs were
checked under a dissecting microscope to confirm the presence of a white spot
indicating germinal vesical breakdown, proper color, jelly coat and cell shape before
use.
De-iellv Xenopus laevis Eggs
Eggs were collected in 0.1 M NaCl solution. The eggs were incubated in 2% cysteine
solution (4g/l cysteine, 200ml dH20, pH 8 w/ pH paper) for 15 minutes. The eggs
were then rinsed 3 times with rinse solution (0.1M NaCl, 0.05M Tris, pH 7). The
eggs were then rinsed twice with 100% MBS and held until ready for use. When
ready for use, the eggs were rinsed with 10%MBS to activate.
25


Xenopus laevis sperm
Xenopus laevis males were obtained from Xenopus One (Ann Arbor, MI) or Xenopus
Express (Homosassa, FL) and maintained on a diet of 1.5 grams of ground beef heart
three times per week. On day of use a male frog was placed on ice for 15-20 minutes
and sacrificed by guillotine. The testicles were removed, cleaned of blood and
mesenteric fat, and placed in 100%MBS at room temperature. The testicles were
placed in a small dish with 2400/il of 100% MBS and minced as small as possible.
Control Fertilization
To make sure the eggs, sperm and solutions were working properly, control
fertilizations were performed. Twenty eggs were placed into a dish with 100% MBS.
The MBS was removed and to activate the eggs, they were rinsed twice with 10%
MBS. Again, the excess MBS was removed and 1ml of 10% MBS was added to the
eggs along with 167^1 of the sperm solution. A timer was started and the number of
eggs with the animal pole facing up was recorded. After 10 minutes, the eggs were
flooded with 5ml of 10% MBS and again, the rotation of eggs with the animal pole
facing up was recorded. The rotation of the eggs was recorded every 1 minute until
all of the eggs had rotated so the animal pole was facing up. If this rotation did not
occur within 20-30 minutes of sperm addition, the solutions were re-made and the
control fertilization was repeated.
26


Initial Immunoprecipitation with
Anti-PLD/KLH Antibody
A mammalian anti-PLD antibody coupled to keyhole limpet hemocyanin (KLH) was
obtained (a gift from Dr. David Brindley at the University of Alberta). Two groups of
100 oocytes each (most were not defolliculated) were placed in 1.7ml v-vials. Excess
superatant was removed and 1ml of 4C lysis buffer (20mM HEPES, l%w/v Triton
X-100, 80mM b-Glycerophosphate disodium salt, ImM Phenylmethlysulfonyl
Flouride, 10/ig/ml Leupeptin, 10^g/ml Aprotinin, 2mM Sodium orthovanadate,
20mM EGTA) was added to each tube. The oocytes were homogenized with 20
strokes of a plastic pestle. The homogenate was vortexed (Vortex Genie, Scientific
Industries Bohemia, NY) on medium speed for 30 seconds. Both tubes were
microfuged (Beckman Microfuge E) for 10 minutes at 4C. The supernatant was
poured off into a clean 1.7ml v-vial and the pellet was discarded. The samples were
precleared with 50/zl of a 50% Protein-A-Sepharose slurry and placed on ice on a
rocker at medium speed at 4 C for 30 minutes. At the end of the incubation, the
samples were microfuged for 5 seconds. The supernatant was poured into a clean
1,7ml v-vial and the pellet was discarded. 10//1 of the anti-PLD/KLH antibody was
added to each tube and they were held for four days at 4C.
After four days, 100/zl 50% Protein-A-Sepharose slurry was added to each tube and
they were incubated for 1 hour. After incubation, the samples were microfuged for 5
seconds. The supernatant was discarded and 500^1 low salt wash buffer (20mM Tris,
27


0.1% Triton X-100, 5mM EDTA, lOOmM NaCl) and incubated on ice on the rocker
at 4C for 1 hour. After incubation, the samples were microfuged for 5 seconds and
the superatent poured. 500^1 low salt wash buffer was added to the pellets and they
were incubated on ice on the rocker at 4C for 1 hour. After 1 hour, the samples were
microfuged 5 seconds the supernatant was poured off and 500/il 20mM HEPES was
added. The samples were incubated on ice on the rocker at 4C for 1 hour. After the
incubation, the samples were microfuged 5 seconds. The supernatant was poured off
and the pellet was saved. Meanwhile, a 10% polyacrylamide resolving gel was
prepared and mounted to the electrophoresis apparatus (California Quality Plastics,
Ontario, CA) in the 4C cold box. 20//1 dH20, 9.45^1 Laemmli buffer and 5.5/^1
mercaptoethanol was added to each pellet. The samples were placed in a beaker of
boiling water for 10 minutes. After a 5 second microfuge, the supernatant (about
30/iil each) off the top of the pellets was loaded onto the gel. Molecular weight
standards (Sigma, SDS-7B) were placed in boiling water for 1 minute and 20^1 were
loaded onto the gel. The gel was loaded with electrode buffer (123.8mM Tris Base,
959. ImM Glycine, 17.3mM SDS) and run overnight at 11mA.
When the leading band was about 1" from the bottom of the gel, the electrophoresis
apparatus was stopped and the gel removed. The Genie apparatus (Idea Scientific
Co., Minneapolis, MN) was prepared and the buffers mixed: Standard buffer
(25.012mM Tris Base, 191.82mM Glycine, 20% v/v Methanol), wash buffer (lOmM
28


Tris Base, lOOmM NaCl, 0.1% w/v Tween-20), blocking buffer (3% BSA, lOmM
Tris Base, lOOmM NaCl, 0.1% w/v Tween-20). The Genie apparatus was assembled
with Polyvinylidene Diflouride (PVDF) (Sigma P-0682) membrane, loaded with
standard buffer and the equipment run for 1.5 hours at 24 V. After 1.5 hours, the
Genie was disassembled and the PVDF membrane soaked in blocking buffer in a
37C water bath with the shaker on for 30 minutes. After 30 minutes the PVDF
membrane was put into a zip-lock bag with the primary antibody solution (7ml
blocking buffer, 14^1 anti-PLD/KLH antibody. The membrane was incubated for 1
hour in the bag on the Belly Dancer (Stovall, Life Sci., Inc., Greensboro, NC) with
steel balls on top to keep the solution moving.
The membrane was placed in a dish and cleaned with the wash buffer for 30 minutes,
with the buffer being changed every 5 minutes. After which, the membrane was
transferred to another zip-lock bag with the secondary antibody solution (30ml
blocking buffer, \/A Anti-Rabbit IgG Alkaline Phosphatase Conjugate). It was
incubated on the Belly Dancer with steel balls for 1 hour. After 1 hour, the membrane
was placed in a dish and cleaned with wash buffer for 30 minutes, with the buffer
being changed every 5 minutes. Finally, the membrane was put into a zip-lock bag
with 20ml BCIP/NBT (Sigma B-6404). After 7 minutes, the color change was
complete and the membrane was rinsed in dH20, and allowed to air dry. The results
can be see in Figures 3.A and 3.B.
29


Std PLD/KLH PLD/KLH Std
65kDa
Figure 2. A
Initial immunoprecipitation with anti-PLD/KLH antibody
Western blot of Xenopus oocytes. Lanes 1 and 4 are molecular weight (MW)
standards. MW as labeled. Lanes 2 and 3 are the sample lanes. 100 oocytes
immunoprecipitated with anti-PLD/KLH antibody. Sample lanes show a main band
at 65 kDa. Electrophoresis performed with 10% polyacrylamide resolving gel.
30


MOLECULAR WEIGHT
main band at 33mm, 65724 Daltons
y=mx+b b(1) is slope, b(o) is Y intercept
Figure 2.B
Plot of molecular weight markers and determination of sample MW.
Initial immunoprecipitation with anti-PLD/KLH antibody.
31


Comparison of Human and Mouse
Anti-PLD Antibodies to Anti-PLD/KLH
Four different polyclonal anti-PLD antibodies were purchased. The purpose of this
experiment was to test the antibodies and to compare to the Brindley anti-PLD/KLH:
The N-terminal region of human PC-specific PLD1 (PLDlhN) (Biosource
International 44-320), an internal segment of human PC-specific PLD1 (PLDlhl)
(Biosource International 44-322), the N-terminal region of mouse PC-specific PLD2
(PLD2mN) (Biosource International 44-324), an internal segment of mouse PC-
specific PLD2 (PLD2mI) (Biosource International 44-326).
Five groups of 100 eggs were collected and dejellied. The five samples were
immunoprecipitated as described above. Each group was homogenized in 1ml 4C
lysis buffer. They were precleared with 50/zl 50% Protein-A-Sepharose slurry and
placed on ice on a rocker at medium speed at 4C for 30 minutes. At the end of the
incubation, the samples were microfuged for 5 seconds. The supernatant was poured
into a clean 1.7ml v-vial and the pellet was discarded. 10/zl of the anti-PLD/KLH
antibody was added to one tube, 10yul of the anti-PLD lhN was added to one tube,
10/^1 of anti-PLD 1 hi was added to one tube, 10/^1 of anti-PLDmN was added to one
tube, and finally, 10/J of anti-PLDml was added to the remaining tube. The samples
were held overnight at 4C.
The following morning, the samples were incubated in 100/zl of 50% Protein-A-
32


Sepharose slurry for 1 hour. They were then washed twice in 500/ri low-salt wash
buffer for 1 hour per wash. Finally, they were washed in 20mM HEPES for 1 hour on
the rocker at 4C. The samples were microfuged for 30 seconds and the supernatant
poured off and discarded. A marker dye of 20/il dH20, 9.45/zl Laemmli and 5.5/ri
mercaptoethanol was added to each sample pellet. The samples were placed in
boiling water for 10 minutes, microfuged for 5 seconds an 40//1 of the supernatant off
the top of the pellet were loaded onto a 10% acrylamide resolving gel off the top of
the pellet. Molecular weight standards were placed in boiling water for 1 minute and
20//1 were loaded onto the gel as shown in Figure 2.C. The gel was loaded with
electrode buffer and run overnight at 8mA.
When the leading band was about 1" from the bottom of the gel, the electrophoresis
apparatus was stopped and the gel removed. The Genie apparatus was prepared and
the buffers mixed as described above. The Genie apparatus was assembled with
PVDF membrane, loaded with standard buffer and run for 1.5 hours at 24 V. After
which, the Genie was disassembled and the PVDF membrane soaked in blocking
buffer in a 37C water bath with the shaker on for 30 minutes. After 30 minutes the
PVDF membrane was cut into strips to separate the antibody lanes and each strip was
put into a zip-lock bag with the primary antibody solution (7ml blocking buffer, 14/ri
the same anti-PLD antibody that was used in the immunoprecipitation.
33


1 2 3 4 5 6 7 8 6 10 11
Figure 2.C
Lane Loading of Gel
See Figure 2.D for final results
34


The membranes were incubated for 1 hour on the Belly Dancer with steel balls on top.
The membranes were then placed into individual dishes and cleaned with the wash
buffer for 30 minutes, with the buffer being changed every 5 minutes. After which,
the membrane strips were transferred to another zip-lock bag with the secondary
antibody solutions (30ml blocking buffer, lyul Anti-Rabbit IgG Alkaline Phosphatase
Conjugate). They were incubated on the Belly Dancer with steel balls for 1 hour.
The membranes were placed into individual dishes and cleaned with wash buffer for
30 minutes, with the buffer being changed every 5 minutes. Finally, the membranes
was put into zip-lock bags with 10ml BCIP/NBT. After 7 to 10 minutes, the color
changes were complete and the membranes rinsed in dH20, and allowed to air dry.
The results can be seen in Figure 2.D.
Verification of Human and Mouse
Anti-PLD Antibodies and Anti-PLD/KLH
In an effort to verify the results from the experiment above, and to obtain a
publishable gel sample, the experiment was repeated using oocytes instead of eggs.
Again, 5 groups of 100 oocytes were collected (most were not defolliculated). They
were homogenized and precleared as described above. Each sample was
immunoprecipitated using one of the anti-PLD antibodies (PLDlhN, PLDlhl,
PLD2mN, PLD2mI and PLD/KLH). The samples were held overnight at 4C.
Electrophoresis and Western Blot procedures were performed as described above.
The results can be seen in Figures 2.E.
35


KDa Ln 1 Ln 2 Ln 3 Ln 4 Ln 5 Ln 6 Ln 7 Ln 8 Ln 9 Ln 10 Ln 11
"r iiSs^^sr;\ i!':.. ;§L
PLD/KLH PLD2ml PLD2mN PLDlhl PLDlhN
Figure 2.D
Compare human and mouse PLD antibodies to anti-PLD/KLH using dejellied eggs
Western blot of PLD immunoprecipitation using dejellied Xenopus eggs. Lanes 1, 3,
5, 7, 9 and 11 are molecular weight (MW) standards. MW as labeled. Lane 2 was
immunoprecipitated with anti-PLD/KLH antibody. Lane 4 used PLD2mI. Lane 6
used PLD2mN antibody. Lane 8 used PLDlhl. Lane 10 used PLDlhN. Main band
at 56kDa. The Pldlhl band is the lightest, the PLD/KLH band is the darkest.
Electrophoresis was performed with 10% polyacrylamide resolving gel.
36


KDa Ln 1 Ln 2 Ln 3 Ln 4 Ln 5 Ln 6 Ln 7 Ln 8 Ln 9 Ln 10
PLD/KLH PLDlhl PLD2mN PLD2ml PLDlhN
Figure 2.E
Verification of human and mouse PLD antibodies and anti-PLD/KLH using oocytes
Western blot of PLD immunoprecipitation using Xenopus oocytes. Lanes 1, 3, 6, 8
and 10 are molecular weight (MW) standards. MW as labeled. Lane 2 was
immunoprecipitated with anti-PLD/KLH antibody. Lane 4 used anti-PLDlhl
antibody. Lane 5 used anti-PLD2mN antibody. Lane 7 used anti-PLD2mI antibody.
Lane 9 used anti-PLDlhN antibody. Main band at 61kDa. Electrophoresis performed
with 10% polyacrylamide resolving gel.
37


Incubation of Oocytes in Progesterone to
Stimulate Meiosis and Identify a Cell-Cycle
Related Change in PLD Levels
One female Xenopus was sacrificed Oocytes were collected and defolliculated and
divided into groups as follows:
50 oocytes = Control
50 oocytes = Control
50 oocytes = 30 minute incubation in progesterone
50 oocytes = 2 hour incubation in progesterone
Four groups of 50 defolliculated oocytes were placed in a 12-well dish. 449/zl OR2
and lyul ethanol were added to 2 control groups. 449//1 OR2 and \/u\ 5mM
progesterone were added to two sample groups. At the 30 minute timepoint, the
supernatant was removed from one sample group and one control group. 1ml cold
lysis buffer was added to both groups and they were homogenized. At 2 hours, the
supernatant was removed from the remaining control group and sample group. 1ml
cold lysis buffer was added and both groups were homogenized.. The control samples
were homogenized at the 30 minute and 2 hour timepoints. After homogenization,
the samples were precleared with a 50% slurry of Protein-A-Sepharose. All of the
samples were then immunoprecipitated with lO^ul PLDlhN following the procedures
described above and held overnight at 4C.
38


The following morning, the samples were incubated in 50^1 Protein-A-Sepharose
(50% slurry) for 1 hour and then washed in low salt wash buffer and HEPES and
loaded onto a gel as described above. The gel used this time was a 14.39%
polyacrylamide resolving gel. The gel was run overnight at 8mA. The gel was
stopped the next morning as soon as the dye front ran off the bottom of the gel.
The Western blot procedure was followed as described above. BCIP/NBT was added
for 4 minutes until a color change appeared and the membranes were rinsed in dH20
and allowed to air dry. The results can be seen in Figure 2.F. The experiment was
repeated and the results can be seen in Figure 2.G.
39


KDo Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8
Std Control Std 30 Min Std Control Std 2 Hour
lOOkDa
53kDa
Figure 2.F
Incubation of oocytes in progesterone to identify a cell cycle related change in PLD
Western blot of PLD immunoprecipitation from Xenopus oocytes. Lanes 1, 3, 5 and
7 are molecular weight (MW) standards. MW as labeled. Lanes 2 and 6 are control
lanes. Controls were groups of 50 oocytes each prepared without progesterone. Lane
4 contained 50 oocytes incubated with 1^1 5mM progesterone for 30 minutes before
immunoprecipitation. Lane 8 contained 50 ooyctes incubated with \p\ 5mM
progesterone for 2 hours before immunoprecipitation. Both control and incubated
lanes show a main band at 53kDa with a minor band at lOOkDa. Electrophoresis was
performed with a 14.39% polyacrylamide resolving gel. Immunoprecipitation was
performed with anti-PLDhl.
40


KDq Lone 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 Lane 9 Lane 10
205 ! '
"if '
Std Control Std 1 hour Std Control Std 30 Min Std Std
Figure 2.G
Incubation of oocytes in progesterone to identify a cell cycle related change in PLD
Western blot of PLD immunoprecipitation from Xenopus oocytes. Lanes 1, 3, 5, 7, 9
and 10 are MW standards. MW as labeled. Lane 2 is the 1 hour control lane. 50
oocytes were incubated in OR2 and homogenized at 1 hour timepoint. Lane 4
contained 50 oocytes exposed to \p\ 5mM progesterone for 1 hour. Lane 6 contained
50 oocytes held in OR2 for 30 minutes before immunoprecipitation. Lane 8
contained 50 oocytes exposed to progesterone for 30 minutes before
immunoprecipitation. Both control and progesterone lanes show a main band at
53kDa. Electrophoresis was performed with a 14.39% polyacrylamide resolving gel.
Immuoprecipitation was performed with anti-PLDhl antibody.
41


Fertilization Induced Change in PLD Levels
at 15 minutes. 30 minutes and 1 hour
Four groups of 50 eggs each were prepared in 100%MBS. The 100% MBS was
removed and the eggs were washed twice in 10%MBS. Excess solution was removed
and 1ml 10% MBS was added to each dish. 167//1 sperm solution was added to each
dish and a timer started. At 15 minutes the first dish was flooded with 5ml 10%MBS
and the eggs were rinsed with 10%MBS twice. Excess solution was removed and the
eggs were transferred to a 1.7ml v-vial and 1ml lysis buffer was added. The eggs
were homogenized with 20 strokes of a plastic pestle and held on ice. At the 30
minute timepoint another dish was flooded with 5ml 10% MBS and washed twice.
The eggs were then homogenized in 1ml lysis buffer and held on ice. At 1 hour one
dish was flooded 10% MBS, washed twice and homogenized in lysis buffer. At 1
hour one control dish that was not exposed to sperm was washed twice with 10%
MBS and homogenized by 1ml lysis buffer.
All tubes were microfuged 10 minutes and supematent was poured off and saved.
The pellets were discarded. The samples were precleared with 50^1 Protein-A-
Sepharose (50% slurry) and incubated on ice for 30 minutes on the Belly Dancer. The
samples were then microfuged 5 seconds, and the supematent saved. 10^1 anti-
PLDhl was added to each sample and held overnight at 4C.
42


The following morning, the samples were incubated for 1 hour in 50/^1 Protein-A-
Sepharose slurry. They were then washed twice in low-salt wash buffer and 20mM
HEPES as described above. The samples were then prepared with loading dye and
loaded onto a 14.39% resolving gel with a 4% stacking gel. The samples were run
overnight at 8mA. The following morning, the Genie apparatus was prepared and the
samples run as previously described. The results can be seen in Figure 2.H. This
experiment was repeated to verify results. The results from the second experiment
can be found in Figure 2.1.
43


((Da Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 Lane 9 Lane 10
57kDa
Std 1 Hour Std 30 Min Std Control Std 15 Min Std Std
Figure 2.H
Fertilization induced change in PLD levels at 15 minutes, 30 minutes and 1 hour
Western blot of PLD immunoprecipitation from fertilized Xenopus eggs. Lanes 1, 3,
5, 7 and 9 are MW standards. Lane 2 contained 50 eggs exposed to Xenopus sperm
for 1 hour. Lane 4 contained 50 eggs exposed to sperm for 30 minutes before
immunoprecipitation. Lane 6 is a control lane, 50 eggs were washed in MBS and
homogenized at 1 hour timepoint. Lane 8 contained 50 eggs exposed to sperm for 15
minutes before'immunoprecipitation. Both control and fertilized lanes show a major
band at 57kDa. Electrophoresis was performed with a 14.39% polyacrylamide
resolving gel. Immunoprecipitation performed with anti-PLDhl antibody.
44


KDa Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 Lane 7 Lane 8 Lane 9 Lane 10 Lane 11
Std Cohntrol 1 Hour Std 30 Min Std 15 min Std Control Std Std
Figure 2.1
Change in PLD levels at fertilization
Western blot of PLD immunoprecipitation from fertilized Xenopus eggs. Lanes 1, 4,
6, 7, 10 and 11 are MW standards. MW as labeled. Lane 2 is a control lane. 50 eggs
washed in MBS and homogenized at 1 hour timepoint. Lane 3 contained 50 eggs
exposed to Xenopus sperm for 1 hour. Lane 5 contained 50 eggs exposed to sperm
for 30 minutes before immunoprecipitation. Lane 9 contained 50 eggs washed in
MBS and homogenized at 15 minutes. Both control and fertilized lanes show a main
band at 43kDa. Electrophoresis was performed with 14.39% polyacrylamide
resolving gel. Immunoprecipitation performed with anti-PLDhl antibody.
45


CHAPTER 4
DISCUSSION
Molecular Weight of Xenopus
Phospholipase D
Initial immunoprecipitation of Xenopus oocytes with anti-PLD/KLH showed one
major band on the Western blot at 78kDa. Two immunoprecipitations with the anti-
PLD/KLH antibodies in comparison to the human and mouse antibodies resulted in
molecular weights of 56kDa for dejellied eggs and 61kDa for oocytes. A
progesterone incubation of oocytes and immunoprecipitation with PLDlhl resulted in
a Western blot with major bands at approximately 57kDa. Experiments in
fertilization before immunoprecipitation resulted in Western blots with major bands at
46kDa and 41kDa.
One progesterone incubation of oocytes and immunoprecipitation with anti-PLDlhl
resulted in two bands. A major band can be seen at about 53kDa with a minor band
at approximately 1 OOkDa. This was perhaps the most interesting of all of the data
accumulated to date. The major band at 53kDa coincides with the results of previous
experiments, however this is the first time a minor band has appeared at 1 OOkDa. As
mentioned in the introduction, mammalian PLD1 is composed of two spliced peptide
fragments. It may be that Xenopus PLD is also composed of two fragments that are
46


approximately 50kDa apiece.
As these experiments have been performed, the samples have been held overnight
during the immunoprecipitation process. It is possible that the normally larger PLD
protein is being degraded by proteases contaminating solution. Although, the lysis
buffer is made with protease inhibitors, specifically aprotinin and leupeptin, it may
not be enough to prevent degradation of the protein. If the proteins are indeed
composed of two spliced fragments, it may be the proteins are weaker at the splicing
point. Previous results have shown smaller proteins. If proteases are breaking the
protein at a weak midpoint that may expose fragile ends that can be more readily
degraded resulting in fragments smaller than the 50kDa size.
In a normal, functioning cell, protein degradation is often a control mechanism. If a
protein is acting as a signaling or control molecule it must be readily degraded should
the cell need to stop its current activity. Since PLD is an enzyme involved in
mitogenic signaling it would stand to reason that it must be readily degraded to
prevent uncontrolled cell growth. However, proteases normally act by degrading
proteins from the ends. It seems most unlikely that proteases could continually break
and degrade PLD in the same manner over a series of Western blots.
Based on these experiments the molecular weight of Xenopus laevis is 56kDa with a
47


standard deviation of 1 Ok (n=7). However, the results of the Western blot shown in
Figure 3.E led us to believe the molecular weight may indeed be lOOkDa. This would
be the expected result given the molecular weights of PLD isozymes in mammals.
The figure of 1 OOkDa is further confirmed by later research in our lab. A second
series of Western blots using a prepackaged protease inhibitor cocktail produced
results very similar to those seen in Figure 2.F here. A major band at 56kDa and a
minor band at 1 OOkDa. It is unlikely protease degradation would still be occuring in
the same way with entirely different protease inhibitors.
Finally, current research in our lab has produced a sequence of the genetic code for
Xenopus PLD. The region sequenced to date is located in the loop region of the
catalytic domain (Figure 1J).
The sequence is 100 nucleotides shorter than human PLD. But this apparent deletion
of 30 amino acids is not enough to account for a molecular weight difference of
almost 50kDa. Unless a major deletion is found in another part of the protein, this
sequence data leads us to believe the molecular weight of Xeopus PLD is indeed
1 OOkDa. If this is indeed the case, the question remains. What protein is the major
band at 56kDa? Have we found a new isoform of PLD? Is this perhaps a related
lipase? Or a breakdown product of the lOOkDa form? Whatever the result, we know
48


the amino acid sequence must be similar to that of PLD because we have been able to
pull the protein with 5 PLD antibodies to different epitopes of the protein.
Densitometry Readings of PLD 1 and PLD2
In addition to identifying the molecular weights of the enzymes, the Western blots can
be used to determine relative amounts of the PLD1 and PLD2 isozymes. Initial
examination of the blots shows much darker bands for the PLD1 antibodies.
Densitometry analysis of the membranes confirms this. The bands for PLD1
antibodies have an average intensity of 799. While bands for PLD2 antibodies have
an average intensity of 215.5.
It would appear from this initial data that PLD1 levels seem to have much higher
basal levels than PLD2. This is converse to currently held belief of the isozyme levels
in mammalian cells.
It is known that PLD2 is constitutively active in a cell. PLD1 is normally found at
very low basal levels until a signaling event occurs. It is with this signaling event that
PLD1 levels increase. The experiments reported here were performed with both
dejellied eggs and oocytes. The levels of both isozymes were measured at basal
levels. It would be interesting to perform experiments using both PLD1 and PLD2
antibodies on eggs after fertilization or oocytes after progesterone incubation.
49


Future Experiments
In many ways the research reported here has resulted in many more questions then
answers. It remains to be seen if the molecular weight of phospholipase D in
Xenopus is 1 OOkDa as predicted or is indeed 56kDa. If this is the case, what is the
band on the Western blots at 1 OOkDa. Sequencing the genetic code can provide the
final answer.
Finally, why does it appear that basal levels of PLD1 are higher than PLD2? It would
be worthwhile to measure relative levels after stimulation of eggs and oocytes.
Fertilization and progesterone incubation before immunoprecipitation with both
antibodies may provide interesting results.
50


APPENDIX A
CHEMICALS, SOLUTIONS and EQUIPMENT
Chemicals
Anti-Rabbit IgG Alkaline Phosphatase Conjugate (Sigma, St. Louis, MO)
BCIP/NBT (Sigma)
BSA (Sigma)
HCG (Calbiochem, LaJolla, CA)
Molecular weight standards (Sigma)
PLD/KLH antibody (Dr. David Brindley at the University of Alberta)
PLDlhN antibody (Biosource International, Camarillo, CA)
PLDlhl antibody (Biosource International)
PLD2mN (Biosource International)
PLD2mI (Biosource International)
PMSG (Calbiochem)
-51-


Solutions
Blocking buffer
Electrode buffer
Low Salt Wash buffer
Lysis buffer
3% BSA
lOmM Tris Base
lOOmMNaCl
0.1% w/v Tween-20
123.8mM Tris Base
959. ImM Glycine
17.3mM SDS
20mM Tris
0.1% Triton X-100
5mM EDTA
lOOmM NaCl
20mM HEPES
1% w/v Triton X-100
80mM b-Glycerophosphate disodium salt
-52-


1 mM Phenylmethlysulfonyl Flouride
10^g/ml Leupeptin
10/zg/ml Aprotinin
2mM Sodium orthovanadate
20mM EGTA
100% Modified Barths 440mM NaCl
Solution 5mM Kcl
50mM HEPES
4.1mM Magnesium Sulfate Hydrate
1.65mM Calcium Nitrate tetrahydrate
2.05mM Calcium Chloride dihydrate
0.202g/l NaHC03
Oocyte Ringers solution 83mM NaCl
0.5mM CaCl2
1 mM MgCl2
lOmM HEPES
-53-


Protein-A-Sepharose
0.9078g Protein-A-Sepharose
slurry
Rinse solution
Standard buffer
Wash buffer
15ml dH20
3.6ml PBS
O.lMNaCl
0.05M Tris
25.012mM Tris Base
191.82mM Glycine
20% v/v Methanol
lOmM Tris Base
lOOmM NaCl
0.1% w/v Tween-20
-54-


Equipment
Vortex Genie (Scientific Industries Bohemia, NY)
Beckman Microfuge E (Beckman Coulter, Inc., Fullerton, CA)
Electrophoresis apparatus (California Quality Plastics, Ontario, CA)
Genie apparatus (Idea Scientific Co., Minneapolis, MN)
Polyvinylidene Diflouride membrane (PVDF) (Sigma)
Belly Dancer (Stovall, Life Sci., Inc., Greensboro, NC)
-55-


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Gomez-Cambronero, J., Keire, Paul, Phospholipase D: A Novel Major Player in
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Camera, A., Lacal, J.C., Activation of intracellular kinases in Xenopus oocytes by
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Ella, K.M., Meier, K.E., Kumar, A., Zhang, Y., Meier, G.P. Utilization of Alcohols
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Exton, J.H., Taylor, S.J., Augert, G., Bocckino, S.B., Cell Signaling Through
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