ABSTRACT
The ability of calcium to turn on the calmodulin-activated enzyme NAD
kinase was tested at different stages in the development of sea urchin embryos
during the first 24 hours after fertilization. It was found that NAD kinase is a stable
enzyme and that its calcium-dependency does not change throughout development.
Tests on NAD kinase from eggs grown in emetine, an inhibitor of protein
synthesis, and actinomycin, an inhibitor of RNA transcription, revealed that the
enzyme is neither translated or resynthesized during the first twenty four hours. A
calcium sensitivity curve was plotted for the enzyme and correlations between the
critical concentrations of free calcium for the activation of NAD kinase and the
actual calcium levels in the eggs are discussed.
.

INTRODUCTION
One of the many calcium-induced changes occurring at fertilization in the
sea urchin egg is the activation of NAD kinase, the enzyme responsible for the
conversion of NAD into NADP. NAD kinase is a calmodulin-dependent enzyme
and is thereby turned on directly by the post-fertilization increase in calcium levels.
Though NAD kinase is responsible for NADP synthesis in all eukaryotic
cells, little is known about the role of this enzyme or its product. There is a small
amount of literature hinting at possible regulatory roles for the enzyme. Mitogen
stimulation of lymphocytes has been shown to cause an increase in NADP levels
(Berger et al, 1987). NAD kinase activity also changes upon photostimulation and
during spore germination in Neurospora (Afanasieva et al, 1982). Finally,
circadian variations in the affinitiy of NAD kinase have been observed in Euglena
(Laval-Marten et al, 1990).
Unpublished work of Robert Sweezy in David Epel's laboratory indicated
an apparent shift in the calcium dependency of NAD kinase at fertilization in sea
urchin eggs and this was the starting point for my project. I wanted to test for
alterations in the enzyme that may occur at fertilization and trace its activity
throughout development in hopes of elucidating any possible regulatory roles of
NAD kinase.
Calcium activation of the enzyme was tested before and after fertilization
and then at various stages in development. Surprisingly, NAD kinase retained its
calcium sensitivity after fertilization. To investigate the turnover rate of NAD
kinase, embryos were grown in emetine, an effective protein synthesis inhibitor in
sea urchin eggs (Hogan and Gross, 1971), and also in actinomycin, an RNA
translation inhibitor (Gross et al, 1964). I found that NAD kinase levels were
unaffected during twenty-four hours incubations in these inhibitors, indicating that
NAD kinase is stable and is not degraded or synthesized during development.
The sensitivity of the enzyme activity to different concentrations of free
calcium was also tested to find out if there was a correspondence between changes
in the intracellular levels of calcium and enzyme activity. NAD kinase was found to
be activated to its maximal level at a calcium concentration between 10-6M and
10-7M.
MATERIALS AND METHODS
Collecting Gametes
Gametes were collected from S. purpuratus by intracoelomic injection of
0.5 MKCl. Released eggs were stored in stirred sea water at 15'C. Sperm was
kept dry at 4'C. All gametes were used within 12 hours of collection. To dejelly
the eggs, the eggs were passed through a 90u Nitex mesh filter three times. The
dejellied eggs were resuspended in fresh sea water and then fertilized by adding a
small sample of sperm freshly dissolved in sea water.
Raising Embryos
Fertilized eggs were suspended in fresh sea water to a final concentration of
1% (vol/vol) and set up with a stirrer in a 15'C water bath. Three samples were
raised for 24 hours; a control group in sea water, one in 25ug/ml solution of
actinomycin in sea water. and one in 10-M emetine in sea water.
Extraction of Enzyme
At three hour intervals, 1Oml of each suspension were pelleted in a hand
centrifuge, washed several times with sea water and then resuspended to
5% (vol/vol) in an homogenization medium containing 300mM glycine/175mM
potassium gluconate/185mM mannitol/SOmM Pipes buffer/2OmM NaclSmM
MgC12/10mM EGTASmM DTT adjusted to pH of 6.8 using KOH. The mixture
was homogenized using a tight Dounce homogenizer until no intact cells were
visible under a microscope. (about 40 strokes) The samples were centrifuged at
20000g for five minutes at 4C and the supernatant was collected and kept at-40C
until the enzyme assay.
Protein Assay
To standardize the amount of protein present in each sample, the Coomassie
blue filter paper assay was used. These values were used to normalize the relative
rates of activity found in the enzyme assay.
Measuring NAD Kinase Activity
The assay mixture consisted of 200mM Pipes buffer, 10mM MgCl, SmM
ATP, 1OmM G6P, 1.0 unit of G6PD, and 0.Iml of the enzyme extract. NAD was
added to 2mM concentration to begin the reaction. After a basal rate had been
established, a solution of calcium acetate was added to 0.SmM final concentration
to stimulate NAD Kinase to its maximal rate. Enzyme activity was measured using
a Perkins Elmer Fluorometer Model 204A at 340 mM excitation and 440mM
emission.
Calcium Sensitivity Assay
For this experiment, different amounts of calcium acetate were added during
the enzyme assay to vary the free calcium levels and observe their effects on NAD
kinase activity. The relationship between the total calcium and the free calcium
varied according to EGTA concentration, pH, temperature, and the presence of
other metal chelators. Chris Patton's chelating program was used to account for
these effects.
RESULTS
NAD Kinase During Normal Development
Aliquots of the developing embryos were taken at three hour intervals and
the enzyme extracts assayed for activity. In all enzyme assays, the rates of activity
as indicated by increase in fluorescence from rising NADPH levels were
standardized using values obtained from the protein assay. Fig.1 shows the rates
of activity before and after stimulation by calcium. As seen, sensitivity to calcium
did not change during development. There was a puzzling variability in rates at the
different time points. Unfortunately, only one time series was done, but it appears
that there no major changes in NAD kinase in the early stages. Possibly, there is an
increase at 18 to 21 hours.
NAD Kinase Activity Düring Development in Actinomycin D
In agreement with Gross et al (1964), embryos developed normally in the
RNA transcription inhibitor Actinomycin up to the blastula stage, but became
arrested at the 18 hour point. There was no apparent effect on NAD kinase level.
nor was there any effect on the ability of the enzyme to be stimulated by calcium.
(Fig 2) Again, puzzling variability in the enzyme activity was seen during
development.
NAD Kinase Activity During Development in Emetine
Emetine is an inhibitor of protein synthesis, and as described by Wagenaar
and Mazia (1978), the fertilized eggs did not divide and were arrested at the single
cell stage. With the lack of proteins needed to undergo mitosis, the eggs gradually
became deformed as time progressed and lysis was seen in some eggs by the 20th
hour. There was no drop in enzyme activity during this twenty hour time period
before or after calcium stimulation (Fig. 3). Again, high variability was seen in the
enzyme activity rates.
Calcium Sensitivity
Testing the NAD kinase activity at different calcium concentrations revealed
the enzyme to be fully activated at free calcium concentrations between 10-M and
10-OM (Fig. 4). There was some activity even at concentrations below 10-7M and
to turn the enzyme off completely, free calcium had to be dropped to 10-9M.
Because of the high pH sensitivity of the EGTA-calcium complex, it was difficult to
pinpoint the exact calcium concentration at which the enzyme reached its maximal
activity. Figure 4 shows a calcium sensitivity curve done on embryos prepared
ten minutes after fertilization. Repeats of this experiment using unfertilized eggs
and embryos one hour after fertilization confirmed these findings. The low rates of
activity at 10 and 10-8M concentrations resembled the off rate activity of the
enzyme while the rates at 10-°M and higher concentrations of calcium were similar
to the on rates.
DISCUSSION
In the three different conditions of raising embryos in this study (control,
actinomycin, emetine), none showed a decrease in the ability of calcium to activate
NAD kinase, nor were there major effects on maximal activity. Unfortunately, the
extreme variations in the rates of activity seen at different time points in all three
conditions have no logical explanation. Though the first set of assays were more
consistent and constant than the two following (Fig. 1,2,3), the rates still varied
widely. Sampling error could account for some of the variation though it cannot
not alone explain the results obtained. Perhaps there was some modification due to
toxic effects of the inhibitors; i.e. release of proteases affecting enzyme activity.
Since there were no logical trends to be deduced, the only significant finding from
the study is that the stimulation of NAD kinase with calcium was stable throughout
development in all three cases. This indicates that NAD is not synthesized or
degraded in the cell during development. This opens up a wide field of
speculations.
During the first 60 seconds after fertilization, NAD kinase is responsible for
converting about half the cell's NAD into NADP (Epel et al, 1981). What turns on
the enzyme is the transient rise in calcium occurring at fertilization. This initial
increase has been documented to last about 5 minutes but only the first minute has a
calcium level high enough to keep the enzyme active. The free intracellular calcium
rises from about 10Onm to 2uM (Poenie, 1985). This corresponds to the Cat
sensitivity curve of NAD kinase (Fig. 4)
There were some problems with trying to come up with the cutoff point for
turning on NAD kinase with calcium. The experiment had been carried out
previously and the crucial concentrations levels were reported to be between 10-7 M
an 10-6 M of free calcium.(Epel et al, 1981) Though the data presented here
confirm the previous findings, using a more sophisticated software program to
account for effects of various chelators as well as pH and temperature, revealed a
similar calcium dependency. More accurate estimates of free calcium were difficult
to obtain since calcium buffering with EGTA was prone to error, in part because
extremely small variations in calcium content would have large consequences for
free calcium estimates. For example, a Sum difference in total calcium could cause
a ten-fold change in free calcium concentration.
Keeping these factors in mind, if the on-off switch for NAD kinase is taken
to be between 10-M and 10M of free calcium, this implies that any changes of
free calcium levels could directly control the NAD kinase. Studies that have traced
the pattern of calcium fluctuations during early development reveal that the cell cycle
shows well defined calcium peaks corresponding to specific events of cell division
and embryo development (Poenie et al, 1985).
Supposing that regulation of NAD kinase does occur, this would mean that
there would be varying levels of NADP or NADPH at different time points in the
cell cyle. One role for NADP relates to the ratio of NADP/NADPH levels in the
egg. This is a current area of research in the lab of David Epel, who believes that
redox regulation could be taking place during development as the NADP/NADPH
ratio changes.
On the other hand, one possibility for consequences of regulation of NAD
kinase activity suggests a mechanism for keeping levels of NADP constant. It has
been discovered that a derivative of NADP, along with other pyridine nucleotide
metabolites, stimulate calcium release (Lee et al, 1987). This apparent relationship
between NADP and calcium hints at a possible positive feedback system, in which
NAD kinase could be reactivated at certain stages in the cell cyle. Depletion of
NADP by conversion to its derivative form would be prevented by stimulation of
NADP production by calcium. In the meantime, the released calcium could
stimulate other processes needed for the cell cycle. The role of NAD kinase would
then be to keep NADP levels constant in the cell.
Another interpretation of the stability of NAD kinase is to consider the tonic
nature of the enzyme. Most enzymes have half lives ranging from three minutes to
twenty-four hours (Bachmair et al, 1986). The finding that NAD kinase is not
degraded or resynthesized yet retains its activity upon calcium stimulation seems to
imply that it is remarkably stable. Sea urchins store their eggs up to six months
before spawning. For the egg to be constantly replacing an enzyme that is
necessary upon fertilization would be a waste of metabolic energy. Since NAD
kinase is such an enzyme, its stability could be a way of saving energy.
ACKNOWLEDGEMENTS
First and foremost, I thank David Epel for his direction, patience and
constant enthusiasm throughout the quarter. I also thank Chris Patton, Rob
Sweezy, and Paul Sun for all their help in the lab. Special thanks go to my
roommate Aimee Sison who put up with all my ups and downs for the past ten
weeks and to Roger Cornwall for the endless supply of Diet Cokes and ice cream
during long hours in lab. Finally, I thank all my fellow 175H spring students for
making this quarter a lot of fun and the rest of the faculty and staff at Hopkins for
making us feel so welcome.
7.





LITERATURE CITED
Afanasieva, T.P., Filippovich, S.Yu., Sokolovsky, V.Yu., and Kritsky, M.S.
(1982) "Developmental Regulation of NAD Kinase in Neurospora crassa".
Archives of Microbiology 133, 307-311.
Bachmair, A., Finley, D., and Varshavsky, A. (1986) "In Vivo Half Life of a
Protein is a Function of its Amino Terminal Residue". Science 234, 179-186.
Berger, S.J., Manory, I., Sudar, D.C. and Berger, N.A. (1987)
"Induction of the
Pyridine Nucleotide Synthesis Pathway in Mitogen-Stimulated Human T-
Lymphocytes". Experimental Cell Research 169, 149-157.
Clapper, D.L., Walseth, T.F., Dargie, P.J., and Lee, H.C. (1987) "Pyridine
Nucleotide Metabolites Stimulate Calcium Release from Sea Urchin Eg
Microsomes Desensitized to Inositol Trisphosphate". Journal of Biological
Chemistry 262, 9561-9567.
Epel, D., Patton,C., Wallace, R.W., and Cheung, W.Y. (1981) "Calmodulin
Activates NAD Kinase of Sea Urchin Eggs: an Early Event of Fertilization". Cell
23, 543-549.
Gross, P.R., Malkin, L.I., and Moyer, W.A. (1964) "Templates for the First
Proteins of Embryonic Development". Proc. National Academy of Science 51,
407.
Hogan, B., and Gross, P.R. (1971) "The Effect of Protein Synthesis Inhibition
on the Entry of Messenger RNA Into the Cytoplasm of Sea Urchin Embryos".
Journal of Cell Biology 49, 692-697
Laval-Marten, D., Carre,I., Barbera, S.J., and Edmunds, L.N. (1990) "Circadian
Variations in the Affinities of NAD Kinase and NADP Phosphatase for Their
Substrates, NAD and NADP, in Dividing and Nondividing Cells of the
Achlorophyllous ZC Mutant of of Euglena Gracilis Klebs (Strain Z)".
Chronobiology International 7, 99-105.
Poenie, M., Alderton, J., Tsien, R.Y., and Steinhardt. R.A. (1985) "Changes of
Free Calcium Levels with Stages of the Cell Division Cycle" Nature 315, 147-149.
Wagenaar, E.B. and Mazia, D. (1978) "The Effect of Emetine on First Cleavage
Division in the Sea Urchin, Stronglyocentrotus purpuratus" in Cell Reproduction:
In Honor of Daniel Mazia (E.R. Dirksen, D.M. Prescott, and C.F. Fox, editors)
539-546.
12
..7
FIGURE LEGEND
FIGURE 1. Relative rate of NAD kinase activity as indicated by appearance of
NADPH in response to calcium during normal development. Each point represents
activity, in relative fluorescent units normalized to equivalent protein, of enzyme
activity in S. purpuratus embryos. Time points correspond to development as
follows; 0: unfertilized, 3: four-cell stage, 6,9,12: multi-cell stage, 18,21: blastula
FIGURE 2. Relative rate of NAD kinase activity in response to calcium during
development in actinomycin. Same conditions as Figure 1. Development also
corresponds to above.
FIGURE 3. Relative rate of NAD kinase activity in response to calcium during
development in emetine. Same conditions as Figure 1 and Figure 2. Development
was arrested at the one cell stage. Cells lysed at the 20th hour.
FIGURE 4. Calcium sensitivity curve for NAD kinase from fertilized eggs. Free
calcium concentrations as predicted by Patton's chelator program.
8
8
—

ubieddy HNo

S
N
ubeddy N
8
8
ueddy N
8
0

9
8
8 8
AV Szu