Cholinephosphotransferase activity during early development of the sea urchin, Arbacia punctulata

DEVELOPMEKTAL

RIOLOG\

31, 234-241

(19i:j)

Cholinephosphotransferase

Activity

during

of the Sea Urchin, Arbacia

Early Development

punctulata

RICHARD D. EWING

Department

of Zoology, Oregon State University, Accepted

September

Corvallis,

Oregon 97331

29, 1972

Cholinephosphotransferase activity was examined during early development of Arbacia embryos. CMP was rapidly incorporated by enzyme preparation from Arbacia embryos into a compound identified as CDP-choline. Activity was dependent upon the presence of Mg*+, Mn?+, or Co*+, and was maximal in phosphate buffer, pH 7.0, containing 3 x lo-’ M MgCl, and 2 x 10m6M CMP. Addition of ATP or egg lecithin had no effect on enzyme activity. The activity was localized in the 1000g and 10,000g pellets, with little or no activity in the microsomal fraction. Upon fertilization, cholinephosphotransferase activity decreased rapidly, detectable changes in activity being observed within 2 min after fertilization. After reaching a minimum activity 2 hr after fertilization, the enzyme activity increased up to the gastrula stage, then decreased once more. Possible interference by competing enzymes was examined and found to be negligible. punctulata

INTRODUCTION

Much of our knowledge of fertilization and cleavage has been derived from extensive studies of the fertilization and early development of sea urchin embryos. The role of membrane synthesis during this period has continually been stressed and many cytological and electron microscopical studies have been carried out on the changes in membranous structures during early development (Runnstriim, 1966; Harris, 1969). However, there have been surprisingly few biochemical studies on membranes in these embryos. Indeed, the biochemistry of one of the major components of membranes, the phospholipids, has received little attention. One of the major phospholipid components of sea urchin membranes, as well as other animal membranes, is phosphatidylcholine (Isono, 1965). Synthesis of this phospholipid occurs primarily by the condensation of the phosphorylcholine portion of CDP-choline with a 1, Z-diglyceride:

CDP-choline + 1,2-digiyceride f phosphatidylcholine

This reaction is catalyzed by the enzyme, cholinephosphotransferase (1,2 - CDP choline : 1 , 2-diglyceride cholinephosphotransferase, EC 2.7.8.2) and proceeds strongly in the direction of phosphatidylcholine synthesis in most systems (Weiss et al., 1958). Studies on the activity of this enzyme in development of the yolkly embryos of Artemiu saline, however, indicated CDP-choline could be rapidly formed from CMP and phosphatidylcholine (Ewing and Finamore, 1970a, b). This suggested that the enzyme may be important in the transfer of phosphorylcholine groups from preformed phosphatidylcholine molecules to sites of phospholipid synthesis involved in membrane biogenesis in the developing embryos. The activity of cholinephosphotransferase (CPT) was therefore examined during early embryonic development of Arbaciu punctulata to determine whether the enzyme was readily reversible, as in the 234

Copyright All rights

0 1973 by Academic Press. Inc of reproduction in any form reserved.

+ CMP

EWING

Cholinephosphotransferase

Artemia system, and whether changes in activity corresponded to developmental events related to membrane biogenesis. MATERIALS

AND

METHODS

Preparation of eggs and sperm. Eggs from Arbacia punctulata were obtained by injection of female urchins with 0.1 ml of 0.2 M acetylcholine and by collection of the eggs by inverting the urchins over beakers of Millipore-filtered sea water. The eggs were washed three times, then suspended in a known volume of Millepore-filtered sea water. A O.Ol-ml sample was taken with a disposable micropipette for direct counting by light microscopy. More than 400 embryos were counted for each sample with a 5% sampling error. Sperm were obtained by injection of male urchins with 0.1 ml of 0.2 M acetylcholine and removal of the sperm from the aboral surface with a pipette. The sperm were centrifuged under standard conditions in graduated centrifuge tubes to determine the number of milliliters of packed sperm. The number of sperm per milliliter of packed sperm was obtained from direct counting of a sample diluted from a known packed volume of sperm. Fertilization was accomplished by mixing 500 ml of egg suspension with 50 ml of diluted sperm to give an approximate final concentration of lo7 sperm and lo4 eggs per milliliter. The extent of fertilization was determined after 60 min and was consistently 96-100%. Embryos were incubated with stirring at 20-22°C and sampled at intervals. Developmental stages were determined by light microscopy. Enzyme preparations. Eggs or developing embryos were removed from sea water by centrifugation at approximately 100 g for 10 min. The supernatant was decanted and the embryos were suspended in a volume of 10% sucrose sufficient to give a concentration of 0.5 x lo6 embryos per milliliter. The suspension was frozen

in Arbacia

235

for 1 hr at - 15°C then homogenized in a Tenbroeck homogenizer. Either freezing or homogenization alone was not sufficient to completely disrupt the membranes of the embryos. Sperm were sedimented at 1000 g for 10 min, the supernatant was decanted, and the pellet was resuspended in distilled water. The sperm suspension was frozen overnight at ~ 15°C then homogenized in a Tenbroeck homogenizer in sufficient sucrose to give a final concentration of 10%. This treatment resulted in the complete disruption of the sperm. Fractionation of intact eggs by centrifugation on a discontinuous sucrose/sea water gradient was accomplished by the method of Tyler and Tyler (1966). Fractions were carefully removed with a pipette, diluted with filtered sea water and centrifuged at 500 g for 10 min. Pellets were resuspended in 10% sucrose and treated as described above for eggs and embryos. Enzyme assays. Assays for cholinephosphotransferase activity were as described earlier (Ewing and Finamore, 1970a). For most assays, activity was based on the formation of 3H-CDP-choline from 3H-CMP. Reaction mixtures were composed of 16 pmoles sodium phosphate, pH 6.8, 10 Fmoles MgCIP, 7.2 nmoles 3HCMP (190,000 cpm), and 0.04 ml of enzyme preparation in a final volume of 0.3 ml. Reactions were run at 37°C and stopped by addition of 0.05 ml 5.0 M perchloric acid. CDP-choline was separated from CMP by ascending paper chromatography on Whatman 3 MM paper, using ethanol/water/O.1 M sodium tetraborate/5.0 M ammonium acetate, pH 9.51 saturated EDTA (220/70/25/20/0.5, v/v) as a solvent. Chromatograms were run for 18 hr with CMP, CDP-choline, and cyt.idine as markers. Ultraviolet-absorbing spots corresponding to these three markers were cut out and placed directly

236

DEVELOPMENTALBIOLOGY

VOLUME 31, 1973

into scintillation vials containing 15 ml of scintillation fluid and counted in a Packard Tri-Carb liquid scintillation spectrometer. Formation of 14C-phosphatidylcholine from CDP-choline-l, 2- 14C was assayed by the method of Goldfine (1969). The product was chromatographed with known standards by thin-layer chromatography on silica gel GF (Brinckmann Co.). Solvent systems were: (1) CH,Cl/MeoH/HAc/ H,O (50/25/g/4, v/v); (2) CHCl,/MeoH/ NH,OH. Chromatograms were developed with iodine vapors and phospholipid spots were scraped into scintillation vials to be counted as described above.

uct were examined to determine that it was indeed CDP-choline. The product was destroyed by incubation with 0.1 M sodium periodate at 37°C for 1 hr, indicating that the ribose moiety of CMP was not reduced to deoxyribose. Enzymatic hydrolysis of the product indicated that the phosphate of CMP was involved in a phosphodiester bond. Thus, bacterial alkaline phosphatase had no effect on migration of the product on paper chromatography, while snake venom phosphodiesterase abolished radioactivity in the CDP-choline region of the chromatogram with a consequent increase in radioactivity in the CMP region. When a mixture of bacterial alkaline phosphatase and snake venom phosRESULTS phodiesterase was incubated with the laFormation and Identification of CDP-Chobeled product, all the radioactivity was line shifted from the CDP-choline and CMP regions of the chromatogram to the cytiIncubation of egg particulate fractions dine region. with 3H-CMP caused rapid formation of a In addition, the radioactive product milabeled compound migrating to a position similar to CDP-choline on paper chroma- grated identically with CDP-choline upon column chromatography on Dowex 1X2 tography. Incorporation of radioactivity ion exchange resin and upon paper chrointo this compound was approximately matography in the following solvents: (1) linear during the first 5 min of the reaction isobutyric acid/water/NH,OH (66/33/l, (Fig. 1). This time interval was used as an v/v) ; (2) butanollacetic acid/water (50/20/ estimate of the initial rate of enzyme ac30, v/v). We therefore concluded that the tivity. product was CDP-choline. The properties of the radioactive prodProperties

FIG. 1. Incorporation of 3H-CMP into CDP-choline with time. Reaction conditions were as described in the text. Enzyme preparation used was 10,000 g pellet from embryos homogenized 2 hr after fertilization.

of the Enzyme

Properties of the cholinephosphotransferase in particulate fractions of Arbacia were examined briefly to obtain optimal assay conditions. The optimal pH using phosphate buffer was 7.0. Activities were markedly lower when other buffers were used. Enzyme activity required the presence of Mg2+, Mn2+, or Co2+ (Table 1). Maximal activity was attained with a Mg2+ concentration of 0.03 M and a CMP concentration of 3 x 10m5M. Half-maximal concentrations of Mg2+ and CPM were 0.013 M and 1.0 x 10m5M, respectively. Addition of 1.0 pmole of ATP or purified egg lecithin to the reaction mixture had no

Cholinephosphotransfemse

EWING

TABLE 1 METAL ION REQUIREMENTS FOR CHOLINEPHOSPHOTRANSFERASE ACTIVITY Reaction

conditions”

Incomplete 0 min 5 min 5 min, + MgCl, 5 min, + MnCl, 5 min, + CoCl,

CDP-choline formation (nmoles/5 min)

0.4 0.5 5.1 3.0 8.0

a Incomplete reaction mixtures contained components as described in Materials and Methods, with the omission of MgCl,. When required, 10 rmoles of MgCl,, MnCll, or CoCIZ were added. Enzyme from the 10,000 g pellet of homogenized sea urchin eggs was used. Reactions were run for 5 min at 37°C then stopped by addition of 0.05 ml of 5 N perchloric acid. CDP-choline formation was assayed as described.

effect on activity whereas addition of 35 pmoles of NaF caused complete inhibition of activity. Formation of ‘S-phosphatidylcholine from CDP-choline-( 1,2- “C) could be readily demonstrated by the method of Goldfine (1969), but the activity was variable from preparation to preparation and consequently was not used as an assay system. Little or no incorporation into other phospholipids was observed. Localization

of Enzyme Activit.v

Enzyme activity was localized primarily in the 1000 g and 10,000 g pellets (Table 2). Little or no activity was observed in the microsomal (100,000 g pellet) or in the 100,000 g supernatant fractions. These results were obtained both with unfertilized eggs and with embryos from the time of fertilization to the pluteus stage. Microsoma1 and supernatant fractions at all stages contained little CPT activity. In an attempt to further localize the enzyme activity, whole unfertilized eggs were sedimented in a discontinuous sucrose/sea water gradient (Tyler and Tyler, 1966). Centrifugation at 12,000 g for 15 min resulted in the appearance of two bands in

237

in Arbacia

TABLE 2 LOCALIZATION OF CHOLINEPHOSPHOTRANSFERASE ACTIMTY Preparation” 1000 g pellet Wash 10,000 g pellet Wash 100,000 g pellet 100,000 g supernatant

CDP-choline formation (nmoles/min/108 eggs) 5.7 0.15 8.7 0.03 0.01 0.05

“Homogenates of unfertilized eggs were centrifuged at 1000 g for 10 min in a refrigerated Sorvall Model RCB-B centrifuge. The supernatant was removed and centrifuged at 10,000 g for 30 min. The supernatant was again removed and centrifuged at 100,000 g for 90 min in a Beckman Model L-2 ultracentrifuge. All pellets were washed once with 10% sucrose. Washed pellets were resuspended in 10% sucrose for assays of cholinephosphotransferase activity.

the gradient and a pellet at the bottom of the centrifuge tube. The upper band was composed of quarter-eggs containing nuclei, the middle band was composed of quarter-eggs containing granular material but no nuclei, and the pellet was composed of half-eggs containing yolk and pigment granules. Assays of the CPT activity of these three fractions showed that 63% of the total activity resided in the upper nucleate fraction, while 37% of the total activity was found in the pellet. The middle band contributed less than 1% of the total enzyme activity. Enzyme Activity

during Development

Under optimal conditions, CPT activities in sperm and eggs of Arbacia were 0.016 and 15.4 nmoles of CDP-choline formed per minute per lo6 sperm or eggs, respectively. Upon fertilization, enzyme activity decreased drastically (Fig. 2). This decrease in activity occurred very rapidly and could be detected in some experiments within 2 min after addition of sperm to the egg suspension. Enzyme activity was maintained at a low level during initial cleavages, then increased to nearly that of

DEVELOPMENTALBIOLOGY

VOLUME 31, 1973

\

.R.

0 \

/ I/

o-

o-o-0

./.-*-

l-

\ /

FIG. 2. Cholinephosphotransferase activity in developing embryos following fertilization. Reaction conditions were as described in the text.

the unfertilized eggs during gastrulation and hatching. At the pluteus stage, the enzyme activity once more dropped to low levels. Since particulate systems contain numerous enzymes competing with CPT for substrates, an examination of some of these enzymes was undertaken to show that the changes in CPT activity during development were not due to changes in the activity of these competing enzymes. Phosphatases hydrolyzing CMP to cytidine were assayed routinely throughout these experiments by paper chromatography. Phosphatase activity was observed in the pellet fractions of’ Arbacia, but was minimized by the use of phosphate buffer and low pH. Phosphatase activity increased 2-fold when tris(hydroxymethyl)aminomethane buffer was used in place of phosphate buffer. Maximal phosphatase activity was observed at pH 9.6. Phosphorylation of CMP to CDP and CTP was not observed upon chromatography of reaction mixtures in an isobutyric acid/water/NH,OH (66/33/l, v/v) solvent with CMP, CDP, and CTP as markers. Phosphodiesterase activity was difficult to measure directly in the present reaction mixtures. However, CDP-choline formation reached a plateau within 30 min incubation and remained at a constant level for at least 50 min (Fig. 3), indicating that di-

FIG. 3. Incorporation of radioactivity into CDPcholine and loss of radioactivity from CMP with time. Enzyme preparation used was the 10,000 g pellet from homogenized unfertilized eggs. CDP-choline (W, CMP (0).

esterase activity was minimal under the conditions used in the assay. CMP decreased in proportion of the amount of CDP-choline formed (Fig. 3) and reached a constant level after about 30 min incubation. About 40% of the CMP added to the reaction mixture was converted to CDPcholine. DISCUSSION

Under the conditions described in the Arbacia system, 40% of the 3H-CMP introduced into the reaction mixture was rapidly converted into CDP-choline. This provides further support for the hypothesis that the phosphatidylcholine in yolk and organelle membranes in developing systems may serve as sources of activated choline which are available for utilization when construction of new membranous structures is required. The localization of the cholinephosphotransferase activity in the 1000 R and 10,000 g pellets is unusual. Cholinephosphotransferase in most systems has been shown to be associated with microsomal fractions (Schneider, 1963; Wilgram and Kennedy, 1963; Ewing and Finamore, 19i’Oa). Yet enzyme activity in Arbacia was consistently absent in microsomal fractions up to the pluteus stage. Centrifu-

EWING

Cholinephosphotransferase

gation of unfertilized eggs suggested that the activity was associated with fractions containing predominantly nuclei and Golgi, or yolk (Anderson, 1969). Egg fractions comprised predominantly of mitochondria contained little or no activity. The most striking behavior of the cholinephosphotransferase activity in Arbacia eggs is the immediate and precipitous decrease which occurs upon fertilization. The reasons for this decrease in activity are all the more interesting because of the increase in in vivo labeling of phosphatidylcholine with ‘*C-glycerol after fertilization (Mohri, 1964). In dealing with enzyme activities during developmental processes, however, one must be cautious of interpretations of changes in enzyme activities because of the complexities of crude enzyme preparations (Moog, 1965). This is even more pressing in particulate enzyme systems such as the present one. It is a source of frustration in these enzyme assays that the lipid substrates cannot be easily controlled. In the reactions described, phosphatidylcholine is present in large quantities in the membrane fractions and is presumed, rather than proved, to be the source of phosphorylated choline for CDP-choline formation. Direct addition of phosphatidylcholine to reactions causes no stimulation of enzyme activity, either because the substrate is already present in excess, or because it is not able to reach the active site of the enzyme. Addition to lecithin to detergent-treated enzyme preparations relieves the inhibition caused by the detergent, but it is not possible to tell from experiments of this sort whether the phosphatidylcholine is actually used as a substrate. It is therefore possible that spingomyelin or an unknown membrane-bound phosphorylcholine donor contributes to the formation of CDP-choline in these assays. Although we have no direct evidence to refute this, there are several lines of indirect evidence which suggest that this is not the

in Arbacia

239

case: (1) Optimal conditions for 3H-CMP incorporation into CDP-choline in Arbacia require a pH of 7.0 and the presence of conditions for MC+> whereas optimal CDP-choline : ceramide choline-phosphotransferase require a pH of 7.5 and the presence of Mn2+ (Sribney and Kennedy, 1958). (2) CDP-choline-l, 2- 14C added to the reaction mixtures is incorporated rapidly only into phosphatidylcholine. (3) The kinetic properties, reaction rates, and apparent equilibrium values are very similar to the CPT activity from Artemiu, in which the reaction components were defined through stoichiometry studies using prelabeled microsomes (Ewing and Finamore, 1970a). Possible errors in measuring enzyme activity could result from any marked change in the concentration of substrate inherent in the enzyme preparation. However, phosphatidylcholine does not seem to undergo significant changes in concentration following fertilization (Mohri, 1964; Isono, 1965). Likewise, CMP concentrations in sea urchin embryos do not seem to be significantly altered following fertilization (Yanagisawa and Isono, 1966), although the changes may be masked by the presence of homarine in the CMP fractions (Hultin et al., 1953). Addition of charcoal to our enzyme preparations to remove residual nucleotides did not stimulate the incorporation of 3H-CMP into CDP-choline, however, as would have been expected if the 3H-CMP were being diluted by unlabeled CMP. A second source of possible error in measuring enzyme activity lies in the competition of other enzymes for the substrates. In the present system there was no extensive phosphorylation or dephosphorylation of 3H-CMP under the conditions used. Hydrolysis of the product, CDP-choline, cannot be easily measured or controlled in this system, however, (Schneider, 1963; Wilgram and Kennedy, 1963). In preparation, an equilibrium the Arbacia

240

DEVELOPMENTALBIOLOGY

between CMP and CDP-choline could be maintained for a considerable period (Fig. 3). This would not be expected if the phosphatidylcholine pool in the enzyme preparation were being constantly diminished. In addition, the properties of the cholinephosphotransferase in Arbacia are very similar to those described in other species (Weiss et al., 1968; Gurr et al., 1965; Pennington and Worsfold, 1969; Ewing and Finamore, 1970a,b; Smith and Law, 1970), whereas changes in properties might be expected if there were major competition for the substrates from another enzyme. Thus, although there are undoubtedly other enzymes in competiton with cholinephosphotransferase for substrates, we feel that this competition is minimal under the conditions used. Finally, there is the possibility that upon fertilization the enzyme is liberated from the phospholipid components in the particulate fractions. Since phosphatidylcholine was not added to the reaction mixtures, the liberated enzyme would be deficient in substrate and hence not be detected. This possibility is extremely interesting in view of the results of Epel et al. (1969) and requires a closer examination. However, we feel that this possibility is unlikely for two reasons: (1) Experiments designed to test this possibility in eggs of Strongylocentrotus purpuratus have indicated that addition of lecithin to supernatant fractions does not stimulate CPT activity in these fractions (unpublished results); (2) the enzyme is very tightly associated with membrane components. Attempts to isolate the enzyme have consistently resulted in its inactivation, and it has, therefore, not yet been purified. Consequently, liberation from phospholipid components upon fertilization seems unlikely. We feel, therefore, that the decrease in cholinephosphotransferase activity following fertilization must be due to changes in the activity of CPT enzyme itself. The high

VOLUME 31, 1973

level of activity found in the unfertilized egg and the sudden decrease in activity following fertilization suggest that these changes may be an integral part of the complex series of reactions which take place immediately following fertilization (Allen, 1939; Endo, 1961; Epel et al., 1969). This work was supported by University of Tennessee Postdoctoral Training Grant No. HD-00296.01 from the National Institute of Child Health and Human Development and by Fertilization and Gamete Physiology Training Grant No. 5TOl-HD00026-09 from the National Institutes of Health at the Marine Biological Laboratory, Wood’s Hole, Massachusetts. REFERENCES ALLEN, R. D. (1939). Fertilization and activation of sea urchin eggs in glass capillaries. Exp. Cell Res. 6, 403-424. ANDERSON, E. (1969). An ultrastructural analysis of the centrifuged whole, half, and quarter eggs of sea urchins. J. Cell Biol. 43, 6a-7a. ENDO, Y. (1961). Changes in the cortical layer of sea urchin eggs at fertilization as studied with the electron microscope. I. Clypeaster juponicus. Erp. Cell Res. 25, 383-397. EPEL, D., WEAVER, A. M., MUCHMORE, A. V., and SCHIMKE, R. T. (1969). B-l, 3-glucanase of sea urchin eggs: Release from particles at fertilization. Science 163, 294-296. EWING, R. D., and FINAMORE, F. J. (1970a). Phospholipid metabolism during development of the brine shrimp, Artemia salina. I. In vitro incorporation of CMP into CDP-choline by a microsomal enzyme system. Biochim. Biophys. Acta 218, 463-473. EWING, R. D., and FINAMORE, F. J. (1970b). Phospholipid metabolism during development of the brine shrimp, Artemia sulk II. Synthesis of phosphatidylcholine by a microsomal enzyme system in nauplii. Biochim. Biophys. Acta 218,473-481. GOLDFINE, H. (1969). Filter paper disk assay for lipid synthesis. In Methods Erzzymol. 14,649-651. GURR, M. I., BRINDLEY, D. N., and HUBSCHER, G. (1965). Metabolism of phospholipids. VIII. Biosynthesis of phosphatidylcholine in the intestinal mucosa. Biochim. Biophys. Acta 98, 486-501. HARRIS, P. (1969). Relation of fine structure to biochemical changes in developing sea urchin eggs and zygotes. In “The Cell Cycle: Gene-Enzyme Interaction” (G. M. Padilla, G. L. Whitson, and I. L. Cameron, eds.). Academic Press, New York. HULTIN, T., LINDVALL, S., and GUSTAFSSON, K. (1953). On the occurrence of picolinic acid betaine in sea urchin embryos. Ark. Kemi 6,477-480. ISONO, Y. (1965). Phospholipids of sea urchin eggs. I.

EWING

Cholinephosphotransferase

Thin-layer and paper chromatographic studies. Sci. Papers Coil. Gen. Educ. Univ. Tokyo 15, 87-94. MOHRI, H. (1964). Utilization of “‘C-labelled acetate and glycerol for lipid synthesis during the early development of sea urchin embryos. Biol. Bull. 126, 440-455. Moot, F. (1965). Enzyme development in relation to functional differentiation. In “The Biochemistry of Animal Development” (R. Weber, ed.). Academic Press, New York. PENNINGTON, R. J., and WORSFELD,M. (1969). Biosynthesis of lecithin by skeletal muscle. Biochim. Biophys. Acta 176, 774-782. RUNNSTR~M, J. (1966). The vitelline membrane and cortical particles in sea urchin eggs and their function in maturation and fertilization. Advan. Morphog. 5, 222-325. SCHNEIDER, W. C. (1963). Intracellular distribution of enzymes. XIII. Enzymatic synthesis of deoxycytidine diphosphate choline and lecithin in rat liver. J. Biol. Chem. 238, 3572-3578.

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SMITH, J. D., and LAW, J. H. (1970). Phosphatidylcholine biosynthesis in Tetrahymena pyriformis. Biochim. Biophys. Acta 202, 141-152. SRIBNEY, M., and KENNEDY, E. P. (1958). The enzymatic synthesis of spingomyelin. J. Biol. Chem. 233, 1315-1322. TYLER, A., and TYLER, B. S. (1966). Physiology of fertilization and early development. In “Physiology of Echinodermata” (R. Boolootian, ed.). Wiley, New York. WEISS, S. B., SMITH, S. W., and KENNEDY, E. P. (1958). The enzymatic formation of lecithin from cytidine diphosphate choline and u-l, P-diglyceride. J. Biol. Chem. 231, 53-64. WILGRAM, G. F., and KENNEDY, E. P. (1963). Intracellular distribution of some enzymes catalyzing reactions in the biosynthesis of complex lipids. J. Biol. Chem. 238, 2615-2619. YANAGISAWA, T., and ISONO, N. (1966). Acid soluble nucleotides in the sea urchin egg. I. Ion-exchange chromatographic separation and characterization. Embryologia 9, 170-183.