Early cyclical changes in polyamine synthesis during sea-urchin development

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1 /. Embryo/, exp. Morph. Vol. 30,1, pp , Printed in Great Britain Early cyclical changes in polyamine synthesis during sea-urchin development By CAROL-ANN MANEN 1 AND DIANE H. RUSSELL 2 From the Department of Zoology, University of Maine, and National Institutes of Health, National Cancer Institute, Baltimore Cancer Research Center SUMMARY The polyamines, putrescine, spermidine, and spermine, undergo dramatic cyclical variation in both synthesis and accumulation during the early cleavage stages of sea-urchin development. Ornithine decarboxylase activity (putrescine synthesis) in developing Strongylocentrotus purpuratus exhibits maxima at and 2 h after fertilization; increases in ornithine decarboxylase activity appear to correspond to the first and second S phases. Putrescine-stimulated S-adenosyl-L-methionine decarboxylase (spermidine synthesis) and spermidine-stimulated S-adenosyl-L-methionine decarboxylase (spermine synthesis) activities reflect rises during prophase-metaphase of the first and second divisions in two species of sea urchins. Cyclical changes in the concentrations of these three amines were evident also. In general, there were drops in the levels of the amines prior to cleavage. These rapid declines in polyamine concentrations may reflect (.1) selective degradation or (2) selective secretion. INTRODUCTION Polyamines have not been studied extensively in marine invertebrates. There have been two reports of the presence of spermine in such, one in an echinoderm, Echinarachinius mirabilis (Ogata & Komada, 1943), and the other in the tunicate Cionia infest inalis (Ackermann & Janka, 1954). We have studied polyamine biosynthesis and accumulation in several species of sea urchins (Manen & Russell, 1973). Gametes of sea urchins as well as their adult tissues contain high amounts of spermine and relatively low amounts of putrescine and spermidine. This is in contrast to the polyamine patterns exhibited by other major groups. For instance, bacteria and amphibians contain substantial amounts of putrescine and spermidine, with spermine either absent or present only in trace amounts (Tabor & Tabor, 1964; Russell, Snyder & Medina, 1969). Spermidine and spermine, present in similar amounts, are the major polyamines detectable in mammalian tissues, with putrescine present in low amounts (Tabor & Tabor, 1964). However, the putrescine concentration increases rapidly when tissues 1 Author's address: University of Maine, Department of Zoology, Orono, Maine, U.S.A. 2 Author's address: National Institutes of Health, National Cancer Institute, Baltimore Cancer Research Center, Laboratory of Pharmacology, Baltimore, Maryland , U.S.A. 16-2

2 244 C.-A. MANEN AND D. H. RUSSELL L-ORNITHINE 0 ORNTTHINE DECARBOXYLASE NH * LH > <-H 2 -CH 2 -CH 2 - CH 2 I PUTRESCINE CH 2 (DIAMINOBUTANE) CH, + ' Decarboxylation CH NH CH 2 -CH 2 -NH 3 + and propylamine From transfer SAM CH 2 -CH 2 -CH 2 -NH 3 + From SAM + -NH + 2 -(CH 2 )3-NH3 + - NH + 3 -(CH 2 ) 4 -NH (CH 2 )3-NH 3 Decarboxylation and SPERMINE.. SPERM1DINE propylamine transfer Fig. 1. Schematic polyamine biosynthetic pathway. undergo growth processes, i.e. in regenerating rat liver (Dykstra & Herbst, 1965;Janne & Raina, 1968), in cardiac hypertrophy (Russell, Shiverick, Hamrell & Alpert, 1971; Feldman & Russell, 1972), and in appropriate mammalian tissues after hormonal stimulation (Pegg & Williams-Ashman, 1968; Russell & Snyder, 1969; Janne & Raina, 1969; Russell, Snyder & Medina, 1970; Russell & Taylor, 1971; Russell & Potyraj, 1972). The polyamine biosynthetic pathway in sea urchins appears to be similar to those reported for yeast (Coppoc, Kallio & Williams-Ashman, 1971), amphibians (Russell, 1971), and mammals (Pegg & Williams-Ashman, 1969; Feldman, Levy & Russell, 1972), and differs from the bacterial systems (Tabor & Tabor, 1964). In general, the precursor for polyamine synthesis is ornithine. The decarboxylation of ornithine results in the formation of putrescine (Fig. 1). Spermidine and spermine are formed from putrescine by the addition of one or two propylamine moieties respectively (Fig. 1). In bacteria, the conversion of putrescine to spermidine involves two separate enzymic reactions (Tabor & Tabor, 1964). The first is the enzymic decarboxylation of S-adenosyl-L-methionine to form carbon dioxide and 5'-deoxy-5'-S'-(3-methylthiopropylamine) sulfonium adenosine (decarboxylated S-adenosyl-L-methionine). This 5-adenosyl-L-methionine decarboxylase required Mg 2+ and contains pyruvate as a prosthetic group. A propylamine transferase then catalyzes the formation of spermidine from putrescine and a propylamine molecule which derives from decarboxylated S-adenosyl-L-methionine. In sea urchins as well as in mammals, at least in crude homogenates, there appears to be a coupling of the decarboxylase

3 Cyclical changes in polyamine synthesis 245 and transferase. Decarboxylated S-adenosyl-L-methionine cannot be separated as a free intermediate, and putrescine or spermidine are required to accept the propylamine molecule (Manen & Russell, 1973, and data in this paper). In contrast to the bacterial system, metal ions are not required nor is pyruvate known to be a cofactor. There may be a pyridoxal phosphate requirement (Feldman et al. 1972). We have reported that the pathway in sea urchins, like the mammalian system, is stimulated by putrescine or spermidine, does not exhibit metal requirements and there is coupled decarboxylation and transfer function. Although there is controversy as to whether purification of S-adenosyl- L-methionine decarboxylase leads to uncoupling of these two functions (Janne & Williams-Ashman, 1971; Janne, Schenone & Williams-Ashman, 1971; Feldman, et al. 1972), the important consideration here is the coupling in crude homogenates. The rate-limiting step in spermidine or spermine synthesis appears to be the activity of -S-adenosyl-L-methionine decarboxylase. Therefore the most accurate estimates of spermidine and spermine synthesis can be obtained from the measurements of putrescine-stimulated S-adenosyl-Lmethionine decarboxylase and spermidine-stimulated S-adenosyl-L-methionine decarboxylase respectively. In a study of changes in polyamine biosynthesis and accumulation during sea-urchin development, we found that during development (i.e. to gastrulation) the spermidine concentration increased markedly, with little change in either putrescine or spermine concentration. However, prior to fertilization spermine concentration can be as much as tenfold above that of spermidine. After gastrulation both spermine and spermidine were elevated. Enzyme activity patterns paralleled the changes detected in the levels of polyamines with the exception of ornithine decarboxylase activity. In this paper we report on studies of the changes in activities of the polyamine biosynthetic enzymes and in the levels of polyamines of sea-urchin eggs within the first 4 h after fertilization. Essentially, synchrony of cell division is exhibited at least through the first two cell cycles. Sharp drops in polyamine synthesis occur prior to cell division, along with marked drops in pool sizes. Therefore, cyclical variations exist in early polyamine synthesis as well as in other early biochemical events that have been studied in sea urchins (Mano, 1970; Lovtrup & lverson, 1969). Labelling experiments reported herein verify the biosynthetic pathway in sea urchins, and studies of partially purified S-adenosyl-L-methionine decarboxylase substantiate the coupling of decarboxylation and transfer activities in both spermidine and spermine formation. The discrepancy stated earlier in this paper between ornithine decarboxylase activity and putrescine levels in sea urchins is explained by the very low K m for putrescine exhibited by partially purified S-adenosyl-L-methionine decarboxylase.

4 246 C.-A. MANEN AND D. H. RUSSELL MATERIALS AND METHODS Materials Ripe Lytechinus pictus and Strongylocentrotus purpuratus were obtained from the Pacific Bio-Marine Supply Co., Venice, California. Spawning was induced by injection of 0-55 M-KCI. Eggs were collected and washed in filtered artificial sea water. Only those cultures with at least 90 % normal development were used in experiments. [Carboxyl- 14 C]5'-adenosyl-L-methionine (7-7 mci/mm), [1-14 C]- DL-ornithine (11-9 mci/mm) and [l-4-14 C]putrescine dihydrochloride (20-29 mci/m.m) were obtained from New England Nuclear. Preparation of enzyme solutions The material was homogenized in 4 vol. of 005 M sodium-potassium phosphate buffer, ph 7-2, containing 0-1 HIM dithiothreitol. These enzymes are inhibited by Tris buffer, as are the mammalian enzymes. The homogenate was centrifuged at 20000# for 20 min, the pellet discarded, and the supernatant used in the assays. Since the supernatant after centrifugation at g for 90 min gave the identical enzyme activities, the 20000# supernatant was used routinely. Protein was determined by the Lowry method (Lowry, Rosebrough, Farr & Randall, 1951) with bovine serum albumin as the standard. Assay for ornithine decarboxylase activity Ornithine decarboxylase activity was determined by measuring the liberation of 14 CO 2 from [l- 14 C]ornithine as described previously (Russell & Snyder, 1968; Russell & Snyder, 1969). Although the substrate concentration (0-1 HIM as L-ornithine used routinely) was non-saturating, the same changes were noted when excess ornithine (2 HIM) was used as substrate in some experiments. Assay for putrescine-stimulated S-adenosyl-L-methionine decarboxylase activity Enzyme activity was determined by measuring the liberation of 14 CO 2 from [carboxyl- 14 C]5'-adenosyl-L-methionine as previously described (Pegg & Williams-Ashman, 1969). Unless otherwise stated, incubation mixtures consisted of 0-1 mm [carboxyl-^cjs-adenosyl-l-methionine, HIM sodiumpotassium phosphate buffer (ph 7-2), 50 /*M pyridoxal phosphate, 2-5 mm putrescine, and ml (3-5 mg protein) of enzyme solution in 0-2 ml. When the formation of [ 14 C]spermidine from unlabelled S-adenosyl-L-methionine and [l,4-14 C]putrescine was estimated as previously described (Russell & McVicker, 1972), there was a stoichiometric relationship between the amount of [ 14 C]-spermidine formed and the evolution of 14 CO 2 when [carboxyl- 14 C].Sadenosyl-L-methionine was added. Therefore, 14 CO 2 evolution from [carboxyl- 14 C]5'-adenosyl-L-methionine was used routinely as a measure of the spermidine biosynthetic rate.

5 Cyclical changes in polyamine synthesis 247 Assay for spermidine-stimulated S-adenosyl-L-methionine decarboxylase activity This assay is identical to that for putrescine-stimulated S-adenosyl-L-methionine decarboxylase except 5 mm spermidine was added instead of 2-5 mm putrescine. Again, there was a stoichiometric relationship between the amount of [ 14 C]spermine formed and the evolution of 14 CO 2 when [carboxyl- 14 C]-Sadenosyl-L-methionine was added. Therefore, 14 CO 2 evolution from [carboxyl- "ClS-adenosyl-L-methionine was used routinely as a measure of spermine bio synthetic rate. Determinations of putrescine, spermidine and spermine concentrations Pools of embryos were homogenized in 4 vol. of 01 N-HCl and subjected to alkaline butanol extraction as previously described (Russell, Medina & Snyder, 19 70).TC A was extracted by ether washes prior to butanol extraction. The butanol was evaporated to dryness in an evapomix and the residue redissolved in 0-2 ml of 0-1 M-HCI. The amines were separated by high-voltage electrophoresis (80 V/cm for 1-5 h) in a 0-1 M citric acid buffer, ph 4-3. Concentrations were determined by staining the chromatography sheet (Whatman 3 MM paper) with a mixture of 1 g ninhydrin, 100 ml acetone, 5 ml glacial acetic acid, 10 ml H 2 O and 100 mg cadmium acetate, drying 60 min at 60 C, eluting the color, and recording the absorbancy at 505 nm. Standards were run in the same range as the samples. Purification of the enzyme The operations described below were done at 0-5 C. Crude extract Freshly spawned unfertilized eggs of L. pictus were homogenized with 4 vol. of 0-05 M phosphate buffer, ph 7-2, containing 1-0 mm EDTA, 0-1 mm dithiothreitol, 3 /(M pyridoxal phosphate and 0-5 M sucrose. Cellular debris was removed from the crude homogenate by centrifugation at g for 20 min. The resulting supernatant solution was recentrifuged at # (in a Beckman L3-5O ultracentrifuge) for 1 h and then passed through cheesecloth to remove the lipid layer. Filtration on Sephadex G-100 A 9 ml portion of the # supernatant solution was applied to a Sephadex G-100 column (45 cm x 4-9 cm 2 ) which had been equilibrated previously with the homogenizing buffer. The enzyme was eluted with the equilibrating buffer under a hydrostatic pressure of 20 cm at a flow rate of 25 ml/h and the eluate was collected in 40 ml fractions. The most active fractions were pooled.

6 248 C.-A. MANEN AND D. H. RUSSELL DEAE-Cellulose chromatography The pooled enzyme fraction was dialyzed for 1 h against two changes of 50 vol. of 0-01 M phosphate buffer, ph 7-2, containing 1 mm EDTA, 0-1 mm dithiothreitol, 3 fm pyridoxal phosphate and 0-5 M sucrose. Then 30 ml of the dialyzed preparation were applied to a DEAE-cellulose column (15 cm x 4-9 cm 2 ) which had been equilibrated previously with the same buffer as used in the dialysis. After the column had been washed with 90 ml of the equilibrating buffer, the active enzyme was eluted with 300 ml of a linear gradient of 0-1 M-KCI in the equilibrating buffer. Fractions (4 ml) were collected, and the most active fractions were pooled. At this stage enzyme activity was stable on storage at 20 C for more than 6 months. Labelling Fertilized eggs were centrifuged gently (270 g for 1 min) and resuspended in 250 ml of sea water with 2 mci of [ 14 C]putrescine (0-1 mmoles). After 30 min the eggs were washed twice with x 50 vol. and cold-pulsed for 10 min with 1 mm putrescine. They were then washed twice and resuspended in 300 ml of sea water. The polyamines were extracted as described above with the exception that the ninhydrin-stained spots were cut out and eluted with 5 ml of methanol for 30 min, after which 10 ml of toluene omnifluor was added and the radioactivity counted on a Beckman LS-150 liquid scintillation counter. Corrections were made for the quench due to the ninhydrin color. RESULTS Ornithine decarboxylase activity {putrescine formation) Earlier we reported a marked elevation in ornithine decarboxylase activity in L.pictus eggs within 1 h of fertilization (Manen & Russell, 1973). When enzymic activity was measured at earlier times, it was found that ornithine decarboxylase activity had increased over fourfold within \ h and within 1 h the activity had dropped to twofold above the level present in unfertilized eggs (Fig. 2). Ornithine decarboxylase activity continued to decline and was below the control level at 3 h post-fertilization. Ornithine decarboxylase activity in fertilized eggs of S. purpuratus exhibits two marked cycles within the first 4 h (Fig. 3), one which has a maximum at \ h, similar to that of L. pictus, and another which is maximal at 2 h, at which time ornithine decarboxylase activity is 14-fold greater than the level found in unfertilized eggs.

7 Cyclical changes in polyamine synthesis Time after fertilization (h) Fig. 2. Ornithine decarboxylase activity in early cleavage stages of L. pictus. Activity was determined by measuring the evolution of 14 CO 2 from ["QL-ornithine (Russell & Snyder, 1968; Russell & Snyder, 1969). Each point represents the mean ± S.E. for four separate determinations. Putrescine-stimulated S-adenosyl-L-methionine decarboxylase (spermidine formation) As previously reported, we found that putrescine-stimulated S-adenosyl-Lmethionine decarboxylase exhibits considerable activity in unfertilized eggs of L. pictus (Manen & Russell, 1973). After fertilization the activity drops markedly with a low point \ h after fertilization, and then cycles with maxima at \\ and 3 h respectively (Fig. 4). This is in contrast to the maxima at \ and 2 h found for ornithine decarboxylase activity in L. pictus. The cycles are precisely 1 h out of synchrony. The same cyclical patterns were detected for putrescine-stimulated S-adenosyl- L-methionine decarboxylase of S. purpuratus (Fig. 5). However, the initial levels

8 250 C.-A. MANEN AND D. H. RUSSELL Time after fertilization (h) Fig. 3. Ornithine decarboxylase activity in early cleavage stages of S. purpuratus. Activity was determined by measuring the evolution of 14 CO 2 from [ 14 C]L-ornithine (Russell & Snyder, 1968; Russell & Snyder, 1969). Each point represents the mean ± S.E. for four separate determinations. of this enzyme are lower in S.purpuratus and there is not the post-fertilization drop in activity. Spermidine-stimulated S-adenosyl-L-methionine decarboxylase (spermine formation) Since the level of spermidine-stimulated S-adenosyl-L-methionine decarboxylase is lower than that of putrescine-stimulated S-adenosyl-L-methionine decarboxylase by a factor of 2, the cycles are not as striking but they are still evident (Figs. 4, 5). There is low activity at \ and 2\ h respectively, and maximal activities at \\ and 3 h in both species of sea urchins studied. Variations in the polyamine pooh during early cleavage stages After fertilization of S. purpuratus eggs, preliminary experiments indicate that all three amines exhibit maximal concentrations 2\ h post-fertilization, followed by marked declines by telophase of the second division. A marked increase in the polyamines between 2 and 3 h after fertilization, followed by a dramatic drop in concentrations, was measured in L. pictus also (Fig. 6).

9 Cyclical changes in polyamine synthesis Putrcscinc-stimulatcd Spcrmidinc-stimulated 4800 X o on e 3 se 3 E d Time after fertilization (h) Fig. 4. Putrescine- and spermidine-stimulated S-adenosyl-L-methionine decarboxylase activity in early cleavage stages of L. pictus. Activity was determined by measuring the 14 CO 2 released from 14 COOH-S-adenosyl-L-methionine in the presence of the appropriate amine (see Materials and Methods). Each point represents the mean ± S.E. for four separate determinations. Therefore, not only do the polyamine biosynthetic enzymes exhibit cyclical patterns, but also the products themselves. There must be either active secretion of polyamines at discrete times or active degradation. Distribution of radiolabel after incubation of embryos with [ u C]putrescine After the incubation of fertilized eggs of L. pictus with [ 14 C]putrescine for \ h, the eggs were washed with sea water containing cold putrescine, then resuspended in sea water alone. Samples were removed at \ h intervals and assayed for radiolabeled amines. Radiolabel is detectable very rapidly, not only in endogenous putrescine but also in spermidine and spermine (Fig. 7). The label is present mainly in putrescine and spermidine. This agrees with the greatly increased accumulations there during early after fertilization. These data indicate further that the polyamine biosynthetic pathway is similar in sea urchins to that already established for other major groups.

10 252 C.-A. MANEN AND D. H. RUSSELL \ r Putrcscinc-stimulatcd Spcrmidine-stimulatcd Time after fertilization (h) Fig. 5. Putrescine- and spermidine-stimulated S-adenosyl-L-methionine decarboxylase activity in early cleavage stages of S. purpuratus. Activity was determined by measuring the 14 CO 2 released, from "COOH-S-adenosyl-L-methionine in the presence of the appropriate amine (see Materials and Methods). Each point represents the mean ± S.E. for four separate determinations. Properties of S-adenosyl-L-methionine decarboxylase from sea urchins Through the use of the double-reciprocal plot, the apparent K ni for putrescine was determined. Saturating levels of -S-adenosyl-L-methionine were used and partially purified S-adenosyl-L-methione decarboxylase preparations from L. pictus were utilized as the enzyme source in these assays. The K m for putrescine under these conditions, 3 x 10~ 5 M, is an order of magnitude lower than the K m for putrescine of this enzyme in rat liver (Table 1). The calculated K m for spermidine obtained from enzyme preparations from L. pictus was 7 x 10~ 4 M. This is similar to the K m calculated for the enzyme partially purified from S. purpuratus (5 x 10~ 4 M). In both cases the values are lower than those obtained from enzyme preparations of rat liver. The lower amounts of putrescine and spermidine necessary to optimize their conversion to spermidine and spermine respectively may account for both the lower amounts of putrescine present in sea urchins and the higher levels of spermine that accumulate.

11 Cyclical changes in polyamine synthesis E 140 o JT 120 o E c E Putrescine Spermidine 1 «D Spermine -, J> Time after fertilization (h) Fig Time after fertilization (h) Fig. 7 Fig. 6. Polyamine pools in early cleavage stages oil.pictus. Pools of embryos were assayed for putrescine, spermidine and spermine by extraction of amines into alkaline-1-butanol and separation by high-voltage electrophoresis (Russell, Medina & Snyder, 1970). Each point represents the mean for two or more duplicate determinations. Fig C content of putrescine, spermidine and spermine in early cleavage stages oil.pictus. Fertilized eggs were incubated with [ 14 C]putrescine for \ h at the start of the experiment (see Materials and Methods). Each point represents the mean for two separate determinations. Table 1. S-adenosyl-h-methionine decarboxylase: comparison of calculated K n values for putrescine and spermidine from rat liver and sea urchin Rat liver* L. p ictus Putrescine 3 x 10-4 M 3 x 10-5 M Spermidine 2 x 10-3 M 7X10-4 M * From Feldman, Levy & Russell (1972).

12 254 C.-A. MANEN AND D. H. RUSSELL DISCUSSION During early cleavage stages in sea urchins there are numerous reports of cyclical variations of metabolic parameters, with these variations occurring at a definite time in relation to cell division (Nagano & Mano, 1968; Lovtrup & Iverson, 1969; Mano, 1970). For instance, protein synthesis in sea urchins in early cleavage is elevated during prometaphase-metaphase and is depressed during anaphase-telophase of the next mitotic division (Mano, 1970). Studies conducted on the cell cycle in a variety of cells capable of being synchronized in some manner indicate that the above-mentioned cell cycle stage specificity of synthesis is a general phenomenon (Friedman, Bellantone & Canellakis, 1972; Mitchell & Rusch, 1972). Preliminary data from our laboratory of the relationship between initiation of polyamine biosynthetic activity as related to the cell cycle indicate that in mammalian tissue culture cells ornithine decarboxylase activity increases early in G x phase and putrescine- and spermidine-stimulated is-adenosyl-l-methionine decarboxylase activities are enhanced during S phase (Heby & Russell, in preparation). Ornithine decarboxylase activity increases rapidly after fertilization, reaching a maximum \ h after fertilization. A rise in ornithine decarboxylase activity therefore appears to slightly precede and extends through the first period of DNA synthesis. This period reportedly initiates in S. purpuratus at 20 min postfertilization and lasts approximately 15 min (Hinegardner, Rao & Feldman, 1964). There may be slight variations in these times attributable to the temperature of the sea water in which the embryos are grown. There is not a G x phase in the cell cycle of this sea urchin (Nemer, 1962; Ficq, Aiello & Scarano, 1963). The second S phase begins in telophase prior to the first cell division and extends into interphase of the next cell cycle. The second rise in ornithine decarboxylase activity again appears to coincide with the second S phase. Putrescine- and spermidine-stimulated S-adenosyl-L-methioninc decarboxylase activities do not appear to increase significantly until after the first S phase - their activities appear to be specific for prophase-metaphase. This correlates with general protein synthesis (Mano, 1970). Another aspect of this study that might be of great importance is the cyclical nature of the levels of the polyamines themselves. This applies to all three amines that are present in sea urchins, i.e. putrescine, spermidine, and spermine. In S. purpuratus there is an increase in all three amines 2\ h after fertilization although this experiment was performed only once and further gametes could not be obtained. L. pictus also exhibits maximal polyamine concentrations 2\ h after fertilization. However, the spermine concentration is always much greater than the concentrations of either putrescine or spermidine (Fig. 6). The rapid declines in the concentrations of these compounds must mean (1) that there are enzymes present for their selective degradation, or (2) these compounds are secreted into the surrounding medium at this time. It would be of value to determine how

13 Cyclical changes in polyamine synthesis 255 these rapid changes occur, as routes of polyamine degradation are almost unknown in mammalian systems. Perhaps an understanding of this mechanism in the sea urchin would facilitate the elucidation of such mechanisms in higher organisms. C.-A. Manen is a University Fellow, University of Maine. This manuscript is a portion of the research completed in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Zoology. REFERENCES ACKERMANN, D. & JANKA, R. (1954). First observation of spermine in invertebrates {Cionia intestinalis). Hoppe-Seyler's Z. physiol. Chem. 296, COPPOC, G. L., KALLIO, P. & WILLIAMS-ASHMAN, H. G. (1971). Characteristics of S-adenosyl- L-methionine decarboxylase from various organisms. Int. J. Biochem. 2, DYKSTRA, W. G. Jr. & HERBST, E. J. (1965). Spermidine in regenerating liver: relation to rapid synthesis of ribonucleic acid. Science, N. Y. 149, FELDMAN, M. J. & RUSSELL, D. H. (1972). Polyamine biogenesis in left ventricle of the rat heart after aortic constriction. Am. J. Physiol. 222, FELDMAN, M. J., LEVY, C. C. & RUSSELL, D. H. (1972). Purification and characterization of.s-adenosyl-l-methionine decarboxylase from rat liver. Biochemistry 11, FICQ, A., AIELLO, F. & SCARANO, E. (1963). Metabolisme des acides nucleiques dans l'ceuf d'oursin en developpement. Expl Cell Res. 29, FRIEDMAN, S. J., BELLANTONE, R. A. & CANELLAKIS, E. S. (1972). Ornithine decarboxylase activity in synchronously growing Don C cells. Biochim. biophys. Acta 261, HINEGARDNER, R. T., RAO, B. & FELDMAN, D. E. (1964). The DNA synthetic period during early development of the sea urchin egg. Expl Cell Res. 36, JANNE, J. & RAINA, A. (1968). Stimulation of spermidine synthesis in the regenerating rat liver: Relation to increased ornithine decarboxylase activity. Acta chem. scand. 22, JANNE, J. & RAINA, A. (1969). On the stimulation of ornithine decarboxylase andrna polymerase activity in rat liver after treatment with growth hormone. Biochim. biophys. Acta 174, JANNE, J. & WILLIAMS-ASHMAN, H. G. (1971). Dissociation of putrescine-activated decarboxylation of S-adenosyl-L-methionine from the enzymic synthesis of spermidine and spermine by purified prostatic enzyme preparations. Biochem. biophys. Res. Commun. 42, JANNE, J., SCHENONE, A. & WILLTAMS-ASHMAN, H. G. (1971). Separation of two proteins required for synthesis of spermidine from S-adenosyl-L-methionine and putrescine in rat prostrate. Biochem. biophys. Res. Commun. 42, LOVTRUP, S. & IversoN, R. M. (1969). Respiratory phases during early sea urchin development, measured with the automatic diver balance. Expl Cell Res. 55, LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. /. biol. Chem. 193, MANEN, C. A. & RUSSELL, D. H. (1973). Spermine is major polyamine in sea urchins: Studies of polyamines and their synthesis in developing sea urchins. /. Embryol. exp. Morph. 29, MANO, Y. (1970). Cytoplasmic regulation and cyclic variation in protein synthesis in the early cleavage stage of the sea urchin embryo. Devi Biol. 22, MITCHELL, J. L. A. & RUSCH, H. P. (1972). Putrescine and spermidine synthesis in Physanun polycephahtm. Fedn Proc. Fedn Am. Socs exp. Biol. 31, 488 (abs.) NAGANO, H. & MANO, Y. (1968). Thymidine kinase, thymidylate kinase and 32pi and [ 14 C]- thymidine incorporation into DNA during early embryogenesis of the sea urchin. Biochim. biophys. Acta 157, NEMER, M. (1962). Characteristics of the utilization of nucleosides by embryos of Paracentrotus lividus. J. biol. Chem. 237,

14 256 C.-A. MANEN AND D. H. RUSSELL OGATA, A. & KOMADA, T. (1943). In the composition of hasunohakashiban (Echinarachinius mirablis). J. pharm. soc. Japan 63, PEGG, A. E. & WILLIAMS-ASHMAN, H. G. (1968). Rapid effects of testosterone on prostatic polyamine-synthesizing systems. Biochem. J. 109, p. PEGG, A. E. & WILLIAMS-ASHMAN, H. G. (1969). On the role of S-adenosyl-L-methionine in the biosynthesis of spermidine by rat prostate. /. biol. Chem. 244, RUSSELL, D. H. (1971). Putrescine and spermidine biosynthesis in the development of normal and anucleolate mutants of Xenopus laevis. Proc. natn. Acad. Sci. U.S.A. 68, RUSSELL, D. H. & MCVICKER, T. A. (1972). Polyamines in the developing rat and in supportive tissues. Biochem. biophys. Ada 259, RUSSELL, D. H., MEDINA, V. J. & SNYDER, S. H. (1970). The dynamics of synthesis and degradation of polyamines in normal and regenerating rat liver and brain. /. biol. Chem. 245, RUSSELL, D. H. & POTYRAJ, J. J. (1972). Spermine synthesis in the uterus of the ovariectomized rat in response to oestradiol-17/?. Biochem. J. 128, RUSSELL, D. & SNYDER, S. H. (1968). Amine synthesis in rapidly growing tissues: Ornithine decarboxylase activity in regenerating rat liver, chick embryo and various tumors. Proc. natn. Acad. Sci. U.S.A. 60, RUSSELL, D. H. & SNYDER, S. H. (1969). Amine synthesis in regenerating rat liver: effect of hypophysectomy and growth hormone on ornithine decarboxylase. Endocrinology 84, RUSSELL, D. H., SNYDER, S. H. & MEDINA, V. J. (1969). Presence and biosynthesis of putrescine and polyamines in amphibians. Life Sci. 8, RUSSELL, D. H., SNYDER, S. H. & MEDINA, V. J. (1970). Growth hormone induction of ornithine decarboxylase in rat liver. Endocrinology 86, RUSSELL, D. H., SHIVERICK, K. T., HAMRELL, B. B. & ALPERT, N. R. (1971). Polyamine synthesis during initial phases of stress-induced cardiac hypertrophy. Am. J. Physiol. 221, RUSSELL, D. H. & TAYLOR, R. L. (1971). Polyamine synthesis and accumulation in the castrated rat uterus after estradiol-17/? stimulation. Endocrinology 88, TABOR, H. & TABOR, C. W. (1964). Spermidine, spermine and related amines. Pharmac. Rev. 16, {Received 1 January 1973, revised 10 March 1973)