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.

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 intestinalis (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 undergo growth processes, i.e. in regenerating rat liver (Dykstra & Herbst, 1965;Jänne & 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; Jänne & 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-methio-nine to form carbon dioxide and 5′-deoxy-5′-S, -(3-methylthiopropylamine) sulfonium adenosine (decarboxylated S-adenosyl-L-methionine). This S-adeno-syl-L-methionine decarboxylase required Mg2+ 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 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).

Fig. 1

Schematic polyamine biosynthetic pathway.

Fig. 1

Schematic polyamine biosynthetic pathway.

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 (Jänne & Williams-Ashman, 1971; Jänne, 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-L-methionine 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 & Iverson, 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 Km for putrescine exhibited by partially purified S-adenosyl-L-methionine decarboxylase.

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-14C]S-adenosyl-L-methionine (7·7 mCi/mM), [1-14C]-DL-ornithine (11·9 mCi/mM) and [l-4-14C]putrescine dihydrochloride (20·29 mCi/mM) were obtained from New England Nuclear.

Preparation of enzyme solutions

The material was homogenized in 4 vol. of 0·05 M sodium-potassium phosphate buffer, pH 7·2, containing 0·1 mM dithiothreitol. These enzymes are inhibited by Tris buffer, as are the mammalian enzymes. The homogenate was centrifuged at 20000g for 20 min, the pellet discarded, and the supernatant used in the assays. Since the supernatant after centrifugation at 100000 g for 90 min gave the identical enzyme activities, the 20000g 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 14CO2 from [l-14C]ornithine as described previously (Russell & Snyder, 1968; Russell & Snyder, 1969). Although the substrate concentration (0·1 mM as L-ornithine used routinely) was non-saturating, the same changes were noted when excess ornithine (2 mM) 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 14CO2 from [carboxyl-14C]S-adenosyl-L-methionine as previously described (Pegg & Williams-Ashman, 1969). Unless otherwise stated, incubation mixtures consisted of 0·1 mM [carboxyl-14C]S’-adenosyl-L-methionine, 25–50 mM sodiumpotassium phosphate buffer (pH 7·2), 50 μ M pyridoxal phosphate, 2·5 mM putrescine, and 0·1-0·15 ml (3·5 mg protein) of enzyme solution in 0-2 ml. When the formation of [14C]spermidine from unlabelled S-adenosyl-L-methionine and [l,4-14C]putrescine was estimated as previously described (Russell & McVicker, 1972), there was a stoichiometric relationship between the amount of [14C]-spermidine formed and the evolution of 14CO2 when [carboxyl-14C]S-adenosyl-L-methionine was added. Therefore, 14CO2 evolution from [carboxyl-14C]S-adenosyl-L-methionine was used routinely as a measure of the spermidine biosynthetic rate.

Assay for spermidine-stimulated. S-adenosyl-L-methionine decarboxylase activity

This assay is identical to that for putrescine-stimulated S-adenosyl-L-methio-nine decarboxylase except 5 mM spermidine was added instead of 2·5 mM putrescine. Again, there was a stoichiometric relationship between the amount of [14C]spermine formed and the evolution of 14CO2 when [carboxyl-14C]S-adenosyl-L-methionine was added. Therefore, 14CO2 evolution from [carboxyl-14C]5’-adenosyl-L-methionine was used routinely as a measure of spermine biosynthetic rate.

Determinations of putrescine, spermidine and spermine concentrations

Pools of embryos were homogenized in 4 vol. of 0·1 N-HCI and subjected to alkaline butanol extraction as previously described (Russell, Medina & Snyder, 1970).TCA 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 H2O 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 mw EDTA, 0·1 mM dithio-threitol, 3 μ M pyridoxal phosphate and 0·5 M sucrose.

Cellular debris was removed from the crude homogenate by centrifugation at 40000 g for 20 min. The resulting supernatant solution was recentrifuged at 100000# (in a Beckman L3-50 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 100000# supernatant solution was applied to a Sephadex G-100 column (45 cm x 4·9 cm2) 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 4·0 ml fractions. The most active fractions were pooled.

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 DIM dithiothreitol, 3 μ M pyridoxal phosphate and 0·5 M sucrose. Then 30 ml of the dialyzed preparation were applied to a DEAE-cellulose column (15 cm × 4·9 cm2) 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-KC1 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 [14C]putrescine (0·1 mmoles). After 30 min the eggs were washed twice with × 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.

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.

Fig. 2

Ornithine decarboxylase activity in early cleavage stages of L. pictus. Activity was determined by measuring the evolution of 14CO2 from [14C]L-ornithine (Russell & Snyder, 1968; Russell & Snyder, 1969). Each point represents the mean ± S.E. for four separate determinations.

Fig. 2

Ornithine decarboxylase activity in early cleavage stages of L. pictus. Activity was determined by measuring the evolution of 14CO2 from [14C]L-ornithine (Russell & Snyder, 1968; Russell & Snyder, 1969). Each point represents the mean ± S.E. for four separate determinations.

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.

Fig. 3

Ornithine decarboxylase activity in early cleavage stages of S. purpuratus. Activity was determined by measuring the evolution of 14CO2 from [14C]L-ornithine (Russell & Snyder, 1968; Russell & Snyder, 1969). Each point represents the mean ± S.E. for four separate determinations.

Fig. 3

Ornithine decarboxylase activity in early cleavage stages of S. purpuratus. Activity was determined by measuring the evolution of 14CO2 from [14C]L-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-L-methionine decarboxylase exhibits considerable activity in unfertilized eggs of L. pictus (Manen & Russell, 1973). After fertilization the activity drops markedly with a low point 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 5. purpuratus (Fig. 5). However, the initial levels of this enzyme are lower in S. purpuratus and there is not the post-fertilization drop in activity.

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 14CO2 released from 14COOH-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.

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 14CO2 released from 14COOH-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.

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 14CO2 released from “COOH-S-adenosyl-L-methioninc in the presence of the appropriate amine (see Materials and Methods). Each point represents the mean ± S.E. for four separate determinations.

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 14CO2 released from “COOH-S-adenosyl-L-methioninc in the presence of the appropriate amine (see Materials and Methods). Each point represents the mean ± S.E. for four separate determinations.

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 respectively, and maximal activities at and 3 h in both species of sea urchins studied.

Variations in the polyamine pools during early cleavage stages

After fertilization of S. purpuratus eggs, preliminary experiments indicate that all three amines exhibit maximal concentrations 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). 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.

Fig. 6

Polyamine pools in early cleavage stages of L. 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. 6

Polyamine pools in early cleavage stages of L. 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.

Distribution of radiolabel after incubation of embryos with [14C]put reset ne

After the incubation of fertilized eggs of L. pictus with [14C]putrescine for , the eggs were washed with sea water containing cold putrescine, then resuspended in sea water alone. Samples were removed at 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.

Fig. 7

14C content of putrescine, spermidine and spermine in early cleavage stages of L. pictus. Fertilized eggs were incubated with [14C]putrescine for i h at the start of the experiment (see Materials and Methods). Each point represents the mean for two separate determinations.

Fig. 7

14C content of putrescine, spermidine and spermine in early cleavage stages of L. pictus. Fertilized eggs were incubated with [14C]putrescine for i h at the start of the experiment (see Materials and Methods). Each point represents the mean for two separate determinations.

Properties of S-adenosyl-L-methionine decarboxylase from sea urchins

Through the use of the double-reciprocal plot, the apparent Km 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 Km for putrescine under these conditions, 3 × 10−5 M, is an order of magnitude lower than the Km for putrescine of this enzyme in rat liver (Table 1). The calculated Km for spermidine obtained from enzyme preparations from L. pictus was 7 ×10−4 M. This is similar to the Km calculated for the enzyme partially purified from S. purpuratus (5 × 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.

Table 1

S-adenosyl-L-methionine decarboxylase: comparison of calculated Km values for putrescine and spermidine from rat liver and sea urchin

S-adenosyl-L-methionine decarboxylase: comparison of calculated Km values for putrescine and spermidine from rat liver and sea urchin
S-adenosyl-L-methionine decarboxylase: comparison of calculated Km values for putrescine and spermidine from rat liver and sea urchin

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; Løvtrup & 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 G1 phase and putrescine- and spermidine-stimulated S-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 Gx 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 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 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.

Ackermann
,
D.
&
Janka
,
R.
(
1954
).
First observation of spermine in invertebrates (Cionia intestinalis)
.
Hoppe-Seyler’s Z. physiol. Chem
.
296
,
279
282
.
Coppoc
,
G. L.
,
Kallio
,
P.
&
Williams-Ashman
,
H. G.
(
1971
).
Characteristics of S-adenosyl-L-methionine decarboxylase from various organisms
.
Jut. J. Biochem
.
2
,
673
681
.
Dykstra
,
W. G.
Jr.
&
Herbst
,
E. J.
(
1965
).
Spermidine in regenerating liver: relation to rapid synthesis of ribonucleic acid
.
Science, N. Y
.
149
,
428
429
.
Feldman
,
M. J.
&
Russell
,
D. H.
(
1972
).
Polyamine biogenesis in left ventricle of the rat heart after aortic constriction
.
Am. J. Physiol
.
222
,
1199
1203
.
Feldman
,
M. J.
,
Levy
,
C. C.
&
Russell
,
D. H.
(
1972
).
Purification and characterization of S-adenosyl-L-methionine decarboxylase from rat liver
.
Biochemistry
11
,
671
677
.
Ficq
,
A.
,
Aiello
,
F.
&
Scarano
,
E.
(
1963
).
Métabolisme des acides nucléiques dans l’œuf d’oursin en développement
.
Expl Cell Res
.
29
,
128
136
.
Friedman
,
S. J.
,
Bellantone
,
R. A.
&
Canellakis
,
E. S.
(
1972
).
Ornithine decarboxylase activity in synchronously growing Don C cells
.
Biochim. biophys. Acta
261
,
188
193
.
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
,
53
61
.
Jänne
,
J.
&
Raina
,
A.
(
1968
).
Stimulation of spermidine synthesis in the regenerating rat liver: Relation to increased ornithine decarboxylase activity
.
Acta chem. scand
.
22
,
1349
1351
.
Jänne
,
J.
&
Raina
,
A.
(
1969
).
On the stimulation of ornithine decarboxylase and RNA polymerase activity in rat liver after treatment with growth hormone
.
Biochim. biophys. Acta
174
,
766
679
.
Jänne
,
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
,
222
229
.
Jänne
,
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
,
758
764
.
Løvtrup
,
S.
&
Iverson
,
R. M.
(
1969
).
Respiratory phases during early sea urchin development, measured with the automatic diver balance
.
Expl Cell Res
.
55
,
25
32
.
Lowry
,
O. H.
,
Rosebrough
,
N. J.
,
Farr
,
A. L.
&
Randall
,
R. J.
(
1951
).
Protein measurement with the Folin phenol reagent
.
J. biol. Chem
.
193
,
265
275
.
Manen
,
C. A.
&
Russell
,
D. H.
(
1973
).
Spermine is major polyamine in sea urchins: Studies of polyamines and their synthesis in developing sea urchins
.
J. Embryol. exp. Morph
.
29
,
331
345
.
Mano
,
Y.
(
1970
).
Cytoplasmic regulation and cyclic variation in protein synthesis in the early cleavage stage of the sea urchin embryo
.
Devi Biol
.
22
,
433
460
.
Mitchell
,
J. L. A.
&
Rusch
,
H. P.
(
1972
).
Putrescine and spermidine synthesis in Physarum polycephalum
.
Fedn Proc. Fedn Am. Socs exp. Biol
.
31
,
488
(abs.)
Nagano
,
H.
&
Mano
,
Y.
(
1968
).
Thymidine kinase, thymidylate kinase and 32pi and [14CJ-thymidine incorporation into DNA during early embryogenesis of the sea urchin
.
Biochim. biophys. Acta
157
,
546
557
Nemer
,
M.
(
1962
).
Characteristics of the utilization of nucleosides by embryos of Paracentrotus lividus
.
J. biol. Chem
.
237
,
143
149
.
Ogata
,
A.
&
Komada
,
T.
(
1943
).
In the composition of hasunohakashiban (Echinarachinius mirablis)
.
J. pharm. soc. Japan
63
,
653
658
.
Pegg
,
A. E.
&
Williams-Ashman
,
H. G.
(
1968
).
Rapid effects of testosterone on prostatic polyamine-synthesizing systems
.
Biochem. J
.
109
,
32
33 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
.
J. biol. Chem
.
244
,
682
693
.
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
,
523
527
.
Russell
,
D. H.
&
McVicker
,
T. A.
(
1972
).
Polyamines in the developing rat and in supportive tissues
.
Biochem. biophys. Acta
259
,
247
258
.
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
.
J. biol. Chem
.
245
,
6732
6738
.
Russell
,
D. H.
&
Potyraj
,
J. J.
(
1972
).
Spermine synthesis in the uterus of the ovariectomized rat in response to oestradiol-17/7
.
Biochem. J
.
128
,
1109
1115
.
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
,
1420
1427
.
Russell
,
D. H.
&
Snyder
,
S. H.
(
1969
).
Amine synthesis in regenerating rat liver: effect of hypophysectomy and growth hormone on ornithine decarboxylase
.
Endocrinology
84
,
223
228
.
Russell
,
D. H.
,
Snyder
,
S. H.
&
Medina
,
V. J.
(
1969
).
Presence and biosynthesis of putrescine and polyamines in amphibians
.
Life Sci
.
8
,
1247
1254
.
Russell
,
D. H.
,
Snyder
,
S. H.
&
Medina
,
V. J.
(
1970
).
Growth hormone induction of ornithine decarboxylase in rat liver
.
Endocrinology
86
,
1414
1419
.
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
,
1287
1291
.
Russell
,
D. H.
&
Taylor
,
R. L.
(
1971
).
Polyamine synthesis and accumulation in the castrated rat uterus after estradiol-17β stimulation
.
Endocrinology
88
,
1397
1403
.
Tabor
,
H.
&
Tabor
,
C. W.
(
1964
).
Spermidine, spermine and related amines
.
Pharmac. Rev
.
16
,
245
300
.