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Glycogen synthase activity of the gonad of the sea urchin Strongylocentrotus intermedius and the starfish asterias amurensis (Echinodermata)
Comp. Biochem. PhysioL Vol. 89B, No. 1, pp. 9-13, 1988 Printed in Great Britain
0305-0491/88 $3.00+0.00 © 1987 Pergamon Journals Ltd
GLYCOGEN SYNTHASE ACTIVITY OF THE G O N A D OF THE SEA U R C H I N STRONGYLOCENTROTUS INTERMEDIUS A N D THE STARFISH ASTERIAS AMURENSIS (ECHINODERMATA) Yu. S. KHOTIMCHENKO,E. A. ZALUTSKAYA,I. I. DERIDOVICH and D. I. KREIMER Institute of Marine Biology, Far East Science Center, Academy of Sciences of the USSR, Vladivostok 690022, USSR (Received 26 February 1987) Abstract--1. The maximal glycogen synthase (GS) activity of the gonad of female and male sea urchins
was observed at pH 7.5. The pH optimum for GS activity of the starfish gonad is in the range from 7.3 to 7.9, but the absolute maximal values were noted at pH 7.7. 2. GS activity of the gonad of sea urchin and starfish depends on both UDPG and G-6-P concentrations. At 5 mM UDPG and 10 mM G-6-P GS reaction attains saturation. 3. GS in the gonads of sea urchin and starfish occurs in two forms: dependent on G-6-P and independent of G-6-P. With an increase in the relative amount of UDPG the proportion of the activity of the G-6-P-dependent form increases. With an increase in the relative amount of UDPG the proportion of the activity of the G-6-P-independent form increases. 4. The properties of GS of the gonad in echinoderms and the character of substrate regulation of GS activity are only slightly different from those in vertebrates, and it is likely that hormonal and neurohormonal regulation of GS activity of the gonad in echinoderms will have some features in common with vertebrates.
INTRODUCTION Reproductive processes in marine invertebrates are regulated by a complex of factors among which environmental signals such as temperature, salinity and oxygen content are of paramount importance (Giese, 1966). They produce their effect on the sex gland of the animals through endogenic mechanisms, primarily nervous and endocrine glands, which are involved in the fine regulation of growth and maturation of gametes at the organ, cellular and subcellular levels. To deeply understand the situation, a knowledge of the peculiarities of metabolism of the main biochemical components of the cell and enzymes participating in this metabolism is required. The most important component of cells of the gonad in echinoderms is glycogen which serves as auxiliary energy material for both adult animals and developing embryos. It was shown that considerable amounts of glycogen accumulate in the gonads and starfishes and sea urchins and there are periods in the sexual cycle of these animals when in the gonad the processes of glycogen synthesis prevail over glycogen decomposition and vice versa (Zalutskaya et al., 1986), which is apparently associated with a concerted action of corresponding enzymes. However, the enzymes of glycogen metabolism in the tissues of echinoderms, particularly in the gonads, have been poorly studied. In this regard the aim of the present work was to investigate the properties of glycogen synthase, an enzyme which, on one hand, participates in glycogen metabolism and, on the other hand, is that link at which the effect of regulators of metabolism, mediators and hormones, can be directed. Glycogen synthase (GS) (E.C. 2.4.1.11) is a key
enzyme regulating glycogen metabolism in mammals. The properties of glycogen synthase, especially from the muscle tissue of mammals, have been the subject of intensive investigations (see reviews Roach, 1981; Cohen, 1982). It has been shown that the activity of the enzyme is determined by interconversion of phosphorylated-dephosphorylated forms catalysed by various GS-kinases and GS-phosphorylases (Cohen, 1982). Phosphorylation decreases the activity of the enzyme, while dephosphorylation, by contrast, increases it. Several regulators of GS are known, the most important of which is glucose-6phosphate (G-6-P). In mammalian tissues (Traut and Lipmann, 1963; Friedman and Lamer, 1963) and in different microorganisms (Rothman and Cabib, 1967; Tellezzin et al., 1969) glycogen synthase is present in two forms: the activity of one of them (D-form) can be detected only in the presence of G-6-P, while the activity of the other (I-form) is practically independent of G-6-P. The I-form of GS is dephosphorylated and the D-form is phosphorylated. In order to characterize the processes of phosphorylation-dephosphorylation of GS, such kinetic parameters of the reaction are widely used as the activaion constant [A]0.5 for G-6-P and the - G - 6 - P / + G-6-P ratio (GS activity in the absence of G-6-P/GS activity in the presence of G-6-P). However, in the liver and skeletal muscles of some fish and amphibians, leucocytes of man, and oocytes and embryos of Misgurmus fossilis the I-form is practically absent (Rossel-Perez and Lamer, 1962; Sevall and Kim, 1970; Yurowitzky and Milman, 1974). In contrast to the situation in vertebrates and some other animals, the literature offers little information on GS in marine invertebrates and none for echinoderms.
10
Yu. S. KHOTIMCHENKOet al.
The present study was designed with the following objectives: E
1. To reveal the presence of GS forms in the gonads of sea urchin and starfish; 2. To establish the relation between GS activity of sea urchin and starfish and U D P G concentration; 3. To establish the relation between the GS reaction of gonad and G-6-P concentration; 4. To establish the relation between the effect of activator and substrate concentrationi 5. To establish the relation between the effect of substrate and the activator concentration; 6. To determine the pH optimum for GS activity of the gonad of sea urchin and starfish; 7. To determine the seasonal changes of GS activity of the gonad of sea urchin.
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7.5
7.9
pH MATERIALS AND METHODS
The sexually mature sea urchins Strongylocentrotus interrnedius Agassiz (shell diameter 6-8 cm) and starfish Asterias amurensis Lfitken (ray length 10-14cm) were sampled in Ussari Bay (Sea of Japan) from January to July 1985-86. The animals were maintained in flow-through sea-water aquaria prior to experiments. The activity of enzyme was determined according to Yurowitzky and Milman (1974) in the following modification: fresh tissues were homogenized in a double volume of 0.05 M Tris-HCl buffer (pH 6-10) with 8.55% saccharose and 2.98% NaCI; 0.1 ml of the homogenate was incubated with 0.1 ml of a mixture containing uridin diphosphate glucose (UDPG), glucose-6-phosphate (G-6-P), 0.05M Tris-HC1 (pH6-10), 1% glycogen and 0.005M EDTA. After incubation (5-15min; +25°C), the reaction was stopped by heating in a boiling water bath (2 min); the mixture was diluted with 0.3 ml of hot water. The denaturated protein was separated by centrifugation: 0.04 ml of 0.02 M phosphoenol pyruvate, 0.04 ml of saturated solution of KCI and MgSO4 and 0.02ml of the suspension of crystalline pyruvate kinase were added to the supernatant and incubated for 30 min at + 25°C. The resultant pyruvate was converted into dinitrophenylhydrasone which was determined spectrophotometrically at 540 nm. For the determination of sex and stage of sexual cycle, pieces of gonads were fixed in Bouin's solution and embedded in paraffin. The cuts 5 #m thick were stained with hematoxylin and eosin and examined with microscope. RESULTS
The maximal GS activity of the gonad of female and male sea urchins was observed at pH 7.5 (Fig. 1), although this value was close to those observed at the pH range of 7.3-7.7. The curve of the relation between GS activity of the starfish gonad and pH had a gentler slope. The optimum pH of reaction was in the range from 7.3 to 7.9, but the absolute maximal values were noted at pH 7.7 (Fig. 2). All the subsequent experiments were performed at the incubation medium pH of 7.5. GS activity of the starfish gonad was detected in the presence and absence of the activator, G-6-P (Table 1). GS activity of the gonads of sea urchin and starfish increased with an increase in the concentration of both G-6-P and UDPG, approaching values close to the maximal at U D P G concentration of 5 mM and G-6-P 10 mM. To establish this we
Fig. 1. pH dependence of GS activity of the gonad of sea urchin Strongylocentrotus intermedius (5 mM UDPG, 5 mM G-6-P).
plotted the curves of GS activity in relation to the activator concentration at various substrate concentrations (Fig. 3) and GS activity in relation to the substrate concentration at various activator concentrations (Fig. 4). In addition to it, the following values were determined graphically: (1) [A]0.~ corresponding to the activator concentration which is necessary for obtaining the half-maximal rate of the reaction, (2) [S]0.5 corresponding to the substrate concentration which is necessary for obtaining halfmaximal rate of the reaction. The [S]0.5 value varied with an increase in the activator concentration (Table 2). An increase of substrate concentration changed, in its turn, the [A]0.5 value, i.e. the activator concentration ensuring the half-maximal rate of the reaction (Table 3). The - G - 6 - P / + G - 6 - P ratio changed in relation to the concentrations of U D P G and G-6-P. At the equimolar concentrations of substrate and activator numerical values of this ratio were in the range from 0.183 to 0.352 for sea urchin (Table 4) and 0.057 to
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E
20 >
t
-L I
I
"71
7.5
I 79
pH
Fig. 2. pH dependence of GS activity of the gonad of starfish Asterias amurensis (5 mM UDPG, 5 mM G-6-P).
Echinoderm glycogen synthase
ll
Table I. GS activity of the gonad of sea urchin S. intermedius and starfish A. amurensis in the presence (+G-6-P) and the absence ( - G - 6 - P ) of aetivator---G-6-P Date; number of animals; sex
Species Strongylocentrotus intermedius
UDPG (mM)
19 February; 4; male 15 March; 3; female 3 June; 3; female 16 April; 3; male 16 April 3; female 3 June; 4; female
Asterias amurensis
G-6-P (mM)
GS activity (nmol pyruvate/min per g) - G-6-P +G-6-P
5
10
117.0 + 19.2
202.3 + 20.6
10
10
286.4 + 14.1
354.6 +_ 18.8
10
10
275.0 +_ 18.4
965.2 _+ 65.5
5
5
25.1 -4-3.1
160.8 _+ 8.1
5
5
21.3 + 3.9
164.4 _+7.9
10
10
24.9 + 2.1
129.3 _+9.8
E E
Table 2. The dependence of the [S]0.5 value of GS reaction of the gonad of sea urchin S. intermedius and starfish A. amurensis on the activator---G-6-P----concentration
x. 160
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g 120
= o-
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-6 =e 8o
a-
Strongylocentrotus intermedius UDPG (raM)
(mM)
10.00 5.00 2.50
0.90 1.05 1.40
0.40
5.00
Aslerias amurensis Concentration [S]o.5 UDPG (mM) (mM) 20.00 5.00 1.25 0.40
0.60 2.00 2.50 5.00
.>_ 4 o
0
5
10 G-6-P,
20
mM
Fig. 3. The curves of GS activity of the gonad of starfish Asterias amurensis in relation to the activator concentration (G-6-P) at various substrate (UDPG) concentrations. (O)--20 mM UDPG; (+)---5 mM UDPG; (I-])---1.25 mM UDPG; (11)---0.40 mM UDPG; (&)---OmM UDPG, The character of the curves of GS activity of the gonad of sea urchin Strongylocentrotus intermedius was n o t different f r o m those o f starfish ( d a t a n o t shown).
Table 3. The dependence of the [A]0.5 value of GS reaction of the gonad of sea urchin S. intermedius and starfish A. amurensis on the substrate--UDPG---concentration Strongylocentrotus intermedius Concentration [A]0.5 UDPG (raM) (raM) 20.00 10.00 5.00 2.50 1.25 0.40
0.45 0.70 1.00 1.25 1.50 2.30
/tsterias amurensis Concentration [A]0s UDPG (raM) (mM) 20.00 10.00 5.00 2.50
0.25 0.45 0.85 2.50
E
"~ 160 c~ 120
-6
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I0
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20
UDPG, m.M
Fig. 4. The curves of GS activity of the gonad of starfish Asterias amurensis in r e l a t i o n to the s u b s t r a t e c o n c e n t r a t i o n ( U D P G ) at v a r i o u s a c t i v a t o r c o n c e n t r a t i o n s (G-6-P). ( 1 1 ) - - 2 0 m M G-6-P; (@)---10 m M G-6-P; (I-"1)---5 m M G-6P; ( A ) - - 2 . 5 m M G-6-P; ( A ) - - - 1 . 2 5 m M G-6-P; ( + ) - 0.40 m M G-6-P; ( O ) - - 0 m M G-6-P. The c h a r a c t e r o f the curves o f G S activity o f the g o n a d o f sea u r c h i n Strongy-
locentrotus intermedius was not different from those of
starfish (data not shown).
0.383 for starfish (Table 5). With an increase of the concentration of U D P G alone the - G - 6 - P / + G-6-P ratio increased and with an increase in the concentration of only G-6-P it decreased (Tables 4, 5). For comparison of GS activities of gonads in different periods of the sexual cycle, we took into account the values of the reaction at the concentrations of substrate and activator which are close to the saturating one (5 mM U D P G and 10 mM G-6-P). For example, we examined four stages of gonad cycle in sea urchin. The first stage---beginning of gametogenesis--occurs in most animals from December to March. The second stage---active gametogenesis I - takes place in April-May, The third stage--active gametogenesis II---continues from the end of May through July until the beginning of August. The fourth stage---ripe gonad--is observed at the time of reproduction of sea urchin in August-September. The histological picture of gonads at these stages was described earlier (Zalutskaya et al., 1986). GS activity increased as the ovary became mature (Table 6). It should be emphasized that GS activity of the gonad
12
Yu. S. KHOTIMCHENKOet al. Table 4. The changes of the - G - 6 - P / + G - 6 - P ratio (GS activity in the absence of G - 6 - P / G S activity in the presence of G-6-P) of the gonad of sea urchin S. intermedius in relation to the G-6-P and U D P G concentrations UDPG (mM) 0.40 1.25 2.50 5.00 10.00 20.00
0
0.40
1.000 1.000 1.000 1.000 1.000 1.000
0.183 0.321 0.407 0.375 0.655 0.722
1.25
G-6-P (mM) 2.50
5.00
10.00
20.00
- G - 6 - P / + G-6-P ratio 0.127 0.106 0.076 0.236 0.178 0.148 0.294 0.237 0.192 0.340 0.281 0.228 0.491 0.399 0.327 0.590 0.508 0.427
0.053 0.137 0.155 0.190 0.285 0.374
0.038 0.111 0.143 0.176 0.269 0.352
Table 5. The changes of the - G - 6 - P / + G - 6 - P ratio of the gonad of starfish A. amurensis in relation to the G-6-P and U D P G concentrations UDPG (mM) 2.50 5.00 10.00 20.00
0
0.40
1.000 1.000 1.000 1.000
0.237 0.332 0.550 0.672
1.25
G-6-P (mM) 2.50
5.00
10.00
20.00
- G - 6 - P / + G-6-P ratio 0.096 0.057 0.038 0.171 0.111 0.083 0.327 0.239 0.193 0.483 0.394 0.341
0.034 0.071 0.175 0.335
0.032 0.069 0.168 0.333
Table 6. Seasonal changes of GS activity in the ovary gland of sea urchin S. intermedius (5 mM UDPG, 10 mM G-6-P) Date 19 15 4 5
Number of animals
Period of the sexual cycle
10 12 12 8
Begining of gametogenesis Active gametogenesis I Active gametogenesis II Ripe gonad
February April June September
of starfish was, on the average, 5-10 times less than that of sea urchin. DISCUSSION
It was shown that glycogen from the gonad of the sea urchin S. purpuratus, in regard to such characteristics as optical rotation, the presence of 1-4 and 1 ~ or-glycoside linkages in a definite ratio, the length of external and internal chains, etc. does not practically differ from glycogen isolated from the liver of rat, bivalve mollusc (Mytilus), nematoda (Ascaris), protozoans (Tetrahymena) and bacteria (Artrobacter) (Doezema, 1969). The greatest amount of glycogen is contained in the digestive tract and gonads, and in starfish, also in the pyloric gland (Giese, 1966: Doezema, 1969; Oudejans and Van der Sluis, 1979; Oudejans et al., 1979). Since among the parts of the body, the gonads (sea urchins and starfishes) and pyloric caeca (starfish) are the greatest in weight, these organs apparently serve as the main depot of glycogen. It was established that the concentration and content of glycogen in these organs is related to the degree of maturity of gonads. In sea urchins the content of glycogen in the ovaries and testes decreased by the spawning period (Greenfield et aL, 1958; Zalutskaya et al., 1986; Zalutskaya, 1987). In starfish the ovarian glycogen concentration remained constant throughout the sexual cycle, but due to a considerable increase of gonad weight by the spawning period the total glycogen content in the gonad substantially increased as the gonad became mature (Oudejans and Van der Sluis, 1979), and vice versa
GS activity (nmol pyruvate/min per g) 202.3 + 334.0 + 790.0 + 41.5 +
20.6 28.9 68.0 5.8
the total glycogen in the digestive gland of starfish decreased by the time of spawning (Oudejans et aL, 1979). To explain the situation, data on the activity of the enzymes of glycogen metabolism are required. In echinoderms, some enzymes of carbohydrate metabolism (phosphorylase, hexokinase, glucose-6phosphate dehydrogenase, phosphoglucomutase, etc.) in the eggs and embryos of sea urchins, in the digestive tract tissue and muscles have been adequately studied to date (Doezema, 1969; Yasumasu et al., 1975; Zammit and Newsholme, 1976; Alevar et al., 1978). However, glycogen synthesizing activity of the gonad in echinoderms has not been investigated before. The method used in our work showed that the gonads of sea urchins and starfish tested are able to efficiently utilize U D P G as a donor of glycose remains for glycogen synthesis, as is the case in vertebrates. It is likely that GS in the gonads of tested animals also occurs in two forms: dependent on G-6-P (D) and independent of G-6-P (I). At the same time, the ratio of the activities of these forms depends on G-6-P and UDPG concentrations. GS activity of the gonad of tested species depends on both U D P G and G-6-P concentrations. At 5 mM UDPG and 10mM G-6-P the GS reaction attains saturation. Under a combined action of G-6-P and U D P G there is an additive effect. With an increase in the relative amount of G-6-P the proportion of the activity of the G-6-P dependent form also increases, and with an increase in the relative amount of U D P G the proportion of the activity of the G-6-P independent form increases. At the same time, a decrease in the activity
Echinoderm glycogen synthase of the G-6-P dependent form may be accompanied by no decrease in a general GS reaction. GS exhibits a high activity over a relatively wide range: from 7.3 to 7.7 in sea urchins and up to 7.9 in starfish. Interestingly, the enzyme content in the gonad of sea urchins was about 5-10 times higher than that of starfish. This may be connected with the fact that most ~,lvco~,en in the ~onad of sea urchins is localized in the auxiliary cells (Zalutskaya et al., 1986) which are absent in the gonad of starfish. Similarly, the concentration of glycogen itself in the gonad of the sea urchin in certain periods of the reproductive cycle is 1-2 orders of magnitude higher than in the gonad of starfish (Zalutskaya et al., 1986). In mammals GS is known to be b o u n d to particulate glycogen in the cell thus forming a c o m m o n complex. Scott and Cooper (1971) proposed that such a complex between glycogen and its metabolizing enzymes may be characteristic of all glycogen-containing cells. It can be assumed that a substantial proportion of GS activity of the sea urchin gonad is contributed by the auxiliary cells. In addition, GS activity (amount) varies with season. It increases as the gonad becomes mature and, at the same time, with increasing the sea-water temperature apparently due to a general activation of metabolism, and decreases sharply at the stage of ripe gonad. Our data indicate that the properties of GS of the gonad in echinoderms and the character of substrate regulation of GS activity are only slightly different from those in vertebrates. If so, it is likely that hormonal and neurohormonal regulation of GS activity of the gonad in echinoderms will have some features in c o m m o n with vertebrates. It is well established that tissues of starfish and sea urchins contain physiological concentrations of biogenic monoamines (dopamine, noradrenaline, tryptamine) (Khotimchenko and Deridovich, 1984), insulin (Wilson and Falkmer, 1965), sex steroid hormones (Voogt and Dieleman, 1984). Further research is needed to determine whether these factors are involved in the regulation of glycogen metabolism and, if so, what the mechanism is.
REFERENCES
Alevar P. M. F., Giacometty D. and Bacila M. (1978) Comparative levels of muscle glycolytic enzymes in mammals, fish, echinoderms and molluscs. Comp. Biochem. Physiol. 60B, 143-148. Cohen P. (1982) The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature 296, 613-620. Doezema P. (1969) Carbohydrate and carbohydrate metabolism of echinoderms. In Chemical Zoology, Vol. 4, pp. 101-122. Academic Press, New York. Friedman D. L. and Lamer J. (1963) Uridine diphosphoglucose (UDPG)
13
Giese A. C. (1976) Reproductive cycle in marine invertebrates. A. Acad. Brasil. sienc. 47, suppl., 49-67. Greenfield L., Giese A. C. and Farmanfarmanian R. A. (1958) Cyclic biochemical changes in several echinoderms. J. exp. Zool. 139, 507-524. Khotimchenko Yu. S. and Deridovich I. I. (1984) Spectrofluorometric determination of indolylalkylamines in radial nerves of the starfish Asterias amurensis (Liitken) and sea urchin Strongylocentrotus intermedius (Agassis). Mar. Biol. Lett. 5, 233-239. Oudejans R. C. H. M. and Van der Sluis I. (1979) Changes in the biochemical composition of the ovaries of the sea star Asterias rubens during their annual reproductive cycle. Mar. Biol. 50, 252-261. Oudejans R. C. H. M., Van der Sluis I. and Van der Plas A. J. (1979) Changes in the biochemical composition of the piloric caeca of female sea stars, Asterias rubens, during their annual reproductive cycle. Mar. Biol. 53, 231-238. Roach P. J. (1981) Glycogen synthase and glycogen synthase kinases. Curr. Topics cell. Regul. 20, 45-105. Rosell-Perez M. and Lamer J. (1962) Uridine diphosphate glucose (UDPG)-glycogen transglucosylase. II. Species variation of glucose-6-phosphate sensitivity of UDPGglycogen transglucosylase. Biochemistry 1, 769-772. Rothman L. B. and Cabib E. (1967) Allosteric properties of yeast glycogen synthetase. 1. General kinetic study. Biochemistry 6, 2098-2107. Scott R. B. and Cooper L. W. (1971) Glycogenolysis and glycogen synthesis in a cell-free system from rat liver. Biochem. biophys. Res. Commun. 44, 1071-1076. Sevall J. S. and Kim K. H. (1970) Purification and properties of hepatic glycogen-synthetase of Ranacatesbeiana. Biochim. biophys. Acta 206, 359-368. Telezin M., Terenzi H., Torres H. (1969) Interconvertible forms of glycogen synthetase in Neurosporacrassa. Biochim. biophys. Acta 191, 765-774. Trzut R. and Lipmann F. (1963) Activation of glycogen synthetase by glucose-6-phosphate. J. biol. Chem. 238, 1213-1221. Voogt R. A. and Dieleman S. J. (1984) Progesteron and oestrone levels in the gonads and pyloric caeca of the male sea star Asterias rubens: a comparison with the corresponding levels in the female sea star. Comp. Biochem. Physiol. 79A, 635~39. Wilson S. and Falkmer S. (1965) Starfish insulin. Can. J. Biochem. 43, 1615-1624. Yasumasu I., Fujiwara A., Shoger R. L. and Asami K. (1975) Distribution of some enzymes concerning carbohydrate metabolism in sea urchin eggs. Expl Cell Res. 92, 445-450. Yurowitzky Yu. G. and Milman L. S. (1974) Glycogen synthase in oocytes and embryos of coach (in Russian, summary in English). Biochim., Moscow 39, 86-95. Zalutskaya E. A. (1987) Glycogen dynamic in the testes of the sea urchin Strongylocentrotus intermedius (in Russian, summary in English). Mar. Biol., Vladivostok (in press). Zalutskaya E. A., Varaksina G. S. and Khotimchenko Yu. S. (1986) Glycogen content in the ovaries of the sea urchin Strongylocentrotus intermedius (in Russian, summary in English). Mar. Biol., Vladivostok NS, 38-44. Zammit V. A. and Newsholme E. A. (1976) The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenase, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphate kinase, glutamat-oxaloacetate transaminase and arglnine kinase in relation to carbohydrate utilization in muscle from marine invertebrates. Biochem. J. 160, 447-462.
0305-0491/88 $3.00+0.00 © 1987 Pergamon Journals Ltd
GLYCOGEN SYNTHASE ACTIVITY OF THE G O N A D OF THE SEA U R C H I N STRONGYLOCENTROTUS INTERMEDIUS A N D THE STARFISH ASTERIAS AMURENSIS (ECHINODERMATA) Yu. S. KHOTIMCHENKO,E. A. ZALUTSKAYA,I. I. DERIDOVICH and D. I. KREIMER Institute of Marine Biology, Far East Science Center, Academy of Sciences of the USSR, Vladivostok 690022, USSR (Received 26 February 1987) Abstract--1. The maximal glycogen synthase (GS) activity of the gonad of female and male sea urchins
was observed at pH 7.5. The pH optimum for GS activity of the starfish gonad is in the range from 7.3 to 7.9, but the absolute maximal values were noted at pH 7.7. 2. GS activity of the gonad of sea urchin and starfish depends on both UDPG and G-6-P concentrations. At 5 mM UDPG and 10 mM G-6-P GS reaction attains saturation. 3. GS in the gonads of sea urchin and starfish occurs in two forms: dependent on G-6-P and independent of G-6-P. With an increase in the relative amount of UDPG the proportion of the activity of the G-6-P-dependent form increases. With an increase in the relative amount of UDPG the proportion of the activity of the G-6-P-independent form increases. 4. The properties of GS of the gonad in echinoderms and the character of substrate regulation of GS activity are only slightly different from those in vertebrates, and it is likely that hormonal and neurohormonal regulation of GS activity of the gonad in echinoderms will have some features in common with vertebrates.
INTRODUCTION Reproductive processes in marine invertebrates are regulated by a complex of factors among which environmental signals such as temperature, salinity and oxygen content are of paramount importance (Giese, 1966). They produce their effect on the sex gland of the animals through endogenic mechanisms, primarily nervous and endocrine glands, which are involved in the fine regulation of growth and maturation of gametes at the organ, cellular and subcellular levels. To deeply understand the situation, a knowledge of the peculiarities of metabolism of the main biochemical components of the cell and enzymes participating in this metabolism is required. The most important component of cells of the gonad in echinoderms is glycogen which serves as auxiliary energy material for both adult animals and developing embryos. It was shown that considerable amounts of glycogen accumulate in the gonads and starfishes and sea urchins and there are periods in the sexual cycle of these animals when in the gonad the processes of glycogen synthesis prevail over glycogen decomposition and vice versa (Zalutskaya et al., 1986), which is apparently associated with a concerted action of corresponding enzymes. However, the enzymes of glycogen metabolism in the tissues of echinoderms, particularly in the gonads, have been poorly studied. In this regard the aim of the present work was to investigate the properties of glycogen synthase, an enzyme which, on one hand, participates in glycogen metabolism and, on the other hand, is that link at which the effect of regulators of metabolism, mediators and hormones, can be directed. Glycogen synthase (GS) (E.C. 2.4.1.11) is a key
enzyme regulating glycogen metabolism in mammals. The properties of glycogen synthase, especially from the muscle tissue of mammals, have been the subject of intensive investigations (see reviews Roach, 1981; Cohen, 1982). It has been shown that the activity of the enzyme is determined by interconversion of phosphorylated-dephosphorylated forms catalysed by various GS-kinases and GS-phosphorylases (Cohen, 1982). Phosphorylation decreases the activity of the enzyme, while dephosphorylation, by contrast, increases it. Several regulators of GS are known, the most important of which is glucose-6phosphate (G-6-P). In mammalian tissues (Traut and Lipmann, 1963; Friedman and Lamer, 1963) and in different microorganisms (Rothman and Cabib, 1967; Tellezzin et al., 1969) glycogen synthase is present in two forms: the activity of one of them (D-form) can be detected only in the presence of G-6-P, while the activity of the other (I-form) is practically independent of G-6-P. The I-form of GS is dephosphorylated and the D-form is phosphorylated. In order to characterize the processes of phosphorylation-dephosphorylation of GS, such kinetic parameters of the reaction are widely used as the activaion constant [A]0.5 for G-6-P and the - G - 6 - P / + G-6-P ratio (GS activity in the absence of G-6-P/GS activity in the presence of G-6-P). However, in the liver and skeletal muscles of some fish and amphibians, leucocytes of man, and oocytes and embryos of Misgurmus fossilis the I-form is practically absent (Rossel-Perez and Lamer, 1962; Sevall and Kim, 1970; Yurowitzky and Milman, 1974). In contrast to the situation in vertebrates and some other animals, the literature offers little information on GS in marine invertebrates and none for echinoderms.
10
Yu. S. KHOTIMCHENKOet al.
The present study was designed with the following objectives: E
1. To reveal the presence of GS forms in the gonads of sea urchin and starfish; 2. To establish the relation between GS activity of sea urchin and starfish and U D P G concentration; 3. To establish the relation between the GS reaction of gonad and G-6-P concentration; 4. To establish the relation between the effect of activator and substrate concentrationi 5. To establish the relation between the effect of substrate and the activator concentration; 6. To determine the pH optimum for GS activity of the gonad of sea urchin and starfish; 7. To determine the seasonal changes of GS activity of the gonad of sea urchin.
E ~
~60
/
120
> Q.
80
o
>, "~
40
g
/ \
#
//
(.9
I
I
J
71
7.5
7.9
pH MATERIALS AND METHODS
The sexually mature sea urchins Strongylocentrotus interrnedius Agassiz (shell diameter 6-8 cm) and starfish Asterias amurensis Lfitken (ray length 10-14cm) were sampled in Ussari Bay (Sea of Japan) from January to July 1985-86. The animals were maintained in flow-through sea-water aquaria prior to experiments. The activity of enzyme was determined according to Yurowitzky and Milman (1974) in the following modification: fresh tissues were homogenized in a double volume of 0.05 M Tris-HCl buffer (pH 6-10) with 8.55% saccharose and 2.98% NaCI; 0.1 ml of the homogenate was incubated with 0.1 ml of a mixture containing uridin diphosphate glucose (UDPG), glucose-6-phosphate (G-6-P), 0.05M Tris-HC1 (pH6-10), 1% glycogen and 0.005M EDTA. After incubation (5-15min; +25°C), the reaction was stopped by heating in a boiling water bath (2 min); the mixture was diluted with 0.3 ml of hot water. The denaturated protein was separated by centrifugation: 0.04 ml of 0.02 M phosphoenol pyruvate, 0.04 ml of saturated solution of KCI and MgSO4 and 0.02ml of the suspension of crystalline pyruvate kinase were added to the supernatant and incubated for 30 min at + 25°C. The resultant pyruvate was converted into dinitrophenylhydrasone which was determined spectrophotometrically at 540 nm. For the determination of sex and stage of sexual cycle, pieces of gonads were fixed in Bouin's solution and embedded in paraffin. The cuts 5 #m thick were stained with hematoxylin and eosin and examined with microscope. RESULTS
The maximal GS activity of the gonad of female and male sea urchins was observed at pH 7.5 (Fig. 1), although this value was close to those observed at the pH range of 7.3-7.7. The curve of the relation between GS activity of the starfish gonad and pH had a gentler slope. The optimum pH of reaction was in the range from 7.3 to 7.9, but the absolute maximal values were noted at pH 7.7 (Fig. 2). All the subsequent experiments were performed at the incubation medium pH of 7.5. GS activity of the starfish gonad was detected in the presence and absence of the activator, G-6-P (Table 1). GS activity of the gonads of sea urchin and starfish increased with an increase in the concentration of both G-6-P and UDPG, approaching values close to the maximal at U D P G concentration of 5 mM and G-6-P 10 mM. To establish this we
Fig. 1. pH dependence of GS activity of the gonad of sea urchin Strongylocentrotus intermedius (5 mM UDPG, 5 mM G-6-P).
plotted the curves of GS activity in relation to the activator concentration at various substrate concentrations (Fig. 3) and GS activity in relation to the substrate concentration at various activator concentrations (Fig. 4). In addition to it, the following values were determined graphically: (1) [A]0.~ corresponding to the activator concentration which is necessary for obtaining the half-maximal rate of the reaction, (2) [S]0.5 corresponding to the substrate concentration which is necessary for obtaining halfmaximal rate of the reaction. The [S]0.5 value varied with an increase in the activator concentration (Table 2). An increase of substrate concentration changed, in its turn, the [A]0.5 value, i.e. the activator concentration ensuring the half-maximal rate of the reaction (Table 3). The - G - 6 - P / + G - 6 - P ratio changed in relation to the concentrations of U D P G and G-6-P. At the equimolar concentrations of substrate and activator numerical values of this ratio were in the range from 0.183 to 0.352 for sea urchin (Table 4) and 0.057 to
E
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~o
_/'f°
°~°~.
E
20 >
t
-L I
I
"71
7.5
I 79
pH
Fig. 2. pH dependence of GS activity of the gonad of starfish Asterias amurensis (5 mM UDPG, 5 mM G-6-P).
Echinoderm glycogen synthase
ll
Table I. GS activity of the gonad of sea urchin S. intermedius and starfish A. amurensis in the presence (+G-6-P) and the absence ( - G - 6 - P ) of aetivator---G-6-P Date; number of animals; sex
Species Strongylocentrotus intermedius
UDPG (mM)
19 February; 4; male 15 March; 3; female 3 June; 3; female 16 April; 3; male 16 April 3; female 3 June; 4; female
Asterias amurensis
G-6-P (mM)
GS activity (nmol pyruvate/min per g) - G-6-P +G-6-P
5
10
117.0 + 19.2
202.3 + 20.6
10
10
286.4 + 14.1
354.6 +_ 18.8
10
10
275.0 +_ 18.4
965.2 _+ 65.5
5
5
25.1 -4-3.1
160.8 _+ 8.1
5
5
21.3 + 3.9
164.4 _+7.9
10
10
24.9 + 2.1
129.3 _+9.8
E E
Table 2. The dependence of the [S]0.5 value of GS reaction of the gonad of sea urchin S. intermedius and starfish A. amurensis on the activator---G-6-P----concentration
x. 160
o4 |
g 120
= o-
•--+'-'--
-6 =e 8o
a-
Strongylocentrotus intermedius UDPG (raM)
(mM)
10.00 5.00 2.50
0.90 1.05 1.40
0.40
5.00
Aslerias amurensis Concentration [S]o.5 UDPG (mM) (mM) 20.00 5.00 1.25 0.40
0.60 2.00 2.50 5.00
.>_ 4 o
0
5
10 G-6-P,
20
mM
Fig. 3. The curves of GS activity of the gonad of starfish Asterias amurensis in relation to the activator concentration (G-6-P) at various substrate (UDPG) concentrations. (O)--20 mM UDPG; (+)---5 mM UDPG; (I-])---1.25 mM UDPG; (11)---0.40 mM UDPG; (&)---OmM UDPG, The character of the curves of GS activity of the gonad of sea urchin Strongylocentrotus intermedius was n o t different f r o m those o f starfish ( d a t a n o t shown).
Table 3. The dependence of the [A]0.5 value of GS reaction of the gonad of sea urchin S. intermedius and starfish A. amurensis on the substrate--UDPG---concentration Strongylocentrotus intermedius Concentration [A]0.5 UDPG (raM) (raM) 20.00 10.00 5.00 2.50 1.25 0.40
0.45 0.70 1.00 1.25 1.50 2.30
/tsterias amurensis Concentration [A]0s UDPG (raM) (mM) 20.00 10.00 5.00 2.50
0.25 0.45 0.85 2.50
E
"~ 160 c~ 120
-6
E >,
.>_
~
I//
5
I0
1
20
UDPG, m.M
Fig. 4. The curves of GS activity of the gonad of starfish Asterias amurensis in r e l a t i o n to the s u b s t r a t e c o n c e n t r a t i o n ( U D P G ) at v a r i o u s a c t i v a t o r c o n c e n t r a t i o n s (G-6-P). ( 1 1 ) - - 2 0 m M G-6-P; (@)---10 m M G-6-P; (I-"1)---5 m M G-6P; ( A ) - - 2 . 5 m M G-6-P; ( A ) - - - 1 . 2 5 m M G-6-P; ( + ) - 0.40 m M G-6-P; ( O ) - - 0 m M G-6-P. The c h a r a c t e r o f the curves o f G S activity o f the g o n a d o f sea u r c h i n Strongy-
locentrotus intermedius was not different from those of
starfish (data not shown).
0.383 for starfish (Table 5). With an increase of the concentration of U D P G alone the - G - 6 - P / + G-6-P ratio increased and with an increase in the concentration of only G-6-P it decreased (Tables 4, 5). For comparison of GS activities of gonads in different periods of the sexual cycle, we took into account the values of the reaction at the concentrations of substrate and activator which are close to the saturating one (5 mM U D P G and 10 mM G-6-P). For example, we examined four stages of gonad cycle in sea urchin. The first stage---beginning of gametogenesis--occurs in most animals from December to March. The second stage---active gametogenesis I - takes place in April-May, The third stage--active gametogenesis II---continues from the end of May through July until the beginning of August. The fourth stage---ripe gonad--is observed at the time of reproduction of sea urchin in August-September. The histological picture of gonads at these stages was described earlier (Zalutskaya et al., 1986). GS activity increased as the ovary became mature (Table 6). It should be emphasized that GS activity of the gonad
12
Yu. S. KHOTIMCHENKOet al. Table 4. The changes of the - G - 6 - P / + G - 6 - P ratio (GS activity in the absence of G - 6 - P / G S activity in the presence of G-6-P) of the gonad of sea urchin S. intermedius in relation to the G-6-P and U D P G concentrations UDPG (mM) 0.40 1.25 2.50 5.00 10.00 20.00
0
0.40
1.000 1.000 1.000 1.000 1.000 1.000
0.183 0.321 0.407 0.375 0.655 0.722
1.25
G-6-P (mM) 2.50
5.00
10.00
20.00
- G - 6 - P / + G-6-P ratio 0.127 0.106 0.076 0.236 0.178 0.148 0.294 0.237 0.192 0.340 0.281 0.228 0.491 0.399 0.327 0.590 0.508 0.427
0.053 0.137 0.155 0.190 0.285 0.374
0.038 0.111 0.143 0.176 0.269 0.352
Table 5. The changes of the - G - 6 - P / + G - 6 - P ratio of the gonad of starfish A. amurensis in relation to the G-6-P and U D P G concentrations UDPG (mM) 2.50 5.00 10.00 20.00
0
0.40
1.000 1.000 1.000 1.000
0.237 0.332 0.550 0.672
1.25
G-6-P (mM) 2.50
5.00
10.00
20.00
- G - 6 - P / + G-6-P ratio 0.096 0.057 0.038 0.171 0.111 0.083 0.327 0.239 0.193 0.483 0.394 0.341
0.034 0.071 0.175 0.335
0.032 0.069 0.168 0.333
Table 6. Seasonal changes of GS activity in the ovary gland of sea urchin S. intermedius (5 mM UDPG, 10 mM G-6-P) Date 19 15 4 5
Number of animals
Period of the sexual cycle
10 12 12 8
Begining of gametogenesis Active gametogenesis I Active gametogenesis II Ripe gonad
February April June September
of starfish was, on the average, 5-10 times less than that of sea urchin. DISCUSSION
It was shown that glycogen from the gonad of the sea urchin S. purpuratus, in regard to such characteristics as optical rotation, the presence of 1-4 and 1 ~ or-glycoside linkages in a definite ratio, the length of external and internal chains, etc. does not practically differ from glycogen isolated from the liver of rat, bivalve mollusc (Mytilus), nematoda (Ascaris), protozoans (Tetrahymena) and bacteria (Artrobacter) (Doezema, 1969). The greatest amount of glycogen is contained in the digestive tract and gonads, and in starfish, also in the pyloric gland (Giese, 1966: Doezema, 1969; Oudejans and Van der Sluis, 1979; Oudejans et al., 1979). Since among the parts of the body, the gonads (sea urchins and starfishes) and pyloric caeca (starfish) are the greatest in weight, these organs apparently serve as the main depot of glycogen. It was established that the concentration and content of glycogen in these organs is related to the degree of maturity of gonads. In sea urchins the content of glycogen in the ovaries and testes decreased by the spawning period (Greenfield et aL, 1958; Zalutskaya et al., 1986; Zalutskaya, 1987). In starfish the ovarian glycogen concentration remained constant throughout the sexual cycle, but due to a considerable increase of gonad weight by the spawning period the total glycogen content in the gonad substantially increased as the gonad became mature (Oudejans and Van der Sluis, 1979), and vice versa
GS activity (nmol pyruvate/min per g) 202.3 + 334.0 + 790.0 + 41.5 +
20.6 28.9 68.0 5.8
the total glycogen in the digestive gland of starfish decreased by the time of spawning (Oudejans et aL, 1979). To explain the situation, data on the activity of the enzymes of glycogen metabolism are required. In echinoderms, some enzymes of carbohydrate metabolism (phosphorylase, hexokinase, glucose-6phosphate dehydrogenase, phosphoglucomutase, etc.) in the eggs and embryos of sea urchins, in the digestive tract tissue and muscles have been adequately studied to date (Doezema, 1969; Yasumasu et al., 1975; Zammit and Newsholme, 1976; Alevar et al., 1978). However, glycogen synthesizing activity of the gonad in echinoderms has not been investigated before. The method used in our work showed that the gonads of sea urchins and starfish tested are able to efficiently utilize U D P G as a donor of glycose remains for glycogen synthesis, as is the case in vertebrates. It is likely that GS in the gonads of tested animals also occurs in two forms: dependent on G-6-P (D) and independent of G-6-P (I). At the same time, the ratio of the activities of these forms depends on G-6-P and UDPG concentrations. GS activity of the gonad of tested species depends on both U D P G and G-6-P concentrations. At 5 mM UDPG and 10mM G-6-P the GS reaction attains saturation. Under a combined action of G-6-P and U D P G there is an additive effect. With an increase in the relative amount of G-6-P the proportion of the activity of the G-6-P dependent form also increases, and with an increase in the relative amount of U D P G the proportion of the activity of the G-6-P independent form increases. At the same time, a decrease in the activity
Echinoderm glycogen synthase of the G-6-P dependent form may be accompanied by no decrease in a general GS reaction. GS exhibits a high activity over a relatively wide range: from 7.3 to 7.7 in sea urchins and up to 7.9 in starfish. Interestingly, the enzyme content in the gonad of sea urchins was about 5-10 times higher than that of starfish. This may be connected with the fact that most ~,lvco~,en in the ~onad of sea urchins is localized in the auxiliary cells (Zalutskaya et al., 1986) which are absent in the gonad of starfish. Similarly, the concentration of glycogen itself in the gonad of the sea urchin in certain periods of the reproductive cycle is 1-2 orders of magnitude higher than in the gonad of starfish (Zalutskaya et al., 1986). In mammals GS is known to be b o u n d to particulate glycogen in the cell thus forming a c o m m o n complex. Scott and Cooper (1971) proposed that such a complex between glycogen and its metabolizing enzymes may be characteristic of all glycogen-containing cells. It can be assumed that a substantial proportion of GS activity of the sea urchin gonad is contributed by the auxiliary cells. In addition, GS activity (amount) varies with season. It increases as the gonad becomes mature and, at the same time, with increasing the sea-water temperature apparently due to a general activation of metabolism, and decreases sharply at the stage of ripe gonad. Our data indicate that the properties of GS of the gonad in echinoderms and the character of substrate regulation of GS activity are only slightly different from those in vertebrates. If so, it is likely that hormonal and neurohormonal regulation of GS activity of the gonad in echinoderms will have some features in c o m m o n with vertebrates. It is well established that tissues of starfish and sea urchins contain physiological concentrations of biogenic monoamines (dopamine, noradrenaline, tryptamine) (Khotimchenko and Deridovich, 1984), insulin (Wilson and Falkmer, 1965), sex steroid hormones (Voogt and Dieleman, 1984). Further research is needed to determine whether these factors are involved in the regulation of glycogen metabolism and, if so, what the mechanism is.
REFERENCES
Alevar P. M. F., Giacometty D. and Bacila M. (1978) Comparative levels of muscle glycolytic enzymes in mammals, fish, echinoderms and molluscs. Comp. Biochem. Physiol. 60B, 143-148. Cohen P. (1982) The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature 296, 613-620. Doezema P. (1969) Carbohydrate and carbohydrate metabolism of echinoderms. In Chemical Zoology, Vol. 4, pp. 101-122. Academic Press, New York. Friedman D. L. and Lamer J. (1963) Uridine diphosphoglucose (UDPG)
13
Giese A. C. (1976) Reproductive cycle in marine invertebrates. A. Acad. Brasil. sienc. 47, suppl., 49-67. Greenfield L., Giese A. C. and Farmanfarmanian R. A. (1958) Cyclic biochemical changes in several echinoderms. J. exp. Zool. 139, 507-524. Khotimchenko Yu. S. and Deridovich I. I. (1984) Spectrofluorometric determination of indolylalkylamines in radial nerves of the starfish Asterias amurensis (Liitken) and sea urchin Strongylocentrotus intermedius (Agassis). Mar. Biol. Lett. 5, 233-239. Oudejans R. C. H. M. and Van der Sluis I. (1979) Changes in the biochemical composition of the ovaries of the sea star Asterias rubens during their annual reproductive cycle. Mar. Biol. 50, 252-261. Oudejans R. C. H. M., Van der Sluis I. and Van der Plas A. J. (1979) Changes in the biochemical composition of the piloric caeca of female sea stars, Asterias rubens, during their annual reproductive cycle. Mar. Biol. 53, 231-238. Roach P. J. (1981) Glycogen synthase and glycogen synthase kinases. Curr. Topics cell. Regul. 20, 45-105. Rosell-Perez M. and Lamer J. (1962) Uridine diphosphate glucose (UDPG)-glycogen transglucosylase. II. Species variation of glucose-6-phosphate sensitivity of UDPGglycogen transglucosylase. Biochemistry 1, 769-772. Rothman L. B. and Cabib E. (1967) Allosteric properties of yeast glycogen synthetase. 1. General kinetic study. Biochemistry 6, 2098-2107. Scott R. B. and Cooper L. W. (1971) Glycogenolysis and glycogen synthesis in a cell-free system from rat liver. Biochem. biophys. Res. Commun. 44, 1071-1076. Sevall J. S. and Kim K. H. (1970) Purification and properties of hepatic glycogen-synthetase of Ranacatesbeiana. Biochim. biophys. Acta 206, 359-368. Telezin M., Terenzi H., Torres H. (1969) Interconvertible forms of glycogen synthetase in Neurosporacrassa. Biochim. biophys. Acta 191, 765-774. Trzut R. and Lipmann F. (1963) Activation of glycogen synthetase by glucose-6-phosphate. J. biol. Chem. 238, 1213-1221. Voogt R. A. and Dieleman S. J. (1984) Progesteron and oestrone levels in the gonads and pyloric caeca of the male sea star Asterias rubens: a comparison with the corresponding levels in the female sea star. Comp. Biochem. Physiol. 79A, 635~39. Wilson S. and Falkmer S. (1965) Starfish insulin. Can. J. Biochem. 43, 1615-1624. Yasumasu I., Fujiwara A., Shoger R. L. and Asami K. (1975) Distribution of some enzymes concerning carbohydrate metabolism in sea urchin eggs. Expl Cell Res. 92, 445-450. Yurowitzky Yu. G. and Milman L. S. (1974) Glycogen synthase in oocytes and embryos of coach (in Russian, summary in English). Biochim., Moscow 39, 86-95. Zalutskaya E. A. (1987) Glycogen dynamic in the testes of the sea urchin Strongylocentrotus intermedius (in Russian, summary in English). Mar. Biol., Vladivostok (in press). Zalutskaya E. A., Varaksina G. S. and Khotimchenko Yu. S. (1986) Glycogen content in the ovaries of the sea urchin Strongylocentrotus intermedius (in Russian, summary in English). Mar. Biol., Vladivostok NS, 38-44. Zammit V. A. and Newsholme E. A. (1976) The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenase, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphate kinase, glutamat-oxaloacetate transaminase and arglnine kinase in relation to carbohydrate utilization in muscle from marine invertebrates. Biochem. J. 160, 447-462.