896
BIOCHEMICAL SOCIETY TRANSACTIONS
of (14C)glucose into glycogen in mantle-tissue slices and the specific activity of glycogen synthase (B. N . Zaba, personal communication). The only other study on seasonal changes in glycogen synthase activity known to us is on frog liver. Castineiras et al. (1977) have shown that the increase in hepatic glycogen content of Rana ridibunda in the autumn can be related to a n increase in the specific activity of glycogen synthase; the percentage I-form remained constant throughout the year. In contrast, both the specific activity and percentage I-form of glycogen synthase varied seasonally in M . edulis. Mantle-tissue slices from winter mussels were incubated for 3h at 20°C in KrebsRinger bicarbonate buffer (pH7.4) in the presence of 5m~-glucose.The activity of the I-form of glycogen synthase increased 5-fold t o the summer level of 20-25 %. The ratio of the I-form activity with 5m~-glucose/controlswas 1.6OkO.17 (n = 9) compared with a ratio of 1.08kO.20(n = 9) for the total (I+D) activity, showing that the glucose treatment stimulates the conversion of the D- to I-form of the enzyme. The I-form activity was significantly higher in the control slices than in winter field mussels but the reason for this difference is not known. The glucose effect was time- and concentration-dependent, with maximum stimulation by 5m~-glucoseover a period of 3h (M. A. Whittle, unpublished results). We believe that the increase in the percentage I-form of glycogen synthase in summer mussels is the result of feeding activity and a concomitant increase in tissue glucose concentration. Work in other laboratories has indicated that the effect of high glucose concentrations o n glycogen synthase activity in bivalve molluscs may be mediated by an insulin-like peptide. Martinez et al. (1973) have isolated insulin-like proteins from the digestive gland of Ostrea edulis L., and Fritsch et al. (1976) have demonstrated insulin-like immunoreactivity in epithelial cells of the intestine associated with the digestive gland in M . edulis. More recently, Plisetskaya e t al. (1979) have used a physiological approach in which glucose and insulin were injected at low doses, and in other experiments antiinsulin serum was used to exclude the endogenous hormone. As a result of these experiments, Plisetskaya e t a / . (1978) have proposed a direct role for insulin, o r a substance closely related to insulin, in the control of glycogen metabolism in the fresh-water bivalves, Anodonta cygnea and Unio pictorum. Castineiras, M. J., Guinovart, J . J., Itarte, E. & Rosell-Perez, M. (1977) Reu. ESP.Fisiol. 33, 311-316 Cook, P. A. & Gabbott, P. A. (1978) Comp. Biochem. Physiol. B 60,419-421 De Zwaan, A. & Zandee, D. I. (1972) Comp. Biochem. Physiol. 43A, 53-58 Fritsch, H. A. R., Van Noorden, S. & Pearse, A. G. E. (1976) Cell. Tissue. Res. 165, 365-369 Gabbott, P. A. (1975) in Proc. Eur. Mar. Biol. Symp. 9th (Barnes, H., ed.), pp. 191-221, Aberdeen University Press, Aberdeen Gabbott, P. A. & Bayne, B. L. (1973) J. Mar. Biol. Assoc. U.K. 53,269-286 Groves, W. E., Davis, F. C . &Sells, B. H. (1968) Anal. Biocheni. 22, 195-210 Houteville, P. & Lubet, P. (1974) C . R. Hebd. Seances Acad. Sci. Ser. D 278,2469-2472 Martinez, de N. R., Garcia, M. C.,Salas, M. & Candela, J. L. R.(1973) Cen. Comp. Endocrinol. 20,305-31 1 Passoneau, J. V. & Rottenberg, D. A. (1973) Anal. Biochem. 51,528-541 Plisetskaya, E., Kazakov, V. K., Soltitskaya, L. & Leibson, L. G. (1978) Gen. Comp. Endocrinol. 35,133-145
Control of NADP+-dependent Isocitrate Dehydrogenase Activity in the Mussel Mytilus edulis L. ERICA J. H. H E A D and PETER A. GABBOTT
NERC Unit of Marine Invertebrate Biology, Marine Science Laboratories, Menui Bridge, Gwynedd LL59 SEH, Wales, U.K. N A D P H is required for fatty acid and sterol synthesis. The control of N A D P H production has been studied in the digestive gland and mantle tissue of Mytilus edulis. 1979
583rd MEETING, CAMBRIDGE
897
In mammalian liver the enzymes involved in NADP+ reduction are the enzymes of the pentose phosphate-pathway, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and ‘malic’ enzyme. Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are present in the digestive gland (Carrion-Angosto et al., 1977; Rodriguez-Segade et af., 1978) and mantle tissue (E. J. H. Head, unpublished work) of M. edulis but ‘malic’ enzyme has been found with only very low activities (de Zwaan & Van Marrewijk, 1973). Another source of NADPH, cytoplasmic isocitrate dehydrogenase (EC 1.1 .I .42), has been found in both mammalian (Gul & Dils, 1969) and insect (see Bailey, 1975) tissues : NADP++isocitrate z C 0 2 + a-oxoglutarate+NADPH Isocitrate dehydrogenase activity has been found in the digestive gland and mantle tissue of M. edulis (Addink & Veenhof, 1975). J. E. Cienfuegos & A. Carrion (personal communication) found that the isocitrate dehydrogenase activity in the digestive gland was at least seven times greater than that of ‘malic’ enzyme. The specific activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and cytoplasmic isocitrate dehydrogenase were measured at bimonthly intervals throughout the year. The results showed that the enzymes of the pentose phosphate pathway increased in activity about 3-fold in the digestive gland (and to a lesser extent in the mantle) during the summer, when high food levels were available. In contrast, isocitrate dehydrogenase activity remained almost constant throughout the year. To study the regulatory properties of isocitrate dehydrogenase, the enzyme from the digestive gland was purified to homogeneity by affinity and ion-exchange chromatography. It was not inhibited by palmitoyl-CoA nor by ADP or ATP. Kinetic experiments were carried out with 5m~-Mg’+present. Initial-rate studies gave Michaelis constants of 3.5 and 3 . 4 for ~ ~NADP+ and total D-isocitrate respectively, and dissociation constants that were indistinguishable from the Michaelis constants. Similar Michaelis values were obtained for the mantle enzyme. Inhibition patterns for the reaction products, a-oxoglutarate and NADPH, and for the isocitrate analogue, tricarballylic acid, were consistent with a rapid-equilibrium random-ordered mechanism as found by Uhr et al. (1974) for isocitrate dehydrogenase from pig heart. The relative initial-reaction rate can be calculated by using the equation given by Fromm (1975), which includes product inhibition by NADPH:
[A], [B] and [Q] are the concentrations of NADP+, total D-isocitrate and NADPH respectively. K, and Kb are the Michaelis constants for NADP+ and total D-isocitrate. K , , is the dissociation constant for the E-NADP+ complex and K , , and K,, are the dissociation constants for the E-isocitrate-NADPH and E-NADPH complexes, where E is the enzyme. Fig. 1 shows the reaction rates for different [NADP+/NADPH] ratios, at constant [NADP++NADPH] of 6 5 , (determined ~ ~ experimentally for the digestive gland). The shape of the curves is dependent on the relative affinities of the enzyme species E and E-isocitrate for NADP+ and NADPH, and it is regions where the response curves are steep that give rise to regulatory interactions (see Atkinson, 1970). Values for the cytoplasmic [NADP+]/[NADPH] ratio in mammalian systems are low, between 0.014 and 0.3 (see Sols & Marco, 1970). This is the range of maximum sensitivity of isocitrate dehydrogenase to changes in the [NADP+]/[NADPH] ratio. Eqn. (1) represents the limiting case in which no a-oxoglutarate is present. In M. edulis the tissue concentrations of a-oxoglutarateare very low (Bayne, 1973),so that the inhibitory effect will benegligible compared with that of NADPH.
Vol. 7
898
BIOCHEMICAL SOCIETY TRANSACTIONS
06
0.8
1.0
[NADP+I/[NADPH] Fig. 1, Calculated variation of relative initial rates for isocitrate dehydrogenase The reaction curves were calculated according to eqn. (I), using values of K, = K , , = 3 . 5 , u ~ ,K b = 3 . 4 p ~ K,, , = 9 . 2 and ~ ~ Kiq= 9 . 7 ~ The ~ . total NADP+NADPH con, was measured by the recycling method of Lowry & Passonneau centration was 6 5 p ~and (1972). D-isocitrate concentrations are indicated on the curves and the shaded area represents the physiological range of D-isocitrate concentrations in the digestive gland of M . edulis. D-Isocitrate concentrations have been determined in individual mussels. I n the digestive gland these lie between the values of K,,, and about 10 K,, with most values in the ~ . NADP+-dependent cytoplasmic dehydrogenases, Sols & Marco range 2 0 - 4 0 ~ For (1970) consider that any utilization of NADPH by biosynthetic pathways will be followed by a fall in N A D P H concentration and a n increased regeneration of NADPH. At saturating concentrations of D-isocitrate and in the presence of 5 m ~ - M g ' + ,isocitrate dehydrogenase behaves in accordance with this prediction. Addink, A. D. F. & Veenhof, P. R. (1975) in Proc. Eur. Mar. Biol. Symp. 9th (Barnes, H., ed.), pp. 109-1 19, Aberdeen University Press, Aberdeen Atkinson, D. E. (1970) Enzymes, 3rd Ed. 1,461-489 Bailey, E. (1975) in Insect Biochemistry and Function, (Candy, D. J. & Kilby, B. A,, eds.), pp. 89-167, Chapman and Hall, London Bayne, B. L. (1973) Neth. J. Sea Res. 7, 399410 Carrion-Angosto, A., Silva Pando, M., Rodrigues-Segade, S. & Ruiz-Amil, M. (1977) Rev. ESP. Fisiol. 33,265-272 De Zwaan, A. &Van Marrewijk, W. J. A. (1973) Comp. Biochem. Physiot. B44,1057-1066 Fromm, H. J. (1975) Initial Rate Kinetics, Springer-Verlag, Berlin, Heidelberg and New York Gul, B. & Dils, R. (1969) Biochem. J. 112,293-301 Lowry, 0. H. & Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, Academic Press, New York and London Rodriguez-Segade, S., Freire, M. & Carrion, A. (1978) Biochem. J. 170, 577-582 Sols, A. & Marco, R. (1970) Curr. Top. Cell. Regul. 2,227-273 Uhr, M. L., Thompson, V. W. & Cleland, W. W. (1974) J. Biol. Chem. 249,2920-2927
Seasonal Changes in the Triacylglycerol Fatty Acids of the Mantle Tissue of the Mussel Mytifus edufis L. MICHAEL J. WALDOCK and DAVID L. HOLLAND NERC Unit of Marine Invertebrate Biology, Marine Science Laboratories, Menai Bridge, Gwynedd LL59 5EH, Wales, U.K.
It has been shown in this laboratory that juvenile bivalves have a limited capacity for elongation and desaturation of fatty acids and that for optimal growth certain fatty acids
1979
BIOCHEMICAL SOCIETY TRANSACTIONS
of (14C)glucose into glycogen in mantle-tissue slices and the specific activity of glycogen synthase (B. N . Zaba, personal communication). The only other study on seasonal changes in glycogen synthase activity known to us is on frog liver. Castineiras et al. (1977) have shown that the increase in hepatic glycogen content of Rana ridibunda in the autumn can be related to a n increase in the specific activity of glycogen synthase; the percentage I-form remained constant throughout the year. In contrast, both the specific activity and percentage I-form of glycogen synthase varied seasonally in M . edulis. Mantle-tissue slices from winter mussels were incubated for 3h at 20°C in KrebsRinger bicarbonate buffer (pH7.4) in the presence of 5m~-glucose.The activity of the I-form of glycogen synthase increased 5-fold t o the summer level of 20-25 %. The ratio of the I-form activity with 5m~-glucose/controlswas 1.6OkO.17 (n = 9) compared with a ratio of 1.08kO.20(n = 9) for the total (I+D) activity, showing that the glucose treatment stimulates the conversion of the D- to I-form of the enzyme. The I-form activity was significantly higher in the control slices than in winter field mussels but the reason for this difference is not known. The glucose effect was time- and concentration-dependent, with maximum stimulation by 5m~-glucoseover a period of 3h (M. A. Whittle, unpublished results). We believe that the increase in the percentage I-form of glycogen synthase in summer mussels is the result of feeding activity and a concomitant increase in tissue glucose concentration. Work in other laboratories has indicated that the effect of high glucose concentrations o n glycogen synthase activity in bivalve molluscs may be mediated by an insulin-like peptide. Martinez et al. (1973) have isolated insulin-like proteins from the digestive gland of Ostrea edulis L., and Fritsch et al. (1976) have demonstrated insulin-like immunoreactivity in epithelial cells of the intestine associated with the digestive gland in M . edulis. More recently, Plisetskaya e t al. (1979) have used a physiological approach in which glucose and insulin were injected at low doses, and in other experiments antiinsulin serum was used to exclude the endogenous hormone. As a result of these experiments, Plisetskaya e t a / . (1978) have proposed a direct role for insulin, o r a substance closely related to insulin, in the control of glycogen metabolism in the fresh-water bivalves, Anodonta cygnea and Unio pictorum. Castineiras, M. J., Guinovart, J . J., Itarte, E. & Rosell-Perez, M. (1977) Reu. ESP.Fisiol. 33, 311-316 Cook, P. A. & Gabbott, P. A. (1978) Comp. Biochem. Physiol. B 60,419-421 De Zwaan, A. & Zandee, D. I. (1972) Comp. Biochem. Physiol. 43A, 53-58 Fritsch, H. A. R., Van Noorden, S. & Pearse, A. G. E. (1976) Cell. Tissue. Res. 165, 365-369 Gabbott, P. A. (1975) in Proc. Eur. Mar. Biol. Symp. 9th (Barnes, H., ed.), pp. 191-221, Aberdeen University Press, Aberdeen Gabbott, P. A. & Bayne, B. L. (1973) J. Mar. Biol. Assoc. U.K. 53,269-286 Groves, W. E., Davis, F. C . &Sells, B. H. (1968) Anal. Biocheni. 22, 195-210 Houteville, P. & Lubet, P. (1974) C . R. Hebd. Seances Acad. Sci. Ser. D 278,2469-2472 Martinez, de N. R., Garcia, M. C.,Salas, M. & Candela, J. L. R.(1973) Cen. Comp. Endocrinol. 20,305-31 1 Passoneau, J. V. & Rottenberg, D. A. (1973) Anal. Biochem. 51,528-541 Plisetskaya, E., Kazakov, V. K., Soltitskaya, L. & Leibson, L. G. (1978) Gen. Comp. Endocrinol. 35,133-145
Control of NADP+-dependent Isocitrate Dehydrogenase Activity in the Mussel Mytilus edulis L. ERICA J. H. H E A D and PETER A. GABBOTT
NERC Unit of Marine Invertebrate Biology, Marine Science Laboratories, Menui Bridge, Gwynedd LL59 SEH, Wales, U.K. N A D P H is required for fatty acid and sterol synthesis. The control of N A D P H production has been studied in the digestive gland and mantle tissue of Mytilus edulis. 1979
583rd MEETING, CAMBRIDGE
897
In mammalian liver the enzymes involved in NADP+ reduction are the enzymes of the pentose phosphate-pathway, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, and ‘malic’ enzyme. Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are present in the digestive gland (Carrion-Angosto et al., 1977; Rodriguez-Segade et af., 1978) and mantle tissue (E. J. H. Head, unpublished work) of M. edulis but ‘malic’ enzyme has been found with only very low activities (de Zwaan & Van Marrewijk, 1973). Another source of NADPH, cytoplasmic isocitrate dehydrogenase (EC 1.1 .I .42), has been found in both mammalian (Gul & Dils, 1969) and insect (see Bailey, 1975) tissues : NADP++isocitrate z C 0 2 + a-oxoglutarate+NADPH Isocitrate dehydrogenase activity has been found in the digestive gland and mantle tissue of M. edulis (Addink & Veenhof, 1975). J. E. Cienfuegos & A. Carrion (personal communication) found that the isocitrate dehydrogenase activity in the digestive gland was at least seven times greater than that of ‘malic’ enzyme. The specific activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and cytoplasmic isocitrate dehydrogenase were measured at bimonthly intervals throughout the year. The results showed that the enzymes of the pentose phosphate pathway increased in activity about 3-fold in the digestive gland (and to a lesser extent in the mantle) during the summer, when high food levels were available. In contrast, isocitrate dehydrogenase activity remained almost constant throughout the year. To study the regulatory properties of isocitrate dehydrogenase, the enzyme from the digestive gland was purified to homogeneity by affinity and ion-exchange chromatography. It was not inhibited by palmitoyl-CoA nor by ADP or ATP. Kinetic experiments were carried out with 5m~-Mg’+present. Initial-rate studies gave Michaelis constants of 3.5 and 3 . 4 for ~ ~NADP+ and total D-isocitrate respectively, and dissociation constants that were indistinguishable from the Michaelis constants. Similar Michaelis values were obtained for the mantle enzyme. Inhibition patterns for the reaction products, a-oxoglutarate and NADPH, and for the isocitrate analogue, tricarballylic acid, were consistent with a rapid-equilibrium random-ordered mechanism as found by Uhr et al. (1974) for isocitrate dehydrogenase from pig heart. The relative initial-reaction rate can be calculated by using the equation given by Fromm (1975), which includes product inhibition by NADPH:
[A], [B] and [Q] are the concentrations of NADP+, total D-isocitrate and NADPH respectively. K, and Kb are the Michaelis constants for NADP+ and total D-isocitrate. K , , is the dissociation constant for the E-NADP+ complex and K , , and K,, are the dissociation constants for the E-isocitrate-NADPH and E-NADPH complexes, where E is the enzyme. Fig. 1 shows the reaction rates for different [NADP+/NADPH] ratios, at constant [NADP++NADPH] of 6 5 , (determined ~ ~ experimentally for the digestive gland). The shape of the curves is dependent on the relative affinities of the enzyme species E and E-isocitrate for NADP+ and NADPH, and it is regions where the response curves are steep that give rise to regulatory interactions (see Atkinson, 1970). Values for the cytoplasmic [NADP+]/[NADPH] ratio in mammalian systems are low, between 0.014 and 0.3 (see Sols & Marco, 1970). This is the range of maximum sensitivity of isocitrate dehydrogenase to changes in the [NADP+]/[NADPH] ratio. Eqn. (1) represents the limiting case in which no a-oxoglutarate is present. In M. edulis the tissue concentrations of a-oxoglutarateare very low (Bayne, 1973),so that the inhibitory effect will benegligible compared with that of NADPH.
Vol. 7
898
BIOCHEMICAL SOCIETY TRANSACTIONS
06
0.8
1.0
[NADP+I/[NADPH] Fig. 1, Calculated variation of relative initial rates for isocitrate dehydrogenase The reaction curves were calculated according to eqn. (I), using values of K, = K , , = 3 . 5 , u ~ ,K b = 3 . 4 p ~ K,, , = 9 . 2 and ~ ~ Kiq= 9 . 7 ~ The ~ . total NADP+NADPH con, was measured by the recycling method of Lowry & Passonneau centration was 6 5 p ~and (1972). D-isocitrate concentrations are indicated on the curves and the shaded area represents the physiological range of D-isocitrate concentrations in the digestive gland of M . edulis. D-Isocitrate concentrations have been determined in individual mussels. I n the digestive gland these lie between the values of K,,, and about 10 K,, with most values in the ~ . NADP+-dependent cytoplasmic dehydrogenases, Sols & Marco range 2 0 - 4 0 ~ For (1970) consider that any utilization of NADPH by biosynthetic pathways will be followed by a fall in N A D P H concentration and a n increased regeneration of NADPH. At saturating concentrations of D-isocitrate and in the presence of 5 m ~ - M g ' + ,isocitrate dehydrogenase behaves in accordance with this prediction. Addink, A. D. F. & Veenhof, P. R. (1975) in Proc. Eur. Mar. Biol. Symp. 9th (Barnes, H., ed.), pp. 109-1 19, Aberdeen University Press, Aberdeen Atkinson, D. E. (1970) Enzymes, 3rd Ed. 1,461-489 Bailey, E. (1975) in Insect Biochemistry and Function, (Candy, D. J. & Kilby, B. A,, eds.), pp. 89-167, Chapman and Hall, London Bayne, B. L. (1973) Neth. J. Sea Res. 7, 399410 Carrion-Angosto, A., Silva Pando, M., Rodrigues-Segade, S. & Ruiz-Amil, M. (1977) Rev. ESP. Fisiol. 33,265-272 De Zwaan, A. &Van Marrewijk, W. J. A. (1973) Comp. Biochem. Physiot. B44,1057-1066 Fromm, H. J. (1975) Initial Rate Kinetics, Springer-Verlag, Berlin, Heidelberg and New York Gul, B. & Dils, R. (1969) Biochem. J. 112,293-301 Lowry, 0. H. & Passonneau, J. V. (1972) A Flexible System of Enzymatic Analysis, Academic Press, New York and London Rodriguez-Segade, S., Freire, M. & Carrion, A. (1978) Biochem. J. 170, 577-582 Sols, A. & Marco, R. (1970) Curr. Top. Cell. Regul. 2,227-273 Uhr, M. L., Thompson, V. W. & Cleland, W. W. (1974) J. Biol. Chem. 249,2920-2927
Seasonal Changes in the Triacylglycerol Fatty Acids of the Mantle Tissue of the Mussel Mytifus edufis L. MICHAEL J. WALDOCK and DAVID L. HOLLAND NERC Unit of Marine Invertebrate Biology, Marine Science Laboratories, Menai Bridge, Gwynedd LL59 5EH, Wales, U.K.
It has been shown in this laboratory that juvenile bivalves have a limited capacity for elongation and desaturation of fatty acids and that for optimal growth certain fatty acids
1979
![Seasonal changes in the triacylglycerol fatty acids of the mantle tissue of the mussel Mytilus edulis L [proceedings].](/assets/img/hold.png)
