Effect of temperature on the adenylate cyclase activity of Mytilus galloprovincialis mantle tissue

Comp. Biochem. Physiol. Vol. 9911,No. 2, pp. 355-357, 1991

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EFFECT OF TEMPERATURE ON THE ADENYLATE CYCLASE ACTIVITY OF M Y T I L U S GALLOPROVINCIALIS MANTLE TISSUE M. J. MANCEBO,*~" M. TREVII~IOand J. ESPlNO~A Department of Physiology, Faculty of Pharmacy, University of Santiago de Compostela, 15706-Santiago de Compostela, Spain

(Received 15 November 1990) Abstract--l. The basal and NaF-stimuiated adenylate cyclase activities of Mytilus galloprovincialis mantle tissue were studied at different temperatures. 2. There are no significant differences in the Km for ATP at 13°C and 20°C in both basal and NaF-stimulated conditions. 3. NaF increases the Vm~ of the enzyme (5-fold) and decreases about 50% the Km for ATP at both temperatures assayed. 4. Activation energy of the enzyme reaction is 33.4 kJ/mol. K in basal conditions and 29.4 kJ/mol. K when NaF is present. The Q~0, at saturating substrate concentrations, is approximately 1.5 and this value is constant in the temperature range studied, 10-30°C. 5. The adenylate cyclase starts being inactivated from 30°C. The enzyme shows greater sensitivity to denaturalization by temperature in NaF-stimulated than in basal conditions.

INTRODUCTION Mussels experience fluctuations in temperature not only seasonally, but also daily and these changes may affect the physiology of the animals. The effects of temperature on enzyme structure and function have been of interest to comparative physiologists and biochemists. M a n y aspects o f enzymatic adaptation to temperature have been extensively reviewed (Hazel and Prosser, 1974; Somero, 1978). The kinetic-catalytic and regulatory properties of enzymes are highly temperature-sensitive. A proper degree o f stability of enzyme structure must be maintained, as well as the rates of enzymatic reaction adjusted to values that correspond to the cells' requirements. In a previous work (Mancebo et al., 1990) we have shown the existence of a membrane-bound adenylate cyclase in the mantle tissue of the mussel Mytilus galloprovincialis. Since the adenylate cyclase is a key enzyme system in the transmembrane signalling processes of the cells, a study concerning the effects of temperature on the adenylate cyclase activity was carried out. This work brings forth new data about the kinetic properties of Mytilus galloprovincialis adenylate cyclase. MATERIALS AND METHODS

Animals Adult female sea mussels, Mytilus galloprovincialis Lmk., were collected from the estuary of Muros (Galicia, NW Spain) and used fresh within I-3 days of collection. *Author to whom correspondence should be addressed. tRecipient of a Reincorporation Postdoctoral Fellowship from the Spanish Ministry of Education and Science. clPe 99/2--H

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Experiments were carried out from August to October 1989.

Chemicals (~32P)-ATP (spec. act. 20-30Ci/mmol) and (2,8-3H)cAMP (spec. act. 32.9Ci/mmol) were obtained from NEN/Dupont (Nemours, Germany). ATP, GTP, cAMP, phosphocreatine, creatine phosphokinase, myokinase and neutral alumina were purchased from Sigma Chemical Co. (St. Louis, USA); MgCI2 and NaF were from Merck (Darmstadt, Germany). Dowez AG 50 W-XR was from BioRad (Richmond, USA). All other reagents were analytical grade.

Adenylate cyclase assays Preparation of a mantle particulate fraction and measurement of adenylate cyclase activity were performed as described previously (Mancebo et aL, 1990). In brief, mantles were removed and quickly homogenized and after subcellular fractionation by centrifugation, the 10,000g pellet was used as the enzyme source. 32p-cAMP formed was chromatographicaUy isolated by the method of Salomon (1974) as modified by Bockaert (1976). Protein concentration was determined according to Lowry et al. (I 951) using bovine serum albumin as standard.

Temperature dependence experiments Enzyme activity was measured following 30 min incubation in the temperature range of 0°C-60°C at saturating substrate concentrations. In dose-response experiments to ATP, incubation was carried out only at 13°C and 20°C for 30 min. Enzyme activity determinations were performed under gentle shaking using a temperature controlled water bath (New Brunswick G-76 shaker coupled, when required, to a cooler unit). NaF, 10 mM, was used as stimulator of the adenylate cyclase activity. Kinetic parameters (K, and Vm~) were drawn from Lineweaver-Burk plots. Activation energy of the enzyme, Ea, was ealeuiated from the Arrhenius plot of log enzyme activity against 1/T (Whitaker, 1972).

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M.J. MANCEBOet al. Arrhenius plot revealed several breaks indicating a phase-transition at 40°C in the presence of NaF; the break-point was shifted to 30°C when basal activity was represented. The activation energy remained essentially the same when comparing the experimental conditions. Q~0 was approximately 1.5 and this value was constant in the temperature range studied (10-30°C).

RESULTS The basal levels of adenylate cyclase activity and the NaF-stimulated activity were compared at two temperatures, 13°C and 20°C, as a function of ATP concentration. The results are shown in Figs 1A and lB. The four curves followed Michaelis-Menten kinetics. Using the Lineweaver-Burk plot we calculated the Km (apparent) for ATP and the Vmax for each condition (Table 1). The temperature rise did not modify the Km for ATP in basal or NaF-stimulated conditions; but an increase in Vmax was observed. The presence of NaF modified the Km for ATP and the Vmax at both temperatures, with a 50% decrease in the Km and a 5-fold increase in the Vm. The adenylate cyclase activity as a function of temperature is presented in Fig. 2. Maximal enzyme activity under the basal and NaF-stimulated conditions was found at 30°C and 40°C, respectively. The adenylate cyclase starts being inactivated at temperatures above 30°C, and in the presence of NaF the enzyme undergoes inactivation at a higher temperature, 40°C. The sensitivity to temperature denaturalization was greater for the NaF-stimulated than for the basal enzyme activity.

DISCUSSION Mussels, as poikilotherms of intermareal zones, are expected to be able to compensate the effects produced by temperature changes in their environment (night-day, high and low tide, summer-winter). The animals used in our study were collected from the estuary of Muros; this estuary, located in the NW of Spain, has a water temperature range fluctuating between 13°C in winter and 20°C in summer. The adenylate cyclase is a complex enzymatic system composed of a receptor, a guanine nucleotide binding regulatory protein and a catalytic unit. We used NaF as a stimulator of the enzyme activity; NaF acts on the regulatory protein enhancing the catalytic activity.

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Fig. 1. Dose-response to substrate concentration. Adenylate cyclase activity was measured in basal conditions and in the presence of 10 mM NaF. Incubation temperatures were 13°C (Fig. 1A) and 20°C (Fig. 1B). Assay conditions, in 50 #I final volume, were: 25 mM Tris-HCl pH 7.5, 1 mM EDTA, 1mM cAMP, 5 mM MgCI2, 10 #M GTP, 3H-cAMP (25,000dpm), ~2P-ATP (4 x 106dpm), 150-250U/mg creatine phosphokinase, 1000-1500 U/rag myokinase, 10 mM ereatine phosphate. Membrane preparations contained 50-70 #g protein. Results, expressed as pmoles cAMP formed min- ~mg protein- ', are the mean of four experiments. Standard errors never exceeded 10% of the values. Inset: Lineweaver-Burk plot of the data.

Mytilus adenylate cyclase activity Table 1. Kinetic parameters versus temperature. Kmand Vm, values were calculated from the Lineweaver-Burk plots shown in Figs IA and lB. Vm~x is expressed as pmoles of cAMP formed rain-~ nag protein 13°C 20°C Basal Km= 0.30 mM Km= 0.45 mM V,,~x= 3.0 +__0.3 Vm,x~ 4.5 +_0.2 NaF (10 mM) Km= 0.16 mM Km= 0.20 mM Vm~x= 15.0+ 1.9 Vmax= 24.2 + 2.3

Results from experiments at 13°C and 20°C showed no significant modification in the Km for ATP, either in basal or stimulated conditions. On the other hand, the Q~0 value, at saturating substrate concentrations, was not modified and it remained constant in the 10-20°C and 20-30°C ranges. It is widely accepted that Ql0 values close to 1 are an indication of instantaneous temperature compensation. The Q,0 of approximately 1.5 obtained for the mantle adenylate cyclase together with the maintenance of the Km at both temperatures suggest certain immediate compensation rather than a "positive thermic modulation", a phenomenon described by Hochachka and Somero (1973) and observed for other enzymes of Mytilus galloprovineialis (O. GarciaMartin, personal communication). Interestingly, the relative independence to temperature changes in the case of enzymes whose substrates are adenylic compounds, such as the adenylate cyclase and its substrate ATP in our studies, was indicated by Hochachka and Somero (1968). Although a change in temperature did not alter the Km for the substrate, NaF caused a decrease in the Km of approximately 50% at 13°C and 20°C. This decrease indicates that, at both temperatures, the enzyme responds to the presence of NaF in the same manner. We cannot conclude the existence of a seasonal adaptation to temperature since a study of the kinetic parameters throughout the year would be necessary. An increase in temperature above 40°C gradually decreased the enzyme activity; since this effect was also observed in stimulated conditions, the structure of the proteins must have been affected causing the

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Fig. 2. Temperature dependence of the adenylate cyclase activity of Mytilus galloprovincialis mantle. Inset: Arrhenius plot of the data. Ea is the activation energy expressed as kJ/mol. K at 2 mM ATP. Eat = 33.4 (Basal O), Ea2 = 29.4 (NaF 0 ) . Data are from three experiments. Standard errors were less than 10% of the reported values.

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denaturalization of the adenylate cyclase. Moreover, it has often been described that temperatures higher than 40°C are lethal for mussels. The Ea values obtained at saturating substrate concentrations are slightly higher than other activation energies for several fish enzymes (Ferracin et al., 1989; Gelman et al., 1989). The break-point in the Arrhenius plot may result from the occurrence of a phase transition in the membrane lipids, with which the adenylate cyclase is strongly associated; this effect has been observed for other integral membrane proteins (Brasitus et al., 1979). In summary, the adenylate cyclase activity in Mytilus galloprovincialis is relatively independent of the temperature, regarding kinetic parameters in basal and NaF-stimulated conditions as expected for an enzyme system located in the plasma membrane. Acknowledgements--The authors wish to thank Dr O. Garcia-Martin (Department of Biochemistry and Molecular Biology, University of Santiago de Compostela, Spain) for his helpful remarks. This work was supported by Grants from the Galician Government (Spain): XUGA 8150589 (Conselleria de Educacibn) and 1/89 (Conselleria de Pesca, Marisqueo y Acuicultura). REFERENCES

Bockaert J., Hunzicker-Dunn M. and Birnbaumer L. (1976) Hormone-stimulated desensitization of hormonedependent adenylyl cyclase: dual action of LH on pig Graafian follicle membranes. J. biol. Chem. 251, 2653-2659. Brasitus T. A., Schachter D. and Mamouneas T. G. (1979) Functional interaction of lipids and proteins in rat intestinal microvillus membranes. Biochemistry 18, 4136-4144. Ferracin A., Annicchiarico M., Coscarella A., Teichner A. and Dell'agata M. (1989) Thermal behavior of A4 lactate dehydrogenase purified from the heterothermic and sympatic vertebrate species, brook lamprey (Lampetra planei ), tench ( Tenca tenca ), smooth ( Triturus vulgaris ) and alpine newt (Triturus alpestris). Comp. Biochem. Physiol. 94B, 435-443. Garcia-Martin O. (1990) Unpublished data. Gelman A., Mokady S. and Cogan U. (1989) The thermal properties of intestinal alkaline phosphatase of three kinds of deep-water fish. Comp. Biochem. Physiol. 94B, 113-116. Hazel J. R. and Prosser C. L. (1974) Molecular mechanisms of temperature compensation in Poikilotherms. Physiol. Rev. 54, 620-677. Hochachka P. W. and Somero G. N. (1968) The adaptation of enzymes to temperature. Comp. Biochem. Physiol. 54B, 620-677. Hochachka P. W. and Somero G. N. (1973) Strategies of Biochemical Adaptation. W. B. Saunders, Philadelphia. Lowry O. H., Rosebrough N. Y., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mancebo M, J., Trevifio M., Crespo C. and Espinosa J. (1990) Adenylate cyclase activity in Mytilus galloprovincialis Link. Characteristics of the enzyme from mantle tissue. J. exp. Zool. (in press). Salomon Y., Londos C. and Rodbell M. (1974) A highly sensitive adenylate cyclase assay. Analyt. Biochem. 58, 541-548. Somero G. N. (1978) Temperature adaptation of enzymes: biological optimization through structure-function compromises. Ann. Rev. EcoL Syst. 9, 1-29. Whitaker J. R. (1972) Principles of Enzymology for the Food Sciences, pp. 332-333. Marcel Dekker, New York.