Skip to main content
SpringerLink
Log in

Pyruvate carboxylase from rainbow trout liver

  • Published:
Journal of comparative physiology Aims and scope Submit manuscript

Summary

A method is described for the partial purification of pyruvate carboxylase from rainbow trout liver. The enzyme has a pH optimum of about 8.0, possesses an absolute requirement for activation by acetylCoA, and prefers MgATP over other nucleoside triphosphates. K+ causes a decrease in the apparentK m for HCO −3 . AcetylCoA activation shows positive cooperativity withK a=0.072 mM andn H=1.78 at pH 7.7, 2.5 mM free Mg2+, 100 mM K+, and saturating concentrations of substrates. A high acetylCoA concentration causes a decrease in the apparentK m values for MgATP and HCO −3 and a biphasic double reciprocal plot with pyruvate as the varied substrate. MgADP and AMP are competitive inhibitors with respect to MgATP. The enzyme shows a ‘U-type’ response to the adenylate energy charge and retains considerable activity throughout a wide range of energy charge values. It is proposed that intramitochondrial acetylCoA concentration and the adenylate energy charge control the rate of pyruvate carboxylation in vivo.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

DTT :

dithiothreitol

PMSF :

phenylmethylsulfonylfluoride

References

  • Ashman LK, Keech DB, Wallace JC, Nielsen J (1972) Sheep kidney pyruvate carboxylase. Studies on its activation by acetylcoenzyme A and characteristics of its acetylcoenzyme A independent reaction. J Biol Chem 247:5818–5824

    Google Scholar 

  • Atkinson DE (1968) The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochem 7:4030–4034

    Google Scholar 

  • Atkinson DE (1976) Adaptations of enzymes for regulation of catalytic function. Biochem Soc Symp 41:205–223

    Google Scholar 

  • Atkinson DE, Fall L (1967) Adenosine triphosphate conservation in biosynthetic regulation.Escherichia coli phosphoribosylpyrophosphate synthase. J Biol Chem 242:3241–3242

    Google Scholar 

  • Atkinson DE, Walton GM (1967) Adenosine triphosphate conservation in metabolic regulation. Rat liver citrate cleavage enzyme. J Biol Chem 242:3239–3241

    Google Scholar 

  • Atkinson DE, Roach PJ, Schwedes JS (1975) Metabolite concentrations and concentration ratios in metabolic regulation. Adv Enz Reg 13:393–411

    Google Scholar 

  • Barden RE, Scrutton MC (1974) Pyruvate carboxylase from chicken liver. Effects of univalent and divalent cations on catalytic activity. J Biol Chem 249:4829–4838

    Google Scholar 

  • Barritt GJ, Zander GL, Utter MF (1976) The regulation of pyruvate carboxylase activity in gluconeogenic tissues. In: Hanson RW, Mehlman MA (eds) Gluconeogenesis: Its regulation in mammalian species. Wiley, New York, pp 3–46

    Google Scholar 

  • Batenburg JJ, Olson MS (1976) Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria. J Biol Chem 251:1364–1370

    Google Scholar 

  • Bilinski E, Gardner LJ (1968) Effect of starvation on free fatty acid level in blood plasma and muscular tissues of rainbow trout (Salmo gairdneri). J Fish Res Board Can 25:1555–1560

    Google Scholar 

  • Feir HM, Suzuki I (1969) Pyruvate carboxylase ofAspergillus niger: Kinetic study of a biotin-containing carboxylase. Can J Biochem 47:697–710

    Google Scholar 

  • French CJ, Mommsen TP, Hochachka PW (1981) Amino acid utilization in isolated hepatocytes from rainbow trout. Eur J Biochem 113:311–317

    Google Scholar 

  • Hayashi S, Ooshiro Z (1977) Gluconeogenesis in perfused eel liver — effect of starvation, aminooxyacetate, D-malate, and hormones. Mem Fac Fish Kagoshima Univ 26:89–95

    Google Scholar 

  • Hayashi S, Ooshiro Z (1979) Gluconeogenesis in isolated liver cells of the cel,Anguilla japonica. J Comp Physiol 132:343–350

    Google Scholar 

  • Hazel JR, Prosser CL (1979) Incorporation of I-14C-acetate into fatty acids and sterols by isolated hepatocytes of thermally acclimated rainbow trout (Salmo gairdneri). J Comp Physiol 134:321–329

    Google Scholar 

  • Idler DR, Bitners I (1958) Biochemical studies on sockeye salmon during spawning migration II. Cholesterol, fat, protein, and water in the flesh of standard fish. Can J Biochem Physiol 36:793–798

    Google Scholar 

  • Keech DB, Utter MF (1963) Pyruvate carboxylase II. Properties. J Biol Chem 238:2609–2614

    Google Scholar 

  • Klungsøyr L, Hageman JH, Fall L, Atkinson DE (1968) Interactions between energy charge and product feedback in the regulation of biosynthetic enzymes. Aspartokinase, phosphoribosyladenosine triphosphate synthetase and phosphoribosyl pyrophosphate synthetase. Biochem 7:4035–4040

    Google Scholar 

  • Layne E (1957) Spectrophotometric and turbidimetric methods for measuring proteins. Meth Enzymol 3:447–454

    Google Scholar 

  • Mahan DE, Mushahwar IK, Koeppe RE (1975) Purification and properties of rat brain pyruvate carboxylase. Biochem J 145:25–35

    Google Scholar 

  • McClure WR, Lardy HA, Kneifel HP (1971) Rat liver pyruvate carboxylase. I. Preparation, properties, and cation specificity. J Biol Chem 246:3569–3578

    Google Scholar 

  • Mommsen TP, French CJ, Hochachka PW (1980) Sites and patterns of protein and amino acid utilization during the spawning migration of salmon. Can J Zool 58:1785–1799

    Google Scholar 

  • Nagai M, Ikeda S (1971) Carbohydrate metabolism in fish. I. Effects of starvation and dietary composition on the blood glucose level and the hepatopancreatic glycogen and lipid contents in carp. Bull Jap Soc Science Fish 37:404–409

    Google Scholar 

  • Randall DJ, Cameron JN (1973) Respiratory control of arterial pH as temperature changes in rainbow troutSalmo gairdneri. Am J Physiol 225:997–1002

    Google Scholar 

  • Reinhart, GD, Lardy HA (1980) Rat liver phosphofructokinase: Kinetic activity under near-physiological conditions. Biochem 19:1477–1484

    Google Scholar 

  • Renaud JM, Moon TW (1980) Starvation and the metabolism of hepatocytes isolated from the American eel,Anguilla rostrata LeSeur. J Comp Physiol 135:127–137

    Google Scholar 

  • Robinson JS, Mead JF (1973) Lipid absorption and deposition in rainbow trout (Salmo gairdneri). Can J Biochem 51:1050–1058

    Google Scholar 

  • Rowan AN, Newshome EA, Scrutton MC (1978) Partial purification and some properties of pyruvate carboxylase from the flight muscle of the locust (Schistocerca gregaria). Biochim Biophys Acta 522:270–275

    Google Scholar 

  • Schulman RG, Brown TR, Ugurbil K, Ogawa S, Cohen SM, Hollander JA den (1979) Cellular applications of31P and13C nuclear magnetic resonance. Science 205:160–166

    Google Scholar 

  • Scrutton MC (1974) Pyruvate carboxylase. Studies of activator-independent catalysis and of the specificity of activation by acyl derivatives of coenzyme A for the enzyme from rat liver. J Biol Chem 249:7057–7067

    Google Scholar 

  • Scrutton MC (1978) Fine control of the conversion of pyruvate (phosphoenolpyruvate) to oxaloacetate in various species. FEBS Lett 89:1–9

    Google Scholar 

  • Scrutton MC, Utter MF (1967) Pyruvate carboxylase IX. Some properties of the activation by certain acylderivatives of coenzyme A. J Biol Chem 242:1723–1735

    Google Scholar 

  • Scrutton MC, Utter MF (1968) The regualtion of glycolysis and gluconeogenesis in animal tissues. Ann Rev Biochem 37:249–302

    Google Scholar 

  • Scrutton MC, White MD (1972) Pyruvate carboxylase from rat liver: Catalytic properties in the absence, and at low concentrations, of acetylCoA. Biochem Biophys Res Commun 48:85–93

    Google Scholar 

  • Scrutton MC, Young MR (1972) Pyruvate carboxylase. In: Boyer PD (ed) The enzymes, vol 6. Academic Press, New York London, pp 1–35

    Google Scholar 

  • Scrutton MC, Keech DB, Utter MF (1965) Pyruvate carboxylase IV. Partial reaction and the locus of activation by acetylCoA. J Biol Chem 240:574–581

    Google Scholar 

  • Seubert W, Remberger U (1961) Reinigung and Wirkungsweise der Pyruvat-carboxylase ausPseudomonas citronellolis. Biochem Z 334:401–414

    Google Scholar 

  • Siess EA, Brocks DG, Lattke HK, Wieland OH (1977) Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate. Biochem J 166:225–235

    Google Scholar 

  • Suarez RK, Hochachka PW (1981a) Preparation and properties of rainbow trout liver mitochondria. J Comp Physiol 143:269–273

    Google Scholar 

  • Suarez RK, Hochachka PW (1981 b) The pyruvate branch point in fish liver mitochondria: Effects of acylcarnitine oxidation on pyruvate dehydrogenase and pyruvate carboxylase activities. J Comp Physiol 143:275–279

    Google Scholar 

  • Tischler ME, Friedrichs D, Coll K, Williamson JR (1977) Pyridine nucleotide distributions and enzyme mass action ratios in hepatocytes from fed and starved rats. Arch Biochem Biophys 184:222–236

    Google Scholar 

  • Titheradge MA, Coore HG (1976) The mitochondrial pyruvate carrier, its exchange properties and its regulation by glucagon. FEBS Lett 63:45–50

    Google Scholar 

  • Titheradge MA, Coore HG (1976) Hormonal regulation of liver mitochondrial pyruvate carrier in relation to gluconeogenesis and lipogenesis. FEBS Lett 71:73–78

    Google Scholar 

  • Titheradge MA, Stringer JL, Haynes RC Jr (1979) The stimulation of the mitochondrial uncoupler-dependent ATPase in isolated hepatocytes by catecholamines and glucagon and its relationship to gluconeogenesis. Eur J Biochem 102:117–124

    Google Scholar 

  • Walton MJ, Cowey CB (1979) Gluconeogenesis by isolated hepatocytes from rainbow troutSalmo gairdneri. Comp Biochem Physiol 62B:75–79

    Google Scholar 

  • Williamson JR (1967) Effects of fatty acids, glucagon, and antiinsulin serum on the control of gluconeogenesis and ketogenesis in rat liver. Adv Enzyme Regul 5:229–255

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Zoology, The University of British Columbia, V6T 2A9, Vancouver, B.C., Canada

    Raul K. Suarez & Peter W. Hochachka

Authors
  1. Raul K. Suarez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter W. Hochachka
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Suarez, R.K., Hochachka, P.W. Pyruvate carboxylase from rainbow trout liver. J Comp Physiol B 143, 281–288 (1981). https://doi.org/10.1007/BF00797709

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00797709

Keywords

Search

Navigation