Malate dehydrogenase of the cytosol. Preparation and reduced nicotinamide-adenine dinucleotide-binding studies.
1. Two methods of preparing pig heart soluble malate dehydrogenase are described. A slow method yields an enzyme composed of three electrophoretically separable subforms. The more rapid method reproducibly gives a high yield of an enzyme that consists predominantly of the least acid subform. 2. The A(1%) (1cm) of the protein was redetermined as 15 at 280nm. By using this value the enzyme molecule was found to contain two independent and indistinguishable NADH-binding sites in titrations with NADH. 3. No evidence was found for the dissociation of the enzyme in the concentration range 0.02-7.2mum. 4. l-Malate (0.1m) tightened the binding of NADH to both pig and ox heart enzyme (2-fold), but, in contrast with the report by Mueggler, Dahlquist & Wolfe [(1975) Biochemistry14, 3490-3497], did not cause co-operative interactions between the binding sites. 5. Fructose 1,6-bisphosphate had no effect on the binding of NADH to the pig heart enzyme, but with the ox heart enzyme the NADH is slowly oxidized. This slow oxidation explains the ;sigmoidal' binding curves obtained when NADH was added to ox heart soluble malate dehydrogenase in the presence of fructose 1,6-bisphosphate [Cassman (1973) Biochem. Biophys. Res. Commun.53, 666-672] without the postulate of site-site interactions. 6. It is concluded that neither l-malate nor fructose 1,6-bisphosphate could in vivo modulate the activity of soluble malate dehydrogenase and alter the rates of transport of NADH between the cytosol and the mitochondrion. 7. Details of the preparation of soluble malate dehydrogenase have been deposited as Supplementary Publication SUP 50080 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1978) 169, 5.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005636:Fructosephosphates / Q000494:pharmacology; D008291:Malate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D008293:Malates / Q000494:pharmacology; D008722:Methods; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D010710:Phosphates / Q000032:analysis; D011485:Protein Binding / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013552:Swine
D005636:Fructosephosphates; D008293:Malates; D010710:Phosphates; D009243:NAD; D008291:Malate Dehydrogenase