Rat liver mitochondrial malate dehydrogenase: purification, kinetic properties, and role in ethanol metabolism.
Malate dehydrogenase was purified from the mitochondrial fraction of rat liver by ion-exchange chromatography with affinity elution. The kinetic parameters for the enzyme were determined at pH 7.4 and 37 degrees C, yielding the following values (microM): Ka, 72; Kia, 11; Kb, 110; Kp, 1600; Kip, 7100; Kq, 170; Kiq, 1100, where a = NADH, b = oxalacetate, p = malate, and q = NAD+. Kib was estimated to be about 100 microM. The maximum velocities for mitochondrial malate dehydrogenase in rat liver homogenates, at pH 7.4 and 37 degrees C, were 380 +/- 40 mumol/min per gram of liver, wet weight, for oxalacetate reduction and 39 +/- 3 mumol/min per gram of liver, wet weight, for malate oxidation. Rates of the reaction catalyzed by mitochondrial malate dehydrogenase under conditions similar to those in vivo were calculated using these kinetic parameters and were much lower than the maximum velocity of the enzyme. Since mitochondrial malate dehydrogenase is not saturated with malate at physiological concentrations, its kinetic parameters are probably important in the regulation of mitochondrial malate concentration during ethanol metabolism. For the mitochondrial enzyme to operate at a rate comparable to the flux through cytosolic malate dehydrogenase during ethanol metabolism (about 4 mumol min-1 per gram liver), the mitochondrial [malate] would need to be about 2 mM and the mitochondrial [oxalacetate] would need to be less than 1 microM.
Wiseman
M S
MS
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
McKay
D
D
Crow
K E
KE
Hardman
M J
MJ
D000818:Animals; D000431:Ethanol / Q000378:metabolism*; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D009243:NAD; D000431:Ethanol; D008291:Malate Dehydrogenase