Abstract
3-D-(-) hydroxybutyrate dehydrogenase (EC 1.1.1.30) from rat-liver mitochondria was purified in the form of the soluble, phospholipid-free apoenzyme by a procedure involving: (1) solubilization of the membrane bound enzyme by controlled digestion of membrane phospholipids with porcine pancreas phospholipase A2; (2) stabilization and separation of the released apoenzyme as a complex with egg-lecithin by gel filtration on Sephadex G-100; and (3) specific displacement of the apoenzyme from the enzyme-lecithin complex by treatment withBothrops atrox venom phospholipase A2 (in the absence of Ca2+ ions) and subsequent separation of the displaced apoenzyme by gel filtration on Sephadex G-100. The method described is adequate for samples containing about 40 mg of mitochondrial protein. The yield in activity is 42% of that present in mitochondria and the degree of purification of the apodehydrogenase is about 170 fold. The purified apodehydrogenase shows one single sharp band when submitted to SDS polyacrylamide gel electrophoresis, with a mobility corresponding to a molecular weight of 38000 daltons. Gel filtration of the apoenzyme on Sephadex G-100 shows two active peaks with molecular weights of 76000 and 38500 daltons, indicating two different states of aggregation, namely, monomer and dimer. The corresponding diffusion coefficients are 7.73 (monomer) and 5.70 (dimer) × 10−7. The apodehydrogenase preparation is devoid of phospholipids and is catalytically inactive. It can be reactivated by addition of egg lecithin or phospholipid mixtures containing lecithin in a suitable physical state. Reactivation occurs after formation of an active apodehydrogenase phospholipid complex.
Published Version
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