ブタ心筋ピルビン酸デヒドロゲナーゼの精製と性質に関する研究

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Pyruvate dehydrogenase [EC 1.2.4.1] was isolated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by the sedimentation equilibrium methods. The enzyme was dissociated into two subunits (α and β), with estimated molecular weights of 41,000 (α) and 36,000 (β), respectively, by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The subunits were isolated by the phosphocellulose column chromatography and their chemical properties were examined. The subunit structure was assigned as α_2β_2. The enzyme contained no thiamine pyrophosphate (TPP), therefore its dehydrogenase activity was completely dependent on added TPP and partially dependent on added Mg^2+ or Ca^2+. The K_m value of pyruvate dehydrogenase for TPP was estimated to be 6.5×10^-5M in the presence of Mg^2+ or Ca^2+. The enzyme showed highly specific activity for both pyruvate and α-ketobutyrate, but it also showed some activity with α-ketovalerate, α-ketoisocaproate, and α-ketoisovalerate. The pyruvate dehydrogenase was strongly inhibited by sulfhydryl inhibitors ; and it contained 27 moles of 5,5'-dithiobis (2-nitrobenzoic acid)-reactive sulfhydryl group and a total of 36 moles of sulfhydryl group. The function of sulfhydryl group was examined and discussed. The reconstitution of the complex by the reassociation of pyruvate dehydrogenase and the yellow fraction is also demonstrated.
1977-04-25