Porcine succinate thiokinase (succinate: coenzyme A ligase (GDP-forming), EC 188.8.131.52) has been purified to virtual homogeneity by modification of the purification method described by Cha ((1969) Methods Enzymol. 13, 62-69). Antibody to the enzyme was also prepared. The antibody reacted almost fully with enzyme inactivated by N-ethylmaleimide or 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), according to the criteria of immunodiffusion and complement fixation. Inactivation and cross-linking (through sulfhydryl groups) of the enzyme by N,N′-o-phenylenedimaleimide or by N,N′-p-phenylenedimaleimide reduced the height of the complement fixation curves significantly, presumably by blockage or alteration of antigenic determinants. Horizontal shifts of the curves in the presence of p-chloromercuribenzoate (PCMB) suggested dissociation of the enzyme into subunits. Titration of the enzyme with 5,5′-dithiobis(2-nitrobenzoic acid) in the presence of sodium dodecylsulfate yielded a maximum of about 12 moles of sulfhydryl groups per mole of enzyme. Labeling of sulfhydryl groups with N-ethyl[14C]maleimide revealed essential sulfhydryl groups on both subunit types with greater incorporation occurring in the larger polypeptide chain.
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