Single subunits of sepharose-bound pyruvate kinase are inactive

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Abstract
Bovine skeletal muscle pyruvate kinase was covalently coupled to Sepharose that had previously been activated by low concentrations of cyanogen bromide. Reaction conditions were chosen such that essentially all tetrameric enzyme molecules were covalently bound via a single subunit. Denaturation of the immobilized enzyme with guanidine hydrochloride followed by removal of noncovalently bound subunits and denaturant resulted in essentially no enzymatic activity remaining bound to the resin. Thus, single immobilized subunits of bovine pyruvate kinase were inactive. Sepharose-bound enzymatic activity could be recovered by adding soluble renaturing enzyme subunits to the immobilized monomers. The former combine noncovalently with the latter, presumably resulting in re-formation of bound tetramers, and an average recovery of 61% of the original matrix-bound activity was observed. While interactions with other enzyme subunits appear to be necessary for catalytic activity of bovine muscle pyruvate kinase, these subunit interactions apparently can be provided by chemically modified subunits. Soluble, renaturing subunits from enzyme that had been inactivated by treatment with trinitrobenzenesulfonic acid were able to interact with matrix-bound single subunits, thereby restoring the enzymatic activity of the latter.