Study of subunit interactions in immobilized D-glyceraldehyde-3-phosphate dehydrogenase.

摘要: Abstract Under conditions which cause dissociation of soluble tetrameric glyceraldehyde-3-phosphate dehydrogenase ( d -glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) into inactive dimers, immobilized apoenzymes from yeast and rat skeletal muscle coupled to CNBr-activated Sepharose via one subunit retain 50% matrix-bound protein with unaltered specific activity. The solubilized dissociated species are inactive. Two molecules NAD+ (NADH) firmly bound the tetramer can prevent dissociation. Immobilized dimer was demonstrated bind molecule coenzyme high affinity. Using various combinations we succeeded in reconstituting tetramers, containing either a matrix-linked or non-covalently dimer. In latter case, completely prevented. This suggests that binding single is suffient stabilize interdimeric contacts provided neighbouring stabilized independently. Such stabilization produced by covalent subunits comprising matrix. structure as whole becomes resistant action dissociating agent. effect appears be cooperative similar NADH. is, most likely, results conformational changes, affecting Any factor, capable preventing these would contacts. conclusion substantiated antibodies, forming complex dimer, evidence obtained present investigation supports isolated represents relatively independent structural functional ‘unit’ enzyme. It catalically active form interactions other than those involved inter-dimeric tetramer. kinetics association dimers have been studied. Association rate constants were determined for homologous (yeast-yeast rat-rat) heterologous (yeast-rat-yeast-rabbit) combinations. antibody shown increase constant association.