[Immobilized active monomers of D-glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscles and their coenzyme-binding properties].
Experimental conditions favouring the dissociation of tetrameric rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase into active monomers were elaborated. The urea-induced dissociation of the tetramer was shown to be a stepwise process (in 2 M urea only dimers are formed; an increase in urea concentration up to 3 M causes the splitting of the dimers into monomers). The specific activity of immobilized monomers in the glyceraldehyde-3-phosphate oxidation reaction does not differ from that of the parent immobilized tetrameric form. The tetrameric enzyme molecule binds the coenzyme with a negative cooperativity (the first two NAD+ molecules bind with KD below 0.1 microM; for the third and fourth molecules the dissociation constant was determined to be equal to 5.5 +/- 1.5 microM (50 mM medinal buffer, 10 mM sodium phosphate, pH 8.2). The cooperativity of NAD+ binding is preserved in the immobilized preparation of tetrameric dehydrogenase. The immobilized monomers bind NAD+ with KD of 1.6 +/- 1.0 microM. The experimental results are consistent with the hypothesis according to which the association of catalytically active subunits into a tetramer changes their coenzyme-binding properties in such a way that the first two NAD+ molecules bind more firmly to a tetramer than to a monomer, whereas the third and the fourth NAD+ molecules bind less firmly.
D000818:Animals; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000302:isolation & purification / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation; D011817:Rabbits
D003067:Coenzymes; D004800:Enzymes, Immobilized; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
Immobilizovannye aktivnye monomery D-glitseral'degid-3-fosfatdegidrogenazy iz skeletnykh myshts krolika i ikh koénzimsviazyvaiushchie svoĭstva.