Studies on the interaction between rabbit liver pyruvate kinase and its allosteric effector fructose 1,6-diphosphate.
Preparation of the L form of rabbit liver pyruvate kinase (EC 2.7.1.40) in the presence of fructose 1,6-diphosphate yielded an enzyme which was kinetically identical with the M or muscle-type form of pyruvate kinase found in liver. Chromatographic and dialysis studies of this complex showed that most of the fructose 1,6-diphosphate molecules were loosely bound to the enzyme, but dilution-dissociation studies and binding experiments established that there was a high initial affinity between the enzyme and fructose 1,6-diphosphate (K(assoc.)=2.3x10(9)), and that binding of the loosely bound fructose 1,6-diphosphate was concentration-dependent and a necessary condition to overcome the co-operative interaction observed with the homotropic effector phosphoenolpyruvate. Preparation of the liver enzyme in the absence of EDTA did not yield a predominantly M form of the enzyme, and incubation of the M form in the presence of EDTA did not convert it into the L form, but resulted in inhibition of enzyme activity. Immunological studies confirmed that the L and M forms in liver were distinct, and that preparation of the L form in the presence of fructose 1,6-diphosphate did not produce an enzyme antigenically different from the L form prepared in the absence of this heterotropic effector.
D000494:Allosteric Regulation; D000818:Animals; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D002247:Carbon Isotopes; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003956:Dialysis; D004492:Edetic Acid; D004789:Enzyme Activation; D005260:Female; D005636:Fructosephosphates / Q000494:pharmacology*; D006598:Hexosediphosphates / Q000494:pharmacology; D007106:Immune Sera; D005779:Immunodiffusion; D007527:Isoenzymes / Q000378:metabolism; D008024:Ligands; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D011485:Protein Binding; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits; D051381:Rats / Q000276:immunology; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors; D014462:Ultrafiltration
D002247:Carbon Isotopes; D005636:Fructosephosphates; D006598:Hexosediphosphates; D007106:Immune Sera; D007527:Isoenzymes; D008024:Ligands; D004492:Edetic Acid; D011770:Pyruvate Kinase