Stoichiometry of slow binding of palmitoyl-CoA to liver glucokinase.
The interaction of palmitoyl-CoA with porcine glucokinase was studied by the gel permeation technique. The finding that glucokinase "bound" up to 60 molecules was unexpected from the specific inhibition of rat glucokinase by long chain acyl-CoA (Tippett & Neet, J. Biol. Chem. (1982) 287, 12839-12845). Sephacryl S-200 gel filtration in the presence of palmitoyl-CoA demonstrated a protein peak without enzyme activity that was eluted earlier than the active enzyme peak, indicating a large molecular weight shift for the inactivated enzyme form and confirming a large number (greater than or equal to 30) of associated palmitoyl-CoA molecules. The binding was also verified by analyzing the absorption characteristics of the inactivated enzyme peak. In the presence of glycerol, the size of the inactivated peak greatly decreased, but the separation between the two peaks remained unchanged. Therefore, the amphiphile bound predominantly to the inactive enzyme and not to the active form, suggesting that the rapid inhibitory interactions between palmitoyl-CoA and glucokinase previously observed are specific. Parallel enzyme activity studies showed that in the time range of the column experiments (4-20 h), both the rat and pig enzyme were greatly inactivated (greater than 90%) in the presence of palmitoyl-CoA (15 microM) in the absence of glycerol. This slow inactivation is different from the immediate specific inhibition previously reported and depends on both enzyme and palmitoyl-CoA concentrations. The presence of up to 20% glycerol slowed this inactivation process. These results demonstrated that even below the critical micelle concentration, partial inactivation of glucokinase occurs in the presence of palmitoyl-CoA over a long period of time.
Lin
S X
SX
Department of Biochemistry, Case Western Reserve University, Cleveland, OH.
Vogel
W K
WK
Neet
K E
KE
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004789:Enzyme Activation / Q000187:drug effects; D005941:Glucokinase / Q000378:metabolism*; D005990:Glycerol / Q000032:analysis; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010171:Palmitoyl Coenzyme A / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine; D013816:Thermodynamics
D000214:Acyl Coenzyme A; D013439:Sulfhydryl Reagents; D010171:Palmitoyl Coenzyme A; D005941:Glucokinase; D005990:Glycerol