The effect of palmitoyl-CoA binding to albumin on the apparent kinetic behavior of carnitine palmitoyltransferase I.
Substrate saturation plots of carnitine palmitoyltransferase I activity from isolated rat liver mitochondria vs. palmitoyl-CoA concentration in the presence of bovine serum albumin have been reported to yield sigmoidal kinetics. Under identical assay conditions we have confirmed these observations as reflected by nonlinear Lineweaver-Burke plots (1/vi vs. 1/[S]) an average Hill coefficient of napp. = 1.98 +/- 0.09 (Mean +/- S.E. from four separate experiments). For these determinations the enzyme activity was plotted against the total [palmitoyl-CoA] in the presence of 0.13% bovine serum albumin. Utilizing the total [palmitoyl-CoA] to determine the kinetic properties of carnitine palmitoyltransferase I would be valid only if the relationship between total and free [palmitoyl-CoA] was linear, which is not the case as we have previously shown. When carnitine palmitoyltransferase I substrate saturation kinetics were reanalyzed using the previously determined free [palmitoyl-CoA]'s, the plots revealed a shift to standard hyperbolic kinetics. This observation was confirmed by an average Hill coefficient of napp. = 1.04 +/- 0.10 (Mean +/- S.E.) and linear Lineweaver-Burke plots. The double-reciprocal plots from these analyses yielded an average S0.5 of 2.55 +/- 0.82 microM (Mean +/- S.E.) palmitoyl-CoA and Vmax of 19.69 +/- 5.48 nmol/min per mg protein. These studies clearly demonstrate the importance of defining the free [palmitoyl-CoA] when analyzing the kinetics of carnitine palmitoyltransferase I in the presence of bovine serum albumin.
Richards
E W
EW
Department of Research, Baptist Medical Centers, Birmingham, AL 35211.
Hamm
M W
MW
Otto
D A
DA
D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D013379:Substrate Specificity
D010171:Palmitoyl Coenzyme A; D012710:Serum Albumin, Bovine; D002334:Carnitine O-Palmitoyltransferase