Importance of acyl-CoA availability in interpretation of carnitine palmitoyltransferase I kinetics.
Bovine serum albumin is generally employed as a substrate depot for the delivery of acyl units to lipid metabolizing enzymes in vitro. Here we test the possibility that albumin alters the availability of substrate to mitochondrial carnitine palmitoyltransferase I and thereby alters its apparent kinetics. Binding competition with palmitoyl-CoA indicates that albumin has 5-6 high affinity sites which avidly bind the substrate, while isolated mitochondria compete favorably for substrate only as the albumin sites become saturated. In contrast to albumin, artificial phospholipid vesicles bind palmitoyl-CoA uniformly. Palmitoyl-CoA distribution between vesicles and mitochondrial membranes appears simply to be a function of the relative size of the two lipid compartments. Both albumin and artificial vesicles reduce the effective concentration of substrate available to the enzyme and in this way reduce apparent affinity. Direct measurement of mitochondrially bound substrate removes this effect and brings the results into agreement with an affinity constant of 6-7 nmol/mg. Changes in gross mitochondrial structure, as indicated by decreased optical density and increased nonpelleting protein, do not begin occurring until levels of mitochondrially bound palmitoyl-CoA are 15 times greater than this. The highly sigmoidal activity profile of carnitine palmitoyltransferase with respect to palmitoyl-CoA (apparent Hill coefficient = 3.0 +/- 0.3) is lost when vesicles are substituted for albumin, suggesting that albumin binding sites contribute to the sigmoidal kinetics in the range of palmitoyl-CoA studied.
Pauly
D F
DF
Department of Medicine, University of Alabama, Birmingham 35294.
McMillin
J B
JB
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D017830:Octoxynol; D010171:Palmitoyl Coenzyme A / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013367:Submitochondrial Particles / Q000201:enzymology
D000214:Acyl Coenzyme A; D003902:Detergents; D010171:Palmitoyl Coenzyme A; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D017830:Octoxynol; D000217:Acyltransferases; D002334:Carnitine O-Palmitoyltransferase