Effects of octylglucoside and triton X-100 on the kinetics and specificity of carnitine palmitoyltransferase.
The effects of octylglucoside on the substrate specificity, kinetics and aggregation state of purified carnitine palmitoyltransferase (CPT) from beef heart mitochondria were investigated and compared to the effects of Triton X-100. Conditions in which CPT can be assayed in the absence of micelles and albumin, thereby eliminating miceller effects on the kinetic parameters, are described. When octylglucoside is substituted for Triton X-100, the specificity of CPT in the forward direction shifts towards the long-chain acyl-CoAs, and large changes in the kinetic constants are observed. The K0.5 for L-carnitine varied as much as 50-fold, depending on the acyl-CoA and detergent used. At pH 8.0 and 200 microM palmitoyl-CoA, the K0.5 for L-carnitine is 4.9 mM in 12 mM octylglucoside and 0.2 mM in 0.1% Triton X-100. Octylglucoside enhances the activity of CPT with long-chain acyl-CoA and lowers the K0.5 for these substrates. At pH 6.0, the K0.5 for palmitoyl-CoA is 24.2 microM in 0.1% Triton X-100, in contrast to 3.1 microM in 12 mM octylglucoside. Octylglucoside is a competitive inhibitor of CPT with octanoyl-CoA as substrate with a Ki of 15 mM. Nonlinear kinetics for both acyl-CoAs and L-carnitine are observed when the concentration of octylglucoside is reduced to less than half of its critical micellar concentration (cmc). Gel filtration of CPT in octylglucoside below its cmc gives a single protein peak with a molecular mass of ca. 660,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)
Fiol
C J
CJ
Biochemistry Department, Michigan State University, East Lansing 48824.
Bieber
L L
LL
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism*; D002417:Cattle; D003902:Detergents / Q000494:pharmacology*; D005960:Glucosides / Q000494:pharmacology*; D006027:Glycosides / Q000494:pharmacology*; D007700:Kinetics; D008823:Micelles; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology*; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology*
D000214:Acyl Coenzyme A; D003902:Detergents; D005960:Glucosides; D006027:Glycosides; D008823:Micelles; D013501:Surface-Active Agents; C018619:octyl-beta-D-glucoside; D011092:Polyethylene Glycols; D017830:Octoxynol; D000217:Acyltransferases; D002334:Carnitine O-Palmitoyltransferase