Evidence for malonyl-CoA-sensitive carnitine acyl-CoA transferase activity in sarcoplasmic reticulum of canine heart.
The formation of palmitoylcarnitine is catalyzed by carnitine palmitoyl-transferase (CPT-I) and this catalysis is the first committed step in beta-oxidation. The malonyl-CoA-inhibited isoform appears to be distinct from latent (CPT-II) activity, which is localized to the matrix side of the mitochondrial inner membrane. Sarcoplasmic reticulum from canine cardiac muscle was fractionated on a discontinuous sucrose density gradient into three major bands, all of which contained Ca(2+)-ATPase activity. Only the fraction that banded at a concentration of 38% surcrose was slightly contaminated by mitochondria. Peroxisomal uricase was low or absent in fractionated SR. All sarcoplasmic reticulum fractions contained malonyl-CoA-sensitive medium- (COT) and long-chain (CPT) carnitine acyltransferase activities. CPT activity decreased in sarcoplasmic reticulum when Triton X-100 was present. Carnitine acyltransferase activities were inactivated by preincubating the sarcoplasmic reticulum with the sulfhydryl reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). In contrast, mitochondrial CPT-II activity was stable in the presence of DTNB and activated by Triton X-100. Western blots of mitochondria and sarcoplasmic reticulum fractions showed that the mitochondrial fractions reacted with antibody to mitochondrial CPT-II but not with SR protein when both were added at comparable specific activities. The data suggest that cardiac SR contains a unique malonyl-CoA-sensitive isoform of CPT, and that synthesis of acylcarnitine may occur in the microenvironment of Ca2+ transport, where the extent of production of acylcarnitine is controlled by cardiac acetyl-CoA carboxylase activity.
McMillin
J B
JB
Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston 77030.
Hudson
E K
EK
Van Winkle
W B
WB
D000818:Animals; D002333:Carnitine Acyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004285:Dogs; D066298:In Vitro Techniques; D008316:Malonyl Coenzyme A / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013379:Substrate Specificity
D008316:Malonyl Coenzyme A; D002333:Carnitine Acyltransferases