Functional studies of yeast-expressed human heart muscle carnitine palmitoyltransferase I.
Long-chain fatty acids are the primary source of energy production in the heart. Carnitine palmitoyltransferase I (CPT-I) catalyzes the first reaction in the transport of long-chain fatty acids from the cytoplasm to the mitochondrion, a rate-limiting step in beta-oxidation. In this study, we report the functional expression of the human heart/skeletal muscle isoform of CPT-I (M-CPT-I) in the yeast Pichia pastoris. Screening of a human heart cDNA library with cDNA fragments encoding the rat heart M-CPT-I resulted in the isolation of a single full-length human heart M-CPT-I cDNA clone. The clone has an open reading frame of 2316 bp with a 5' untranslated region of 38 bp and a 256-bp 3' untranslated region with the poly(A)+ addition sequence AATAAA. The predicted protein has 772 amino acids and a molecular mass of 88 kDa. Northern blot analysis of mRNAs from different human tissues using the human M-CPT-I cDNA as a probe revealed an abundant transcript of approximately 3.1 kb that was only present in human heart and skeletal muscle tissue. Expression of the human M-CPT-I cDNA in P. pastoris, a yeast with no endogenous CPT activity, produced an 80-kDa protein that was located in the mitochondria. Isolated mitochondria from the M-CPT-I expression strain exhibited a malonyl-coenzyme A (CoA)-sensitive CPT activity that was detergent labile. The I50 for malonyl-CoA inhibition of the yeast-expressed M-CPT-I was 69 nM, and the Kms for carnitine and palmitoyl-CoA were 666 and 42 microM, respectively. The I50 for malonyl-CoA inhibition of the heart enzyme is 30 times lower than that of the yeast-expressed liver CPT-I, and the Km for carnitine is more than 20 times higher than that of the liver CPT-I. This is the first report of the expression of a heart CPT-I in a system devoid of endogenous CPT activity and the functional characterization of a human heart M-CPT-I in the absence of the liver isoform and CPT-II.
Zhu
H
H
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000, USA.
Shi
J
J
de Vries
Y
Y
Arvidson
D N
DN
Cregg
J M
JM
Woldegiorgis
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002331:Carnitine / Q000378:metabolism; D002334:Carnitine O-Palmitoyltransferase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008316:Malonyl Coenzyme A / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism
D012333:RNA, Messenger; D011994:Recombinant Proteins; D010171:Palmitoyl Coenzyme A; D008316:Malonyl Coenzyme A; D002334:Carnitine O-Palmitoyltransferase; D002331:Carnitine