Functional characterization of the human heart skeletal muscle isoform of carnitine palmitoyltransferase i expressed in the yeast pichia pastor1s

Carnitine palmitoyltransferase I (CPTI) catalyzes the first reaction in the transport of long chain fatty acids from the cytoplasm to the mitochondrion, a rate limiting step in 0-oxidation. Screening of a human heart cDNA library with PCR-amplified cDNA fragments of the rat heart M-CPTI cDNA resulted in isolation of a single full-length clone for human heart MCPTI cDNA with an open reading frame of 2316 bp. The predicted protein contains 772 amino acids and encodes a protein of 88 kTJa. Northern blot analysis of mRNA from different human tissues, using a fragment of the human heart M-CPTI cDNA as a probe, revealed an abundant transcript of - 3.1 kb that was only expressed in human heart and skeletal muscle. Human heart M-CPTI was expressed in Pichia pastoris that has no endogenous CPT activity, and the expressed protein was located in the mitochondria. Isolated mitochondria from the CPTI expression strain, but not the control strain, exhibited malonyl-CoA-sensitive CPT activity that was detergent labile. The I50 for malonyl-CoA inhibition of the yeastexpressed M-CPTI was 69 nM and the Kms for carnitine and palmitoyl CoA were 666 -4M and 42 -4M, respectively. This is the first report of functional and kinetic characterization of a human heart skeletal muscle isoform MCPTI in the absence of the liver isoform, L-CPTI, and CPTII.