Cloning, sequencing, and expression of a cDNA encoding rat liver mitochondrial carnitine palmitoyltransferase II.

摘要: We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase II (CPT II). Beginning with purified protein CNBr fragments were generated sequenced. Corresponding oligonucleotides used to screen library constructed in plasmid cloning vector, pcDV. The clone ultimately obtained consisted 62 nucleotide 5'-untranslated region, single open reading frame 1,974 bases predicting 658 amino acids (Mr = 74,119), 3'-untranslated segment 260 nucleotides followed by poly (A) tail. identity was confirmed findings that (a) encoded all three peptides found original protein; (b) fourth peptide synthesized from portion deduced acid sequence immunize rabbit resulted generation an antibody recognized pure CPT on Western blot; (c) vitro transcription translation (ligated into pBlue-script KS (+] specifically immunoprecipitated anti-CPT having Mr slightly greater than mature II; (d) transfection COS cells subcloned expression pCMV4, 6-fold induction mitochondrial catalytic activity. It seems likely de novo enzyme gains entry mitochondrion via targeting is subsequently cleaved. probably associates (relatively loosely) inner membrane through limited number spanning domains. predicted shows strong those two other acyltransferases, namely, peroxisomal octanoyltransferase porcine choline acetyltransferase.