Characterization of rat cytochrome P-450MC synthesized in Saccharomyces cerevisiae.
Rat cytochrome P-450MC cDNA was expressed in Saccharomyces cerevisiae AH22, SHY3 and NA87-11A cells under the control of the yeast ADH1 promoter and terminator. Although the three yeast strains transformed with the constructed expression plasmid, pAMC1, contained approximately three copies of the plasmid, the levels of both P-450MC mRNA and the corresponding protein in the AH22 cells carrying plasmid pAMC1 were 1.4- to 1.7-fold and 2-fold higher than in the other two strains, respectively. The P-450MC protein was purified from the microsomal fraction of AH22 cells carrying pAMC1 by a rapid purification method. The apparent molecular weight, chromatographic behavior, spectral properties, substrate specificity and immunochemical properties of the purified P-450MC protein were indistinguishable from those of rat liver P-450MC-I and P-450MC-II (Sasaki, T., et al. (1984) J. Biochem. 96, 117-126). The NH2-terminal amino acid sequence of the purified protein up to 10 residues was the same as those of P-450MC-I and P-450MC-II. In addition, HPLC analysis of the microsomal fraction of AH22 cells containing pAMC1 indicated that the synthesized P-450MC protein corresponds to P-450MC-II, but not P-450MC-I. With another purification method, we obtained the cleaved P-450MC protein which lacked the NH2-terminal 30 amino acids of intact P-450MC. The spectral properties and monooxygenase activities towards benzo(a)pyrene and 7-ethoxycoumarin of the cleaved P-450MC were nearly the same as those of intact P-450MC.
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification; D008748:Methylcholanthrene; D008970:Molecular Weight; D010957:Plasmids; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013057:Spectrum Analysis; D013379:Substrate Specificity
D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System