THE ISOLATION AND COMPARISON OF MULTIPLE FORMS OF CYP2B FROM UNTREATED AND PHENOBARBITAL-TREATED RABBIT LIVER-MICROSOMES

At low levels of phenobarbital induction two forms of isoenzyme 2 (LM2 ; CYP2B4) were obtained during purification of cytochrome P450 from ra bbit liver microsomes. At high levels of induction only one form (LM2A ) was present. Although the two purified forms (LMS2 and LM2B) were ve ry similar they differed in: (a) peak elution on CM-Sepharose, (b) wav elength maximum of the reduced P450-CO spectrum, and (c) metabolism of several substrates, where the activities of LM2B ranged from 0.6 to 2 .65 times that of LM2A. A third LM2 fraction (2C) was isolated from un treated rabbit liver and, although homogenous on sodium dodecyl sulfat e-polyacrylamide gel electrophoresis, appeared to be a mixture of LM2B and a form of P450 LM2 other than LM2A. LM2A was not found in the unt reated rabbit liver microsomes. On CM-Sepharose the elution of fractio n 2C overlapped that of LM2B. The apparent molecular weight and immuno response to anti-LM2A IgG were the same for fraction 2C as for LM2A an d LM2B. Peptide mapping using trypsin showed no difference between LM2 A and LM2B, but consistently revealed at least one extra band with fra ction 2C. After CNBr cleavage and high-pressure liquid chromatography separation of the LM2A and LM2B fragments the peptide beginning with P ro(347) of LM2A (peak 4A) eluted 1/2 min later than that of LM2B (peak 4B) indicating a difference in the fragments, although partial NH2-te rminal amino acid sequences and molecular masses were the same. The co rresponding CNBr fragment of fraction 2C splits into two peaks (4C:1 a nd 4C:2) with retention times corresponding to 4B and 4A, respectively . The mass of 4C:1. was the same as that of 4B, while the mass of 4C:2 markedly differed from that of 4A and 4B. Both fragments had the same partial NH2-terminal amino acid sequence as 4A and 4B. After comparin g the physicochemical properties as well as catalytic activities of th ese isolated and purified LM2 forms with the cDNA-expressed forms 2B-B 0, 2B-B1, 2B-B2, and 2B-Bx [see R. Ryan ct al. (1993) Arch. Biochem. B iophys. 304, 454-463], the data suggest that LM2A is the form designat ed as 2B-B0 (LM2), LM2B is 2B-Bx, and fraction 2C is a mixture contain ing 2B-B1 and 2B-Bx. This is the first isolation and identification of the three isozymic LM2 proteins from rabbit liver. (C) 1995 Academic Press, Inc.