The membrane topology of the mammalian P450 cytochromes has been studied intensively by computational approaches, proteolysis, chemical modification, genetic engineering, and immunochemistry. Initial results for the cytochromes of the endoplasmic reticulum appeared to indicate a polytopic, four to eight transmembrane anchor model with an active site buried in the membrane. However, recent findings show that the microsomal P450s are bound to the endoplasmic reticulum by only one or two transmembrane peptides located at the NH2-terminal end, and that the active site is part of a large cytoplasmic domain that may have one or two additional peripheral membrane contacts. The membrane-bound state is viewed as rather rigid, and the plane of the heme lies between perpendicular and parallel to the plane of the endoplasmic reticulum. The mitochondrial P450 cytochromes lack a hydrophobic NH2 terminus in the mature form, and thus differ from the microsomal isozymes in this significant way. However, although the exact topology of cytochrome P450 in the inner mitochondrial membrane remains to be elucidated, certain features are clearly comparable to those of microsomal P450. Therefore, the membrane topology of the P450 gene superfamily may follow a similar pattern.
The complete amino acid sequence of phenobarbital-induced isozyme 2 of rabbit liver microsomal cytochrome P450 (P-450LM2) is presented. The polypeptide consists of 491 residues with a calculated Mr of 55,755. The rabbit isozyme is 77% identical to the corresponding rat cytochrome, P-450b, as deduced from eDNA, with 96% of the hydrophobic, 88% of the anionic, and 83% of the cationic positions conserved. The secondary structure of isozyme 2 was predicted and a model was developed for the membrane topology of this cytochrome. Of the two highly conserved cysteinyl peptides in P-45OLM2, P-450b, and bacterial P- After the isolation of P-450LM2* in an electrophoretically homogeneous state (1), we reported that it contained a single NH2-terminal amino acid sequence (2). The composition and sequence of this region provided an example of a mature protein having retained a "signal peptide" of the type ordinarily removed from preproteins by biological processing (3). More recently, we reported additional sequence data on rabbit P-450 isozyme 2 (4), which showed that it is about 80% identical to the corresponding rat protein, P-450b, the sequence of which was deduced from cloned cDNA (5), and we compared highly conserved cysteine-containing regions from rabbit isozyme 2, rat isozyme b, and P-450C,, from Pseudomonas putida (6).We report here the complete amino acid sequence of P-450LM2, along with methodology that should be generally effective with other hydrophobic proteins. We include a correction to our previous report (4), in which Cys-436 was mistakenly identified as Glu, and offer compelling evidence for the identity of positions and residues at variance with the conclusions of Heinemann and Ozols (7) Chemical Modification. In most cases, prior to protein fragmentation cysteine residues were reduced with 3 equivalents of dithiothreitol in 6 M guanidinium chloride/0.5 M N-ethylmorpholinium acetate, pH 8.2, and alkylated with a 1.4-fold excess of 4-vinylpyridine (8), with both reactions at 220C for 4 hr. Several reagents were tested to provide stable groups to block enzymic digestion at Lys residues. The best results were obtained with acid anhydrides, which were added to a solution or fine suspension of the intact protein or fragment in N-methylmorpholine/H20, 1:1 at 00C, followed by stirring at 22TC for 1 hr. The benzenetricarboxylic derivative of isozyme 2 was by far the most soluble at neutral pH, so it was used for the earlier tryptic digestions. Reaction with succinic anhydride tended to produce a more uniform product and a more easily analyzed Lys derivative, so it was employed for most of the later studies. With smaller peptides or when neutral solubility was unimportant, acetic anhydride was most convenient.Digestion. Standard methods were used for cleavage with CNBr, trypsin, chymotrypsin, S. aureus protease (9), proteinase A (10), and clostripain (11). For digestion with endoproteinase Lys-C an enzyme-to-substrate ratio of 1:50 (wt/wt) was used and the mixture was incubated in 0. (10 ,m, 3.9 X 300 ...
The native molecular weight of affinity-purified cytochrome P450 102 from barbiturate-induced Bacillus megaterium has been studied by sedimentation methods and HPLC size-exclusion chromatography. Sedimentation velocity experiments yielded an s020,w = 9.244 S for the holocytochrome, but the diffusion coefficient was unexpectedly large and varied widely with centrifugal field, ionic strength, and protein concentration. Addition of 50 mM DL-dithiothreitol (DTT) caused a small decrease in the value of s020,w, but D20 still did not behave as expected. The sedimentation coefficients were consistent with a molecular weight of about 200,000, and the diffusion coefficients indicated molecular aggregation. Sedimentation equilibrium analyses suggested that the native enzyme was a mixture of monomer, dimer, trimer, and tetramer. However, after incubation of P450 102 with DTT, sedimentation equilibrium demonstrated that the enzyme was dimeric (molecular weight 236,000). HPLC size-exclusion chromatography of the cytochrome showed the presence of four peaks, which corresponded to 1.45-mer, 2.06-mer, 3.02-mer, and a higher molecular weight fraction; aggregated forms accounted for about 52% of the P450 102. Incubation of the enzyme with DTT caused a shift toward the 1.45-mer, but dimer, trimer, and the high molecular weight peak still persisted; the shift was not attributable to disulfide bond reduction. The 1.45-mer was determined to be a monomeric species of significantly asymmetric geometry. Together, the results indicated that cytochrome P450 exists with monomer, dimer, trimer, etc. in equilibrium, contrary to the expectation that this soluble P450 would be monomeric.(ABSTRACT TRUNCATED AT 250 WORDS)
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