Abstract
Two cytochrome P-450 preparations, a constitutive isozyme, form 3b, and a phenobarbital-induced isozyme, form 2, were isolated from rabbit liver microsomes and compared by peptide mapping following digestion with trypsin and by partial sequence analysis. The NH2-terminal sequence of form 3b differed from form 2 in 15 out of 18 amino acids, but both forms have an NH2-terminal methionine residue followed by an acidic residue. Comparisons of many of the tryptic peptides of the two forms by means of high pressure liquid chromatography, as well as amino acid composition and sequence analysis, indicated that peptides from these forms, with one exception, are different. A tridecapeptide, differing only in a methionine (form 3b)/isoleucine (form 2) replacement was isolated from both forms. The amino acid sequence of this peptide is as follows: Met-Pro-Tyr-Thr-Asp-Ala-Val-Ile/Met-His-Glu-Ile-Gln-Arg. Taken together, these data indicate that forms 2 and 3 represent dissimilar gene products. The observation that these two cytochromes share an analogous peptide suggests that this tridecapeptide may contribute structural information necessary for common functional properties.
Highlights
The manner of preparationand characterization of the cytochromes, form 2 [13] and form 3b’ ( l l ), are given in the references
Prior to trypsin digestion or NH2-terminal amino acid sequence analysis, cytochrome P-450 samples were dialyzedagainst 0.2 M NH4acetate, pH 7.4, and lyophilized.Trypsin digestion of cytochromes P450 was performed as previously described [14].The lyophilized proteins were dissolved in 8 M urea, 2 mM NH4HC03 buffer, pH 8.1, and the urea concentration was reduced to 2 M by the addition of water
These contrasting properties of the electrophoretically distinct forms of cytochrome P-450 could conceivably arise as the result of post-translational modifications of a single gene product or through the expression of specific genes codingfor the individual cytochromes
Summary
Cation [11].it will be designated as 3b in the present paper since it appears to be similar to LMsI, described by Koop and Coon [12] as determined by direct comparison in our two laboratories and in order to distinguish it from other forms which have been designated as 3,e.g. LM3, [28]. Phenylthiohydantoin derivatives of amino acids were identified by reverse phase chromatography on a C-18 column using a 5-45% linear gradient of 0.1% aqueous acetic acid/ methanol. High performance liquid chromatography on an alkylphenyl column using a 1844% linear gradient of 0.06% aqueous propionic acid/methanol was used to identify those phenylthiohydantoin derivatives not resolved on the ‘2-18column. Seryl and threonyl phenylthiohydantoins were identified by amino acid analysis after back conversion to alanine or a-aminobutyric acid by hydrolysis in hydroiodic acid vapor ( 14)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
