The general purpose of this project is to understand primary structure and the mechanism of proteolytic degradation of membranous hepatic cytochromes. Among the specific objectives of this study is the characterization of the protease responsible for cleaving the membranous segment of cytochrome b5 at some 13 residues from the COOH terminus of the molecule, yielding preparations that bind to the liposome vesicles. The protease is present in detergent extracts of hepatic microsomes and is functional at pH 8.0 when non-ionic detergent concentration is above 2.7%. Because of the high specificity of this cleavage it is unlikely that we are dealing with nonspecific lysosomal proteinases. We will continue the primary structure studies on the phenobarbital, methlycholanthrene and tetrachlorodibenzo-p-dioxin induced forms of cytochrome P-450. To date I have isolated and determined the complete primary structure of cytochrome b5 from four different animal species. An effective methodology has been developed for cleaving the membranous segment into large fragments suitable for automated sequence analysis. Such an approach will be extended to the cytochrome P-450 preparations. Using cytochrome b5 as the model protein we designed experiments which identify the peptide segments that are embedded in the membrane. In these experiments cytochrome b5 of known primary structure is bound to synthetic vesicles on a preparative scale and the vesicles are treated with proteolytic enzymes. Automated sequence analysis of partially delipidated vesicles clearly identified the membranous segment of the molecule and demonstrated that membranous segment of cytochrome b5, in a liposome, is resistant to tryptic or chymotryptic digestion. Similar experiments will be extended to cytochrome P-450 preparations. Such studies, together with sequence data of these cytochromes and their reductases, will provide a basis for designing experiments which will increase our understanding membrane structure and function.