Primary structure of porcine pepsin. I. Purification and placement of cyanogen bromide fragments and the amino acid sequence of fragment CB5.

K C Chen,N Tao,J Tang

doi:10.1016/s0021-9258(19)41279-9

Abstract

Fragments resulting from the cyanogen bromide cleavage of reduced and aminoethylated porcine pepsin were purified. Only four of the five fragments theoretically present could be accounted for in major yield when the cyanogen bromide reaction was carried out at room temperature. The NH2-terminal fragment, CB2, contained an internal homoserine which was not cleaved to any significant extent. The amino acid sequence around this internal homoserine was determined by isolating and partially determining the sequence of an alpha-chymotryptic peptide. Cleavage at this methionine was increased by 50% when the cyanogen bromide reaction was carried out at 37 degrees. The NH2- and COOH-terminal sequences of five major fragments were determined. The placement of these fragments in the native pepsin molecule was demonstrated. The amino acid sequence of one of the fragments, CB5, was determined. This fragment contains 44 residues with an internal disulfide bridge. The COOH-terminal methionine of this fragment was connected to another 37-residue cyanogen bromide fragment of known sequence. Together these two fragments formed the COOH-terminal 81 residues of porcine pepsin.

Highlights

From the Laboratory of Protein Studies, Oklahoma Medical Research Foundation, and the Department of Biochemistry and Molecular Biology, University of Oklahoma School of Medicine, Oklahoma City, Oklahoma 73104
Fragments resulting from the cyanogen bromide cleavage of reduced and aminoethylated porcine pepsin were purified
The NH,terminal fragment, CB2, contained an internal homoserine which was not cleaved to any significant extent

Summary

Introduction

The amino acid sequence around this internal homoserine was determined by isolating and partially determining the sequence of an a-chymotryptic peptide Cleavage at this methionine was increased by. The amino acid sequence of one of the fragments, CB5, was determined This fragment contains 44 residues with an internal disulfide bridge. The COOH-terminal methionine of this fragment was connected to another 37.residue cyanogen bromide fragment of known sequence. Together these two fragments formed the COOH-terminal 81 residues of porcine pepsin. The enzymic mechanism and the catalytic groups of pepsin, as well as other acidic proteases, must differ strikingly from those of alkaline proteases The latter groups are much better understood, largely due to the available information on their primary and tertiary structures

Methods

Results

Discussion

Conclusion