CHICKEN PEPSIN: STRUCTURE AND HOMOLOGY WITH OTHER ACID PROTEINASES

Abstract

The amino acid sequence of chicken pepsin (CP) has been studied to provide a basis for the elucidation of (a) the catalytic mechanism of CP at the molecular level, and (b) the evolutionary relationship of CP with other carboxyl proteinases. For more efficient. preparation of the enzyme CP was isolated directly from acid extracts of chicken proventriculi mucosa by covalent and affinity chromatography; these procedures were based on the coupling of CP either via its SH-group to mercurial Sepharose or to Sepharose with immobilized A. lumbricoides pepsin inhibitor as affinant. The latter support was used to advantage also for affinity chromatography purification of other carboxyl proteinases. As starting material for sequence studies the zymogen (CPG) was isolated by anion exchange chromatography of alkaline extracts of the mucosa. The process of CPG activation to CP starts (at the amino acid sequence level) by scission of the bond between Phe (26) and Leu (27). The 26-residue N-terminal activation peptide released inhibits to different degrees CP, calf chymosin, and hog pepsin. Sequence studies carried out by automatic degradation of CPG and CP derivatives and by the classical approach based on overlapping peptides permitted a partial structure of CPG to be derived. The latter accounts for 322 residues arranged in 19 segments of interchangeable order. The alignment of this structure with those of hog pepsinogen and calf prochymosin shows sequential homologies in the terminal parts of the molecule, around half-cystines and the reactive aspartic acid residues.