Inhibition of pepsin by zymogen activation fragments. Spectrum of peptides released from pepsinogen NH2 terminus and solid phase synthesis of two inhibitory peptide sequences.

Exposure of porcine pepsinogen to controlled conditions of pH and temperature led to the time-dependent release of several peptide fragments totaling 44 amino acid residues from the NH2 terminus with formation of active pepsin.The activation process was quenched at various times by an increase in pH and the protein was removed by passage through DEAE-cellulose to give a mixture of activation fragments.Fractionation of these peptides on CM-cellulose or CM-Sepharose yielded a set of purified peptides for each set of activation conditions.The appearance of specific "early" products was noted for the first time.The isolated peptides were tested as inhibitors of pepsin catalysis by a milk clotting assay.The peptide corresponding to sequence 1-16 of the zymogen (peptide (1-16)) was found to have the strongest interaction with pepsin at pH 5.5.Other peptides also inhibit, and & values are derived for each from plots of per cent inhibition uersus inhibitor concentration.Two of the peptides have been synthesized by solid phase procedures.The activity of synthetic peptide (1-16) and synthetic peptide (25-41) was equivalent to that of the corresponding natural peptide.In order to assess the importance of various functional groups in peptide (l-16), chemical modification studies were carried out.Reaction of 1,2+yclohexanedione with the arginine residues gave a derivative that, after purification, retained its full capacity to inhibit pepsin.Since this derivative will retain a charge at pH 6.6, either charge is a more important requirement for inhibition than size or the arginine residues are not essential for the interaction. Catalyticallyinert porcine pepsinogen is converted to enzymatically active pepsin by loss of the first 44 NH&xrninal residues.Leu-Val-Lys-Val-Pro-Leu-Val-Arg-Lys-Lys-10 Ser-Leu-Arg-Gln-Asn-Leu-Ile-Lys-Asp-Gly-20 Lys-Leu-Lys-Asp-Phe-Leu-Lys-Thr-His-Lys-30 His-Asn-Pro-Ala-Ser-Lys-Tyr-Phe-Pro-Glu-Ala-Ala-Ala-Leu-pepsin 44This highly basic sequence (9 Lys, 2 Arg, 2 His) most likely