Intramolecular Activation of Porcine Pepsinogen

Abstract

Conversion of pepsinogen to pepsin at acid pH involves an intramolecular reaction in which the unproteolyzed zymogen cleaves itself. This conclusion is based upon experiments in which pepsinogen, at low concentrations, was activated in the presence of substrate, hemoglobin. Under these conditions, the activation of pepsinogen is independent of pepsinogen concentration, and addition of pepsin does not enhance the rate of autoactivation. The activity of Sepharose-bound pepsinogen was independent of the average amount of pepsinogen bound per g of Sepharose, suggesting that the activation process is not caused by neighboring molecules mutually activating. Apparently, in more concentrated solutions that were previously employed for autoactivation, the concentration of pepsinogen was such that the reaction could be catalyzed by one of the products (an autocatalytic reaction). When the activation process is performed at low pepsinogen concentration in the presence of substrate, or in columns of Sepharose-bound pepsinogen, the results indicate that reaction between a trace of pepsin and pepsinogen or between molecules of pepsinogen is not a prerequisite for activation. Amino-terminal studies on Sepharose pepsinogen yielded leucine as the amino-terminal acid. Exposure of the Sepharose pepsinogen to acid yielded leucine and isoleucine as amino-terminal amino acids. Treatment of base-denatured pepsinogen with acid failed to yield isoleucine as the new amino-terminal. These results suggest that cleavage of the peptide bond which results in exposure of a new amino terminus involves an intramolecular enzymatic process. Precipitin tests with antipepsinogen and antipepsin sera which had been passed through Sepharose-pepsinogen columns exposed to various conditions indicated that Sepharose-bound pepsinogen retained its antigenic determinants upon conversion to pepsin.