Activation and release of a trypsin-like proteinase from bovine lens α-crystallin

Abstract

Trypsin-like activity was demonstrated in extracts of lens outer cortex by measuring the hydrolysis of α-N-benzoyl-arginine- p -nitroanilide. This activity had a sharp temperature optimum at 34°C, two pH optima at 6·–6·7 and 8·0, and required an incubation period of 15–20 hr to activate the enzyme. The capsule-epithelium fraction had the highest specific activity while nuclear extracts had the lowest. Bovine lens outer cortical extract was fractionated by Agarose A-1·5 m chromatography into three peaks of enzyme activity with molecular weights of 2·5×10 4 (BAPNAase I), 6−7×10 4 (BAPNAase II) and 7−9×10 5 (BAPNAase III). The isolated BAPNAase III peak required an activation period of about 20hr before enzyme activity could be demonstrated. This activation required the presence of both 5 m m -MgCl 2 and 60 m m -NaCl and resulted in a partial inactivation of the endogenous trypsin inhibitor at the time the BAPNAase activity was induced. After a 40 hr incubation period BAPNAase III samples showed the release of a low molecular weight proteinase and the cleavage of α-crystallin into polypeptides of lower molecular weight. The released proteinase had trypsin-like specificity, hydrolyzed the protein substrate, azocoll, and was inhibited by benzamidine and sulfhydryl blocking agents. These data suggest that BAPNAase III represents a complex between a trypsin-like enzyme and the endogenous lens inhibitor, TI H . A 20 hr incubation in the presence of high cation levels results in the release of the bound proteinase which then chromatographs in the region of BAPNAase I.