Activation of proteinases with trypsin-like specificity from bovine and human lenses

Abstract

Proteinases with apparent trypsin-like substrate specificity were present in extracts prepared from both bovine and human lenses. The enzymes from both sources required an incubation period of 10–25 hr at 34°C in the presence of 60 m m -NaCl or 5 m m -MgCl2 before any hydrolytic activity could be observed. Assays for BAPNA hydrolysis by bovine lens cortical extracts using this assay system showed one pH optimum at pH 6·5 and another at pH 8·0. The pH 8·0 activity was separated from the pH 6·5 activity by precipitation at pH 5·0. The precipitate, α1 crystallin, contained the pH 8·0 activity and still required an incubation period of 10–15 hr to activate the enzyme. Fractionation of bovine lens α1 crystallin by Agarose A-1·5 m gel filtration separated the pH 8·0 activity into three peaks eluting at molecular weights of 25000, 70000 and 900000 daltons. Fractionation of human lens cortical extracts by Agarose A-1·5 m gel filtration showed two peaks of pH 8·0 activity eluting at molecular weights of 25000 and 900000 daltons. Bovine lens α1 crystallin was activated by incubation with either magnesium or calcium at a concentration of 5 m m or sodium or potassium at 100 m m . No activity was induced, however, when extracts were incubated in the presence of either 5 m m -dithiothreitol or 1 m m -EDTA. These reagents appear to inhibit the activation of the enzyme, since fully active enzyme was no longer sensitive to either of these compounds. The activated enzyme has trypsin-like specificity, but was not inhibited by any of the classical trypsin inhibitors except benzamidine. This enzyme is clearly distinct from the bovine lens neutral proteinase and has not been reported previously, presumably because it exists in normal lens cortex in an inactive or inhibited state. The data suggest that this enzyme is active only in the presence of high cation levels and in the absence of thiol reducing agents. This situation, while not present in normal lens, does occur during cataract formation.