| Popis: |
Isolation and purification of trypsin-like proteases from the pyloric caeca of the starfish Dermasterias imbricata resulted in the separation of two different proteins which possessed tryptic-like activity. Both enzymes were homogeneous as judged by polyacrylamide electrophoresis and both have molecular weights of 25,000 to 26,000 as determined by gel filtration. The enzymes were inhibited by diisopropyl phosphorofluoridate and Nα-tosyl-l-lysylchloromethane, but not by soybean trypsin inhibitor. Enzyme stability, as affected by temperature and pH, showed marked differences between the two enzymes. Hydrolysis of benzoyl-dl-arginine-p-nitroanilide hydrochloride by each enzyme was optimal at pH 8.0 to 8.5. The starfish enzymes hydrolyze this synthetic substrate at rates 3.2- and 1.2-fold faster than bovine pancreatic trypsin does. Proteolytic digestion of glucagon and amino acid analysis of the resulting peptides revealed a cleavage specificity for both starfish proteases similar to that of bovine pancreatic trypsin. The enzymes appear to exist as inactive precursors in the pyloric caeca, with spontaneous activation taking place on incubation at 20°. The activation was accelerated by addition of exogenous pancreatic trypsin and calcium. However, neither of the purified starfish enzymes showed dependence on calcium ion concentration for enzymatic activity or stability and were unaffected by ethylenediaminetetraacetate. |
