Abstract
The insulin-like activity of proteases was assayed in terms of the glycogen-increasing effect on hemidiaphragms isolated from mice. This effect was observed with Pronase, subtilisin BPN', Sfericase, Dispase II, trypsin, α-chymotrypsin, and elastase, but not with pepsin, kallikrein, or lysozyme. Pronase, a mixed protease preparation from Streptomyces griseus, lost its insulin-like activity on treatment with diisopropyl fluorophosphate (DFP), whereas no loss of activity was observed with 1-chloro-3-tosylamide-7-amino-2-heptanone (TLCK) or L-(1-tosylamide-2-phenyl)-ethyl chloromethyl ketone (TPCK). An insulin-like activity-possessing protease (ILAPP) was partially purified from TLCK and TPCK-pretreated Pronase by affinity chromatography on soybean trypsin inhibitor-conjugated carriers. This enzyme migrated as a single band with a faint sub-band in disc electrophoresis, though it sedimented as a single peak on ultracentrifugal analysis. It hydrolyzed succinyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide and acetyl-L-alanyl-L-alanyl-L-alanine methyl ester at relatively high rates at pH 9, whereas the hydrolytic activities towards casein and elastin-Congo Red were low. It was strongly inhibited by DFP and phenylmethane sulfonyl fluoride (PMSF) but not by ethylenediaminetetraacetic acid, p-chloromercuribenzoic acid, monoiodoacetic acid, N-ethylmaleimide, or dithiothreitol. HgCl2 was inhibitory at a concentration of 10-3M. It is suggested that ILAPP is a DFP- and PMSF-sensitive alkaline protease and that the proteolytic activity of ILAPP is responsible for the insulinlike activity of Pronase.
