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. 1984 Feb;114(2):250–263.

A high-molecular-weight trypsinlike protease in the skin sites of delayed hypersensitivity in guinea pigs.

T Nakamura, T Yamamoto, T Ishimatsu, T Kambara
PMCID: PMC1900333  PMID: 6198919

Abstract

A trypsinlike protease was extracted from the delayed hypersensitivity skin sites in guinea pigs. Extractable amounts of the enzyme were chronologically paralleled with the gross appearance of the inflammation, and the maximum activity from the inflamed sites at 24-36 hours was about 20 times stronger than that from normal skin, suggesting a potential role in the pathogenesis of the delayed hypersensitivity reaction. The enzyme, which suitably hydrolyzed t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide, was partially purified by isoelectric focusing or by gel filtration. The enzyme demonstrated a single peak of activity on the former column with an apparent isoelectric point of 4.2, and in the latter it showed an apparent molecular weight of 600,000 (600K-protease). When incubated with 3H-diisopropylfluorophosphate, the enzyme lost all amidolytic activity and yielded a single band of radioactivity in polyacrylamide disk gel electrophoresis in the presence of sodium dodecyl sulfate, and a single peak of radioactivity in gel filtration, both having an apparent molecular weight of 31,000-33,000 (31K-protease). That the 600K-protease might be a complex with alpha 2-macroglobulin was ruled out. The 31K-protease was separated from the 600K-protease by gel filtration in the presence of 6 M guanidine hydrochloride, and was renatured to an active form. An apparent isoelectric point of the 31K-protease observed was 9.4, suggesting that the 600K-protease may be a complex of 31K-protease with an acidic carrier molecule(s). Both proteases, 31K- and 600K-protease, had identical substrate specificity, a pH profile of amidolytic activity, and susceptibility to exogenous protease inhibitors. However, when sensitivities to intrinsic protease inhibitors in guinea pig plasma, two kinds of trypsin inhibitor, and alpha 2-macroglobulin were compared, the 600K-protease was at least 100 times more resistant than the 31K-protease. It was supposed that one of the pathophysiologically significant functions of the complex formation might be to maintain the enzyme activity longer in vivo.

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Selected References

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