tosylarginine-methyl-ester and benzamidine

Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.

Topics: Ammonium Sulfate; Animals; Benzamidines; California; Cecum; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Enzyme Inhibitors; Enzyme Stability; Fishes; Pacific Ocean; Phenylmethylsulfonyl Fluoride; Pylorus; Tosylarginine Methyl Ester; Trypsin; Trypsin Inhibitor, Kunitz Soybean

Evidence from a number of laboratories has suggested that the mechanism of insulin action involves the release of an intracellular mediator polypeptide from the plasma membrane. It has been proposed that activation of a protease with trypsin-like specificity is involved in release of the putative mediator. In an effort to assess the potential role of such a protease in intact cells, the present study tested the effects of a variety of low-mol-wt protease inhibitors on insulin's metabolic action in isolated rat epididymal fat cells. The protease inhibitors studied included p-aminobenzamidine, benzamidine, phenylguanidine, diisopropylfluorophosphate, leupeptin, and the competitive substrate N-alpha-tosyl-L-arginine methylester. Leupeptin was devoid of activity. Most of the other inhibitors used were able to interfere with insulin-stimulated metabolism if used in sufficiently high concentrations, concentrations considerably higher than those required for inhibition of known proteases or inhibition of intracellular processes in a previously described system which involves a trypsin-like enzyme. Moreover, they displayed various activities unrelated to protease inhibition that could explain their effects on insulin action better than protease inhibition. While none of the data on individual inhibitors were by themselves convincing enough to either confirm or reject the hypothesis concerning the involvement of a protease with trypsin-like specificity in insulin action, taken together our results do weaken the hypothesis considerably and in particular render the involvement of an extracellular trypsin-like enzyme improbable.

Topics: Adipose Tissue; Animals; Benzamidines; Glucose; Guanidines; In Vitro Techniques; Insulin; Insulin Antagonists; Isoflurophate; Leupeptins; Molecular Weight; Rats; Tosylarginine Methyl Ester; Trypsin Inhibitors