Characterization of active derivatives produced by acetamidination and selective autolysis of bovine trypsin.
A commercial preparation of bovine trypsin was treated with methyl acetimidate-HCl, and most of the lysine residues were converted to trypsin-resistant residues retaining their cationic charges. The modified preparation was then fractionated by ion-exchange chromatography on SE-Sephadex C-50 into two active components, amidinated alpha- and amidinated beta-trypsins. The latter component (Am-beta-trypsin), which consisted of a single polypeptide chain, was allowed to autolyze at pH 7.8, 25 degrees C for 3.5 h and a new active component named Am-delta-trypsin was isolated from the autolysate. Several lines of experimental evidence indicated that Am-delta-trypsin was derived by primary cleavage of the bond Arg105-Val106. Cleavage at Arg55-Leu56, on the other hand, appeared to lead to inactivation of Am-beta-trypsin. The kinetic properties of the catalytic hydrolyses of synthetic substrates and the affinity to Gly-Gly-Arg immobilized on Sepharose were compared among Am-delta-, Am-beta-, and Am-alpha-trypsins. Am-delta-trypsin resembled Am-beta-trypsin in these properties, but did not resemble Am-alpha-trypsin which had a cleavage at Lys131-Ser132.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D004950:Esterases / Q000378:metabolism; D006868:Hydrolysis; D007097:Imines; D007202:Indicators and Reagents; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D013379:Substrate Specificity; D013820:Thermolysin; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D007097:Imines; D007202:Indicators and Reagents; D004950:Esterases; D014357:Trypsin; D013820:Thermolysin