Abstract
The ability of trypsinogen to catalyze its own activation was studied at pH 8.1 in the presence of Ca++ ions. The lag phase portion of the classical S-shaped curve of activation was shortened by increasing the trypsinogen concentration but was extended by pretreatment of the zymogen with diisopropyl fluorophosphate or soybean trypsin inhibitor. Despite these pretreatments, however, full activation was achieved. In confirmation of the proposed catalytic activity of trypsinogen, acetyltrypsinogen, which cannot be activated by trypsin or acetyltrypsin, was able to activate chymotrypsinogen at a rate about 10-5 that of trypsin and to hydrolyze p-toluenesulfonyl arginine methyl ester at a very slow rate. Pretreatment of the acetyltrypsinogen with diisopropyl fluorophosphate and soybean trypsin inhibitor did not affect the rate at which it carried out these two reactions, thus excluding the presence of trypsin or acetyltrypsin. The conclusion is drawn that the activities observed were not due to contamination of the zymogen preparations with active enzyme but were due to an activity inherent in the zymogens. The S-shaped curve of trypsinogen activation is now interpreted as two separate phases: a slow initial formation of trypsin by self-activation of the zymogen, followed by a rapid acceleration of the reaction due to catalysis by the product, trypsin.
Highlights
The ability of trypsinogen to catalyze its own activation was studied at pH 8.1 in the presence of Ca++ ions
The conclusion is drawn that the activities observed were not due to contamination of the zymogen preparations with active enzyme but were due to an activity inherent in the zymogens
A classical S-shaped profile [11] was obtained with an initial lag period during which no tryptic activity with respect to TAMe could be detected. The length of this lag phase increased from approximately 30 to 3600 min on decreasing the trypsinogen concentration from 7.2 to 0.1 mg per ml (Table I)
Summary
Materials-Bovine trypsinogen (crystallized once, Lot TGOGL), bovine chymotrypsinogenA (crystallized five times, LotCGC OEA), bovine trypsin A (crystallized five times, Lot. CGC OEA), bovine trypsin The zymogens were dialyzed against 1 mM HCl and lyophilized before use. The trypsin was further purified on SE-Sephadex by the method of Papaioannou and Liener [8]. DFPl was a gift from Merck, Sharp and Dohme. L-arginine methyl ester, was synthesized by standard methods [9] and benzoyl-L-tyrosine ethyl ester (Lot D4304), the chymotrypsin substrate, was a product of Schwarz/Mann, Orangeburg, New York. All other chemicals were of reagent grade, and deionized water was used throughout
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