Cowper’s Gland Secretion in Rat Semen Coagulation, II. Identification of the Potentiating Factor Secreted by the Coagulating Glands

Abstract

The in vitro coagulation of the basic protein fraction from the seminal vesicles of the rat by an acetone extract from Cowper’s gland secretion at pH 5.9 is potentiated by incubation of the substrate from the seminal vesicles with coagulating gland secretion. This reaction is termed the interaction reaction. The rate of coagulation for the interaction reaction was found to be dependent upon the duration of the incubation period, but maximum coagulation was independent of incubation time. Extraction of vesiculase, an enzyme that coagulates the basic protein fraction from the seminal vesicles optimally at pH 7.4, from coagulating gland secretion by double sequential ammonium sulfate and acetone precipitation yielded a 25-fold purification of the enzyme and demonstrated that maximum potentiating activity resided in the fraction containing maximum vesiculase activity. Disc gel electrophoresis demonstrated that this extraction procedure reduced the number of bands from six in the crude secretion to two in the acetone extract. Versene, added early in the incubation period, inhibited coagulation and versene inhibition was reversed by the simultaneous addition of calcium chloride. Heating the vesiculase extract previous to incubation also resulted in no potentiation. Urea (8.0 M) failed to solubilize the coagulum produced during the interaction reaction and the vesiculase mediated reaction, pH 7.4, but readily solubilized the clot produced by reaction of the seminal vesicle basic protein fraction with only Cowper’s secretion extract, pH 5.9. Moreover, extracted vesiculase demonstrated fibrinase activity when tested in a bovine fibrinogen-thrombin system. These results suggest that vesiculase is the potentiating factor from the coagulating glands and that its mechanism of action may involve the covalent joining of the basic protein monomers from the seminal vesicles by a transamidase reaction.