PZ-peptidase activity from ejaculated boar spermatozoa.

Protein constituents of the boar spermatozoon were fractionated in three components, the hypotonic soluble fraction, the detergent-soluble fraction, and the detergent-insoluble fraction. When all these fractions were assayed spectrophotometrically using the PZ-peptide as substrate, a high value of PZ-peptidase specific activity was observed in the first fraction. Electrophoretic analysis at pH 8.3 of the protein content from the hypotonic soluble fraction revealed the existence of multiple molecular forms capable of hydrolysing the PZ-peptide. The major form was characterized by a surprisingly high value of electrophoretic mobility, index of the presence of numerous negatively charged residues. Biochemical and ultrastructural analyses showed that the hypotonic soluble fraction did not contain intrinsic, and specifically acrosomal, sperm enzymes.

Fractionation of of proteolytic enzymes by affinity chromatography on sepharose aminocaproyl proflavin.

The serine proteinases trypsin, chymotrypsin, elastase, and acrosin bind to the proflavin resin, the sulfhydryl proteinases ficin, bromelain, and papain are retarded by the resin, whereas most proteins and enzymes tested are not bound. Elution of the bound activities is accomplished NaCl or by variation from the pH optimum of the enzyme. Commercially available enzymes that are bound or retarded are easily further purified by the column. The acrosin activity of sperm acrosomal extracts is separated into bound and unbound activities. Acrosin is purified 120-fold from sperm acrosomal extracts in a single step, yielding a specific activity of 96.

A role for esterases in the fertilization process.

The corona penetrating enzyme of sperm is shown to be esterolytic in nature. This is the first known function of the several sperm esterases and indicates the cementing substance between cells of the corona radiata contains an essential ester linkage.