Proteolysis by collagenase preparations alters the macromolecular composition of the porcine zona pellucida.
The zona pellucida (ZP) from pig oocytes was isolated using two different methods. In the first method, the ZP was isolated using sieving procedures. In the second method, an enzymatic step with collagenase was used in addition to sieving procedures. Several commercial collagenase preparations were tested. The macromolecular composition of the ZP isolated by these two methods was determined by two-dimensional polyacrylamide-gel electrophoresis after disulfide bond reduction. The ZP prepared by the sieving method contained four glycoprotein families with apparent molecular weights of 25,000, 55,000, 65,000, and 90,000. The ZP obtained using the enzymatic method was distinctly different, lacking the highest molecular-weight family (90,000) and containing at least three new constituents with apparent molecular weights of 70,000, 40,000, and less than 25,000. Commercial collagenase preparations, when analyzed by two-dimensional polyacrylamide-gel electrophoresis to assess homogeneity, contained numerous protein components. The trypsin-like protease concentration in the collagenase preparations was determined to be 3.4-42 X 10(-8) M as determined by activity measurement using benzoyl-DL-arginine-beta-naphthylamide as substrate or the active site titrant p-nitrophenyl-p'-guanidinobenzoate. Thus, the ZP prepared by the enzymatic method, using collagenase preparations, had an altered macromolecular composition, thereby rendering the ZP unsuitable for most structural, immunological, or sperm-binding studies.
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006023:Glycoproteins / Q000032:analysis; D046911:Macromolecular Substances; D003012:Microbial Collagenase / Q000378:metabolism*; D008970:Molecular Weight; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D006023:Glycoproteins; D046911:Macromolecular Substances; D014357:Trypsin; D003012:Microbial Collagenase