Isolation and Preliminary Characterization of a Purified Pig Zona Antigen (PPZA) from Porcine Oocytes

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Abstract
A protocol was developed for isolation of a purified pig zona antigen (PPZA) under nondissociating conditions. Heating of isolated zona-encased porcine oocytes at 73 degrees C for 20 min resulted in optimal solubilization of zona antigen activity (ZAA) as assessed by radioimmunoassay. Subsequent fractionation of solubilized proteins by ammonium sulfate precipitation, ultrafiltration, gel filtration, and ion exchange chromatography yielded PPZA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of PPZA yielded a major diffuse band at 58,000 Mr which stained for protein and carbohydrate and which possessed ZAA. Two-dimensional gel electrophoresis confirmed the identity of the 58,000 Mr glycoprotein and one of the three major charge heterogeneous glycoprotein families of the porcine zona pellucida. These experiments established that the principal macromolecular and antigenic component of PPZA is a 58,000 Mr glycoprotein originating from the zona pellucida. The nature of PPZA antigenic determinants recognized by a rabbit antiserum to PPZA was studied by radioimmunoassay. ZAA of PPZA was sensitive to the action of mercaptans and proteases, indicating a contribution of polypeptide chain to PPZA antigenicity. Loss of ZAA upon periodate oxidation of PPZA also implicated carbohydrate residues. PPZA retains antigenic determinants of the intact zona pellucida as assessed by reactivity with antiserum to intact porcine zonae. Likewise, rabbit antiserum to PPZA reacts avidly with intact porcine zonae. These results demonstrate that PPZA is a suitable target antigen for further studies on immunocontraception.