ALTERATIONS IN THE PLASMA MEMBRANE PROTEINS OF MAMMALIAN SPERMATOZOA DURING EPIDIDYMAL MATURATION

Abstract

Biochemical investigation of rat sperm has revealed the presence of a 32,000 Mr glycoprotein, susceptible to labeling by galactose oxidase/{lcub}('3)H{rcub}NaBH(,4), which is present on caudal but apparently lacking from caput sperm. A glycoprotein isolated from rat caudal epididymal fluid is also labeled by galactose oxidase/{lcub}('3)H{rcub}NaBH(,4) and migrates with an apparent molecular weight of 32,000. Antibodies raised against this protein cross react with the 32,000 Mr membrane derived protein. These results suggest that the origin of the membrane protein may be by absorption from epididymal fluid.;To gain insight into the mechanism of acquisition of this maturation associated membrane glycoprotein, we have characterized its hydrodynamic properties. Our results show that the membrane glycoprotein has properties which are inconsistent with those of a soluble protein, and are more characteristic of an integral membrane protein. Though these findings do not preclude the possibility that the protein is secreted from the epididymis, and subsequently binds to caudal sperm, they do suggest that if both proteins are related, then the mechanism of insertion into the membrane must be complex.;In an attempt to overcome some of the difficulties encountered in studying sperm, we have developed a method for the selective retrieval of plasma membrane proteins by covalently "tagging" the intact cell with 2-iminobiotin. Retrieval is based upon the specific interaction between 2-iminobiotin, avidin, anti-avidin antibody and insoluble protein A from S. aureus. The pH dependent interaction of 2-iminobiotin with avidin makes recovery possible. At high pH, the free base form of 2-iminobiotin retains high affinity specific binding to avidin, whereas at acidic pH values, the salt form of the analog interacts poorly with avidin. The use of this selective retrieval system permits study of plasma membrane proteins without prior isolation of purified membrane vesicles. The practicality of both the preparative (affinity chromatography using immobilized avidin) and analytical (immunoprecipitation with anti-avidin antibody) selective retrieval systems has been established utilizing models.