This proposal extends previous work which uses purified plasma membranes and membrane vesicles (PMV) to determine the mechanisms of Ca2+ transport by boar spermatozoa. The major objective seeks to isolate the proteins involved in Ca2+ uptake and Ca2+ efflux. Transport proteins will be identified by several methods: 1) by identifying and isolating proteins phosphorylated by Ca2+-dependent plasma membrane ATPase and by Ca2+-dependent plasma membrane PM protein kinase, 2) by identifying Ca2+ binding proteins directly on two-dimensional PAGE transblots of PM polypeptides and indirectly by isolating PM from intact cells that have transported 45Ca2+, 3) by identifying calmodulin (CM) binding proteins using 125I-CM overlays of 2-D PAGE gels containing separated PM proteins, and 4) by separating CM binding proteins by affinity chromatography. The enzymatic properties of (Ca2+ + Mg2+)ATPase in PMV and isolated proteins will be compared to the properties of transport in PMV and after reconstitution in liposomes. Changes in the phosphorylation of proteins identified to be involved in transport and their surface location and organization during epididymal transit and after capacitation will be determined. Specific antibodies to proteins identified to be involved in Ca2+ transport or specific binding to PM proteins will be used to localize transport proteins and to determine whether activation of sperm and fertilization in vivo can be prevented by antibodies directed at these proteins. The transport capability of proteins, isolated by preparative isoelectric focusing, preparative 2-D PAGE (and immunoabsorption chromatography) will be determined by reconstituting transport in liposomes. These transport mechanisms are viewed as critical to understanding the process of capacitation and the acrosome reaction.
| Peterson, R N; Chaudhry, P; Tibbs, B (1989) Calcium-binding proteins of boar spermatozoan plasma membranes: identification and partial characterization. Gamete Res 23:49-60 |
| Peterson, R N; Gillott, M; Hunt, W et al. (1987) Organization of the boar spermatozoan plasma membrane: evidence for separate domains (subdomains) of integral membrane proteins in the plasma membrane overlying the principal segment of the acrosome. J Cell Sci 88 ( Pt 3):343-9 |
