Nine calcium binding protein spots were Sorafenib Tosylate IC50 carefully excised from complementary stained gels and PVDF membranes, and identified by mass spectrometry and or Edman degradation analysis. The nine calcium binding proteins thus identified in detergent urea extracts of human sperm are given in Figure 2. The Inhibitors,Modulators,Libraries protein with the highest relative 45Ca binding capacity was identified as calmodulin, the major calcium binding com ponent of the mammalian sperm cytosol. Computer comparison of 2D images of calcium bind ing spots with images of proteins vectorially labelled with radioiodine and images of 2D gels where the pro teins had been visualized by Coomassie or silver stain ing, allowed identification of calcium binding proteins exposed on the sperm surface. Five calcium binding pro teins.

HYOU1, HSPA5, HSPA2, SAP and 80K H were found to be accessible to Iodo Bead catalyzed radiolabel ling. The three calcium binding HSP70 chaperones HYOU1, HSPA5 and HSPA2 were recently shown to be accessible to biotin labelling on the surface of motile human sperm. The 80 kDa calcium binding surface protein migrating at a pI of 4 was Inhibitors,Modulators,Libraries identified as 80K H, a phosphoprotein containing two calcium binding helix loop helix structures. Radiolabelling of 80K H was highly reproducible, although the amount of iodine iso topes incorporated into the protein was sparse. 80K H contains several potential threonine and tyrosine phosphorylation sites, and increased phos phorylation of the protein was observed following in vitro capacitation of human sperm.

Efficient induction of in vitro capacitation was confirmed by the significant increase in tyrosine phosphorylation of CABYR, fibrous sheath protein 95 and valosin contain ing protein p97. The capacitation induced phosphorylation of 80K H did not alter the proteins 45 Ca binding capacity. Densitometry of the autoradiograms showed that the abundant Inhibitors,Modulators,Libraries surface protein SAP accounts for more than six per cent of the 45Ca binding capacity in the acidic and neutral pH range of the human sperm proteome, thus identifying SAP as the surface labelled constituent that binds relatively most 45 Ca in the overlay assay. Immuno staining of the PVDF membranes used for 45Ca and 125I autoradiography confirmed that the 26 kDa surface labelled calcium binding protein was SAP. In addition to the major 45Ca binding form, a slightly more basic and at least one slightly more acidic form of the SAP antigen was revealed Inhibitors,Modulators,Libraries by the Western blot analysis.

SAP is a glycoprotein with a single Inhibitors,Modulators,Libraries N glycosy lation site, at Asn 32, which in the native protein con tains a typical complex biantennary oligosaccharide chain. Structural variants of R115777 SAP which lack one or both terminal sialic acid residues have been found in human plasma and urine, suggesting that the charge variants of human sperm SAP might be due to micro heterogeneity of the glycan structure.