Abstract
Adsorption of caltrin on a cation exchanger during purification transformed it from inhibitor to enhancer of calcium uptake. Ether extracts of acidified preparations of bovine seminal plasma transformed enhancer caltrin back to inhibitory caltrin; this capacity of the ether extracts was lost after incubation with an anion exchanger, indicating that anions in the extract could be responsible for the reversal of caltrin activity. Of the anions identified in the ether extract of acidified bovine seminal plasma, phosphatidylserine converted enhancer caltrin to the inhibitory form at pH 7.4. Citrate at millimolar concentrations lowered calcium uptake of sperm in the presence of enhancer caltrin to near control levels. Cardiolipin at concentrations comparable to its natural occurrence in the seminal plasma prevented enhancer caltrin from stimulating the sperm cells to take up calcium above their usual capacity. Dipalmitoylphosphatidylglycerol, phosphatidylcholine derived from bovine brain, phosphatidylethanolamine from bovine heart, and other phospholipids with transition temperatures higher than the assay temperature had no effect on the activity of enhancer caltrin, while dimyristoylphosphatidylcholine had an effect on enhancer caltrin similar to that of citrate. Phosphatidylinositol from soybean was also capable of lowering caltrin-stimulated calcium uptake in bovine sperm to control levels. Data on enhancer caltrin fluorescence in the presence of phosphatidylserine from bovine brain suggest conformational changes in the protein due to binding of the phospholipid. In comparison, the phosphatidylcholine from bovine brain appeared not to alter enhancer caltrin.
Highlights
Bovine Seminal Plasma Constituents Modulate the Activity of Caltrin, the Calcium-transport Regulating Protein of Bovine Spermatozoa*
Ether extracts of acidified preparations of bovine seminal plasma transformed enhancer caltrin back to inhibitory caltrin; this capacity of the ether extracts was lost after incubation with an anion exchanger, indicating that anions in the extract could be responsible for the reversal of caltrin activity
We found that the activity of caltrin as inhibitor of calcium uptake in bovine epididymal spermatozoa was sensitive to the conditions employed in its isolation (San Agustin et al, 1987) as suggested earlier (Singh, 1980)
Summary
WI and was always kept at ambient temperature during transit and prior to use. Purification of Cc&in-Homogeneous caltrin was obtained from bovine seminal plasma using either the urea/DTT phenyl-Sepharose method. Method or the Preparation of Homogeneous Cultrin by Urea Denaturation and DTT Reduction of Coeluting Bovine Seminal RNase-The first three steps of caltrin purification were as described by Rufo et al (1982, and see Table I), except that the column buffer of step 3 was 10 mM. Uncoiled bovine seminal RNase eluted ahead of caltrin (San Agustin, 1988). Bovine seminal plasma components were added to the medium 5 min after caltrin and 10 min before the cell suspension (final cell count, 4 x lo cells/ml). Caltrin was always kept on ice prior to addition to the assay medium and calcium uptake of the spermat,ozoa was always determined at 30 “C. Caltrin was always kept on ice prior to addition to the assay medium and calcium uptake of the spermat,ozoa was always determined at 30 “C. Removal of cell suspension aliquots (at least 225 ~1) from the assay medium at particular times, vacuum filtration of the aliquots to separate the cells from the medium, washing of the filtered cells, and subsequent counting for radioactivity accumulated before (San Agustin et al, 1987)
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