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Several lines of evidence have demonstrated conclusively the presence of cAMP-dependent protein kinase (ecto-RC) activity on the external surface of goat cauda-epididymal intact spermatozoa. The intact-cell ecto-kinase that caused transfer of the terminal phosphate of exogenous ATP to the serine and threonine residues of exogenous histone was specifically activated by cAMP. As well, the ecto-kinase caused phosphorylation of the synthetic peptide Kemptide. The isolated spermatozoa, before or after incubation with reaction mixture for ecto-kinase assays, were approximately 99.5% viable, as shown by the analyses of ethidium bromide fluorescence and the cytosolic marker enzymes lactic dehydrogenase and 3-phosphoglycerate kinase. The ecto-kinase activity was not due to contamination of epididymal plasma and damaged cells or to protein kinase that may have leaked from the cells. There was little uptake of ATP and histone by the cells. The intact-cell kinase activity was strongly (80-90%) inhibited by treatment with membrane nonpenetrating surface probes: p-chloromercuriphenylsulfonic acid (2 microM), diazonium salt of sulfanilic acid (DSS, 0.5 mM), and proteases such as trypsin, chymotrypsin, and pronase (each 125 micrograms/mL). Disruption of sperm plasma membrane by sonication or Triton X-100 (0.2%) caused about a fivefold increase of the intact sperm kinase activity. Highly purified sperm plasma membrane (PM) possessed ecto-kinase activity that was resolved into type I and II kinases by DEAE-cellulose chromatography, the type I isoenzyme being the major (approximately 70%) enzymic species. Treatment of the intact spermatozoa with DSS prior to isolation of PM caused a marked loss of the activities of both the isoenzymes, indicating thereby the "ecto" nature of the PM-bound type I and II kinases. Preparations of vigorously forward-motile spermatozoa with 100% intactness had approximately fourfold higher specific activity of the ecto-kinase than the "composite" cells from which the former cells were isolated. However, the profiles of the type I and II ecto-kinases of the composite, as well as forward-motile spermatozoa, were nearly identical. The data are consistent with the view that ecto-kinases may have role in the regulation of flagellar motility.

An improved Ficoll density gradient centrifugation method has been described for the isolation of goat caput-epididymal immature spermatozoa of a high purity and intactness. The method consists of layering freshly extracted sperm suspension on the top of a Ficoll gradient comprising 2.5 ml each of 2%, 4%, 6%, and 8% Ficoll-400 in a modified Ringer's solution and centrifugation at 300 g for 3 min in a swing bucket table centrifuge. Spermatozoa, free from fat globules and blood cells, sedimented at the bottom of the 8% Ficoll layer. The plasma membrane of the isolated cells showed a high degree of intactness (approximately 96%) as assessed by lactic dehydrogenase marker enzyme and ethidium bromide-fluorescence methods.

Highly purified plasma membranes (PMs) isolated by aqueous two-phase polymer methods from goat sperm undergoing epididymal maturation, have been analyzed for the isoenzymes of cyclic AMP-dependent protein kinase (RC). The mature and the immature spermatozoa showed profound differences in the profile of the isoenzymes of RC solubilized from the isolated PMs with 0.1% Triton X-100. The immature sperm PM consists of only type I RC in contrast to the mature sperm membrane which possesses both the type I and II isoenzymes. The type II kinase represents nearly 30% of the total membrane-bound RC of the mature cells. The analysis of the surface topography of these isoenzymes of the maturing spermatozoa by using diazonium salt of sulfanilic acid as the surface probe shows that the PM-bound RC(s) are oriented primarily on the external surface of these intact cells. The data demonstrate that type II RC is a maturation-specific ecto-kinase as it appears on the sperm surface specifically during the maturation of spermatozoa in the epididymis.

Goat epididymal intact spermatozoa have been shown to possess on the external surface specific receptors that bind with high affinity to exogenous [8-3H]cyclic AMP. The ecto-cyclic AMP-receptor activity was not due to contamination of broken or "leaky" cells, if any. The binding reaction of [3H]cyclic AMP with the receptors was extremely rapid. Uptake of the labeled cyclic AMP to the sperm cytosolic fraction was undetectable. There was little leakage of cyclic AMP-receptors from intact spermatozoa during the binding assays. The binding reaction was proportional to cell concentration, specific and saturable at 250 nM cyclic AMP. The binding of the labelled cyclic nucleotide was nearly completely displaced at saturating concentrations (2.5 microM) of the unlabelled nucleotide. The ecto-receptors showed high specificity for binding to cyclic AMP. The Kd of the binding sites was approximately 1.7 X 10(-8) M. The binding interaction was highly sensitive to treatment with proteolytic enzymes: trypsin, chymotrypsin, or pronase (125 micrograms/ml). Sonication caused a nearly 450% increase of the ecto-receptor activity. The specific activity of the ecto-cyclic AMP-receptor was approximately twofold higher in the vigorously forwardly motile spermatozoa than in the "composite" cells, suggesting that the ecto-receptors may have a role in modulating flagellar motility.

Phosphatidyl inositol has been found to inhibit strongly the activity of a cyclic AMP-independent protein kinase located on the external surface of goat epididymal intact spermatozoa. Phosphatidyl inositol at a concentration as low as 10 micrograms/ml inhibited nearly 50% of the ecto-kinase activity for the phosphorylation of the exogenous protein substrate: casein. Phosphatidyl ethanolamine at a relatively high concentration (125 micrograms/ml) inhibited slightly (approx 25%) the activity of the enzyme whereas other phospholipids: phosphatidyl serine and choline, diacyl glycerol, phosphatidic acid and myo-inositol-2-phosphate had no appreciable effect on the kinase activity. Phosphatidyl inositol has also served as a potent inhibitor of the phosphorylation of sperm ecto-phosphoproteins by the endogenous kinase activity of intact spermatozoa. By thin layer chromatography it has been shown that the observed inhibitory effect of the phospholipid was not due to any impurities or degraded products of phosphatidyl inositol. Phosphatidyl inositol inhibited the kinase activity noncompetitively with respect to casein and Mg2+ but uncompetitively with respect to ATP. The results raised the possibility that phosphatidyl inositol-mediated high affinity inhibition of protein kinase(s), may constitute a novel mechanism for the regulatory actins of the phospholipid in mammalian cells.

A quantitative method based on fluorescence generated by the binding of ethidium bromide (EB) to DNA has been developed for estimation of the intactness of the plasma membrane of a mammalian cell type (goat epididymal spermatozoon). The method consists of mixing of sperm preparations with EB in a modified Ringer's solution followed by immediate measurement of fluorescence intensity at 365-580 nm (excitation-emission). The data were corrected for non-specific values of fluorescence due to intact cells only. The percentage of damaged cells in a sperm population was calculated by comparing the corrected fluorescence values of the cell preparations with those of the sonicated cells. The values of sperm intactness obtained by this method (99.5 +/- 0.3) compared well with those obtained by the widely used "marker enzyme" method (97 +/- 0.8) based on estimation of lactic dehydrogenase in the extracellular medium. The validity of this method has been confirmed by using cells of defined intactness i.e. preparations of vigorously forward-motile spermatozoa that showed nearly 100% intactness. The method can detect as low as 0.5% "leaky" or damaged cells in a cell preparation. The "EB-fluorescence" method is simpler and more rapid and reliable than the conventional "marker enzyme" method for estimation of cellular intactness.

Goat cauda-epididymal intact spermatozoa have been shown to possess an ecto-cyclic AMP-independent protein kinase activity on the external surface that causes phosphorylation of the serine and threonine residues of exogenous phosvitin. The enzyme is neither a tyrosine kinase nor a catalytic subunit of the cyclic AMP-dependent protein kinase. It is not activated by Ca2+, calmodulin and phosphatidylserine. The intact-cell enzyme is capable of phosphorylating a variety of proteins including sperm plasma membrane-bound phosphoprotein(s). The enzymic activity of the intact spermatozoa was not due to contamination of broken or "leaky" cells. The kinase activity of the whole cells was strongly inhibited by the non-penetrating surface probes: p-chloromercuriphenylsulphonic acid (10 microM) and proteases (125 micrograms/ml). The specific activity of the ecto-kinase increased nearly 100% during vigorous forward progression of spermatozoa.