Edmund R. Casillas

Cyclic AMP-dependent protein kinases of bovine epididymal spermatozoa.

Abstract Cyclic AMP-dependent protein kinase (cAMP-PrK) activity, some 100-fold greater than that in extracts of other bovine tissues, has been found in sonicates of bovine epididymal spermatozoa. A less than 20-fold purification of the enzyme yields a preparation migrating as three components, two of which possess protein kinase activity, by polyacrylamide gel isoelectric focusing. Bands with identical pl values and activities are found in crude sonicates. Following salt precipitation, cAMP-PrK is separated into two components (Peaks I and II) on DEAE-cellulose. Kinetic constants for cyclic nucleotides, ATP, and protein substrates are reported for purified Peak II enzyme. The results indicate that cyclic AMP-dependent protein kinases are present at high levels in the sperm cytosol and suggest an involvement in motility.

Activation of monkey spermatozoal adenyl cyclase by thyroxine and triiodothyronine

Thyroxine and triiodothyronine (T3) stimulated the conversion of ATP to 3′,5′-AMP (cAMP) by ejaculated rhesus monkey (Macaca mulatta) spermatozoa. Compounds structurally related to thyroxine, other than T3, had no effect on cAMP accumulation. The response to T3 concentration was biphasic, with maximal stimulation occurring at a hormone level of 4 μM. The action of T3 on cAMP accumulation appears to be a direct effect on adenyl cyclase since T3, at concentrations employed in this study, had no effect on the breakdown of cAMP by intact spermatozoa. Anaerobic spermatozoal fructolysis was found to respond to varying concentrations of T3 in a biphasic manner similar to that found for adenyl cyclase.

Adenyl cyclase activity and cyclic 3 5-AMP content of ejaculated monkey spermatozoa.

Abstract Adenyl cyclase activity was measured in washed ejaculated spermatozoa of the rhesus monkey ( Macaca mulatta ) by monitoring the conversion of [ 14 C]ATP to [ 14 C]cyclic 3′,5′-AMP. Thyroxine and triiodothyronine increase the accumulation of cyclic 3′,5′-AMP; other hormones, including androgens, gonadotropins, prostaglandins, and catecholamines had no effect. Manganese ion, over the range 0.5–20 m m , stimulated adenyl cyclase activity 2- to 50-fold, but in parallel studies inhibited fructolysis and motility. Testosterone, dihydrotestosterone, and epinephrine stimulated the accumulation of cyclic 3′,5′-AMP 60–70% after preincubation of the cells with triiodothyronine while androstenedione and progesterone had no effect under the same conditions. Spermatozoal intracellular cyclic 3′,5′-AMP, measured using cyclic 3′,5′-AMP-dependant protein kinase, averaged 103 ± 28 pmoles/10 9 spermatozoa. Intracellular cyclic 3′,5′-AMP content was not altered by treatment with single hormones but combinations of triiodothyronine and dihydrotestosterone, in some instances, increased cyclic 3′,5′-AMP levels almost 2-fold. Manganese ion, at concentrations which stimulate adenyl cyclase activity, decreased the intracellular content of cyclic 3′,5′-AMP by 40% after incubation for 1 hr at 37 °.

Casein kinase ii activity and polyamine stimulated protein phosphorylation of cytosolic and plasma membrane proteins in bovine sperm

A highly purified preparation of sperm cytosolic protein kinase was obtained by repeated chromatography with phosphocellulose. The preferred substrate of the enzyme was casein and the activity was not stimulated by added Ca2+, calmodulin, or cAMP. With casein as substrate, both ATP and GTP served as phosphate donors and the activity was inhibited by low micromolar heparin and stimulated by low millimolar spermine and spermidine. These properties are characteristic of casein kinase II from other cells. Endogenous protein substrates of the enzyme in sperm cytosolic fractions and in plasma membranes were demonstrated by incubating the preparations with [gamma-32P]GTP, under conditions unfavorable to other protein kinases, and analyzing the products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Spermine greatly enhanced the phosphorylation of three (55, 92, and 106 kDa) proteins in both cytosolic and plasma membrane preparations. Our results indicate that polyamines play a role in modulating the phosphorylation state of proteins in sperm and may further regulate sperm function through this mechanism.

Calmodulin-stimulated cyclic nucleotide phosphodiesterases in plasma membranes of bovine epididymal spermatozoa

Cyclic nucleotide phosphodiesterase in the plasma membranes of bovine epididymal spermatozoa was stimulated by added Ca2+ and calmodulin. The rate of hydrolysis and responsiveness toward calmodulin was greater for cAMP than for cGMP. The kinetic analysis of the activity revealed two forms of phosphodiesterase with apparent Km values of 7.5 and 95 microM for cAMP. Calmodulin stimulated both of the activities by increasing the Vmax without affecting the Kms. The activity response with respect to Ca2+ concentration appears to be biphasic in both the absence and presence of added calmodulin. Trifluoperazine inhibited the Ca2+- and calmodulin-sensitive enzyme activity in a dose-dependent manner. The calmodulin-stimulated phosphodiesterase activity in the sperm plasma membranes can be solubilized and absorbed to a Calmodulin-Sepharose affinity column in the presence of Ca2+.

Manganese and manganese-ATP interactions with bovine sperm adenylate cyclase☆

The adenylate cyclase of mammalian spermatozoa shares some of the properties of the isolated catalytic component from somatic cell adenylate cyclases. One of these properties is the large apparent stimulation by Mn2+. We have used the direct linear plot according to Eisenthal and Cornish-Bowden to explore whether this apparent stimulation is due to direct stimulation by Mn2+ or due to complexation of free ATP, a postulated inhibitor of cyclase activity. We have observed the activity of the particulate adenylate cyclase from bovine caudal epididymal spermatozoa as a function of calculated equilibrium values for the concentrations of Mn2+, free ATP, and the enzymes substrate, the manganese-ATP complex. Direct linear plots for activity and substrate concentration over the apparent inhibitory concentration range of free ATP give the pattern expected for a hyperbolic substrate response. By contrast, direct linear plots in which Mn2+ concentration varies over its apparent stimulatory range show that as Mn2+ concentration increases, activities are higher than would be predicted for a hyperbolic substrate response. We conclude that for particulate bovine sperm adenylate cyclase, free ATP is not strongly inhibitory, and Mn2+ is a positive effector, reaching half-maximal stimulation at 0.2 mM. The unique nature of the sperm adenylate cyclase and its possible regulation by Mn2+ under physiological conditions is discussed.

Enzymic control of fructolysis in primate spermatozoa

Abstract 1. 1. The present report is concerned with the enzymic control of fructolysis in ejaculated monkey (Macaca mulatta) spermatozoa. Determination of levels of fructolytic intermediates and confactors and calculation of mass-action ratios for each enzymic step has shown that the potential for control in vivo occurs at the levels of hexokinase, phosphofructokinase, and pyruvate kinase. 2. 2. Parallel studies with spermatozoa cell-free extracts, capable of converting either Glc-6-P or Fru-6-P to lactate, have provided supportive evidence for a regulatory role for phosphotfructokinase. First, extracts catalyze the degradation of Fru- i ,6-P 2 at a rate several-fold faster than Fru-6-P. Secondly, the regulatory properties of the soluble “fructolytic” system have been shown to be nearly identical to sperm phosphofructokinase. The following properties are shared by both systems. Reaction rates respond in a biphasic manner to varying ATP levels. ATP-inhibited states are relieved by ADP, 5′-AMP, 3′,5′-AMP, or Pi acting singly or in combination. Combinations of Pi+3′,5′-AMP, Pi+ADP, or 3′,5′-AMP+ADP are synergistic in their relief of severely inhibited states. Reaction rates are also inhibited by citrate with the inhibition being relieved by Pi and 5′-AMP. Finally, determination of the levels of fructolytic intermediates under varying conditions induced by nucleotides or citrate have localized the above effects at the level of phosphofructokinase. 3. 3. Fructolytic rates in intact cells have been shown to be only marginally affected by compounds shown to effect either phosphofruktokinase or the soluble fructolytic system. Compounds studied included ATP, Pi, 3′,5′-AMP, and N6,O2-dibutyryl 3′,5′-AMP. 4. 4. The above considerations, coupled with determination of intracellular levels of ATP, ADP, and AMP, have led to the suggestion that fructolysis in ejaculated cells is under adenylate control and that the intensely glycolytic nature of ejaculated mammalian spermatozoa reflects the relatively low [ATP]/[ADP]+[AMP]ratio found.

Isotypes of protein kinase C in bovine sperm

Protein kinase C is present in bovine epididymal sperm. The enzyme was partially purified by gel filtration on Sephacryl S-300. The Ca2+/phosphatidylserine-dependent histone phosphotransferase activity elutes from the gel filtration column in a manner corresponding to a Mr approximately 80 kDa. The activity peak also corresponds with [3H]phorbol 12,13-dibutyrate binding activity. Immunoblot analysis of the partially purified enzyme with isozyme-specific monoclonal antibodies revealed the presence of alpha-, beta-, and gamma-subspecies of protein kinase C. Indirect immunofluorescence showed that the antibodies against alpha-, beta-, and gamma-subspecies produced prominent staining of the postacrosomal region of the sperm head. In addition, beta-subspecies antibodies produced minor staining of the midpiece and gamma-subspecies antibodies produced a minor staining of the acrosomal region.

Purification and characterization of polyamine-stimulated protein kinase (casein kinase II) from bovine spermatozoa

Casein kinase II from bovine epididymal spermatozoa was purified to apparent homogeneity by repeated chromatography with phosphocellulose and gel filtration with sephacryl S-200. The purified enzyme exhibited a molecular mass of 130 kDa by gel filtration and displayed three polypeptide bands with molecular masses of 26, 33, and 36 kDa by SDS-polyacrylamide gel electrophoresis. Antibodies raised against calf thymus casein kinase II cross reacted with the three sperm polypeptides. Incubation of the holoenzyme with either [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of the 26-kDa subunit. The enzymatic activity with casein as substrate was strongly inhibited by nanomolar heparin and greatly stimulated by micromolar spermine. With casein as substrate, the specific activity of the pure enzyme (0.5 mumol/min/mg protein) was comparable to that of casein kinase II from other sources. Endogenous substrates of the kinase were demonstrated by incubating sperm cytosolic extracts with [gamma-32P]GTP, under conditions that limit the expression of other protein kinases, and analyzing the products by SDS-PAGE and autoradiography. Similar results were obtained when sperm extracts, suitably diluted to minimize endogenous casein kinase II, were incubated with [gamma-32P]GTP and aliquots of pure sperm casein kinase II. Low concentrations (50 microM) spermine strongly enhanced the phosphorylation of 92- and 106-kDa cytosolic proteins. Our results clearly show that casein kinase II is present in spermatozoa and that it shares many of the properties of the enzyme from other sources. Further, they indicate that the enzyme plays a role in mediating the phosphorylation state of sperm proteins.

The intracellular localization and properties of carnitine acetyltransferase from ram spermatozoa

Abstract Although spermatozoa possess a very active carnitine acetyltransferase, there is no satisfactory explanation for such a high activity. In order to help elucidate possible roles for carnitine acetyltransferase in spermatozoa, we examined the intracellular location and properties of carnitine acetyltransferase from ejaculated ram spermatozoa. The spermatozoa were disrupted by hypotonic treatment with 10 m m phosphate buffer (pH 7.4), followed by mild sonication. The resulting homogenate was separated by sucrose step-gradient centrifugation into soluble, plasma membrane, acrosomal membrane, and mitochondrial fractions. These fractions were characterized by electron microscopy and marker enzyme assays. The particulate fractions were made soluble by treatment with 0.1% deoxycholate and then were assayed for carnitine acetyltransferase activity. Carnitine acetyltransferase activity was found exclusively in the mitochondrial fraction with a specific activity of 0.151 μmol CoASH · min −1 · mg −1 . The apparent K m values for acetyl-CoA and l -carnitine were 1.1 × 10 −5 and 1.3 × 10 −4 m respectively.