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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35--45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.
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PMID:Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin. 701 13

Differences in the metabolism of testicular and cauda epididymal sperm have been demonstrated for several species, but the region(s) of the ram epididymis in which changes in metabolism occur is not known. In these experiments respirometric and radioisotopic methods were used to monitor metabolic activity of ram sperm isolated from six regions of the epididymis. Effects of exogenous carnitine on metabolism of the sperm also were studied. Sperm from the caput and corpus epididymidis had similar rates of glucose and oxygen consumption and of lactate and CO2 production. However, the magnitude of each parameter was severalfold higher for cauda sperm. In addition, cauda sperm produced 25 times more acetate than caput or corpus sperm. The amount of energy derived from utilization of endogenous substrates was similar for sperm from all regions of the epididymis. Exogenous D,L-carnitine (5 mM) had no effect on the metabolism of caput or corpus sperm. However, when cauda sperm were incubated with carnitine, they consumed less glucose and produced less lactate and more acetate; thus they produced the same amount of ATP from less glucose than did control aliquots incubated without exogenous carnitine. Since the rate of ATP synthesis was equivalent for both incubations, we believe this change in metabolism reflects an increased efficiency of glucose utilization. This increased efficiency may be vital for sperm motility.
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PMID:Changes in metabolism of ram sperm associated with epididymal transit or induced by exogenous carnitine. 713 17

This study provides evidence for the occurrence of a biochemical function for the polyamines, spermidine and spermine, in bovine epididymal spermatozoa. Methods are described for the detection and isolation of a protein kinase from spermatozoa which catalyzed transfer of phosphate from [gamma-32P]ATP to a unique endogenous protein of Mr = 70,000 in a reaction that was polyamine-dependent. Enzymatic phosphorylation of the 70,000-dalton protein by the purified protein kinase was sharply activated, at times more than 80-fold, by the polyamines, spermidine and spermine. Spermidine and spermine, together in equimolar combination, synergistically activated the protein kinase when compared with all other possible combinations of putrescine, spermidine, or spermine. The polyamine-dependent protein kinase was resolved by phosphocellulose chromatography into a catalytic component of Mr = 19,000 and a complex comprised of the catalytic component associated with the natural phosphate-acceptor protein of Mr = 70,000. This protein kinase was not activated by cyclic nucleotides or calcium ion.
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PMID:A polyamine-dependent protein kinase from bovine epididymal spermatozoa. 726 52

Rats were fed diets containing various amounts of added thiram, a dithiocarbamate fungicide. As thiram feeding resulted in decreased appetite, control rats not receiving thiram were pair-fed to the experimental ones. On d 30 of the experiment the animals were weighed and sacrificed, and the following organs were weighed: liver, kidneys, heart, epididymal and perirenal fat pads, testes, seminal vesicles, tibia, adrenals, and thyroid. Liver concentrations of lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, ATP, and ADP were determined by enzymatic-spectrofluorimetric assay. For each parameter studied and each thiram dosage, values for treated rats were compared to those for control rats and the probability under the null hypothesis was computed. These probabilities were transformed into probits, logits, or "Weibull transforms" and plotted against the logarithms of the respective doses. Models were fitted to the data by linear regression techniques. Finally, the dose inducing the least significant difference (LSD dose), and the dose considered "safe" at P = 0.95, 0.99, and 0.999 were calculated. Significant pesticide-induced changes in the following parameters were found: food intake; weights of the whole body, kidneys, epididymal and perirenal fat pads, testes, and seminal vesicles; and liver beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios. As the models did not differ in fit to the experimental data or in computed LSD doses, they were discriminated on the grounds of their underlying theoretical assumptions and their prediction of safe doses in a long-term study. The log-probit model was rejected for the former reason, and it was shown that the Weibull model foresees a nonnegligible risk of change, with thiram feeding at low doses, for too many parameters. The analysis resulted in the selection of the log-probit model for further use. Weight of fatty tissues was the most sensitive parameter and, using the log-probit model, the predicted no-effect dose at the 95 percent confidence level was 38 ppm thiram in the diet.
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PMID:Dietary no-effect level of a dithiocarbamate fungicide, thiram, evaluated from measurement data on rats. I. Choice of the model of the dose-response relationship. 739 1

An outwardly rectifying conductance was observed in primary cultured human epididymal cells under the patch-clamp whole-cell configuration in KCl pipette and bath solutions. Removal of Cl- from intracellular and extracellular solutions did not affect this conductance, but substitution of K+ with N-methyl-D-glucamine in both solutions completely blocked the current. The current magnitude was also found to be affected by intracellular but not extracellular K+ concentrations. The conductance could be inhibited by the cation channel blockers, barium and tetraethylammonium chloride. Single-channel recordings from the same cell population also revealed the presence of a conductance of about 150 pS with voltage dependence and blocker sensitivity similar to those observed for the whole-cell current. This cation conductance was not affected by changes in solution osmolarity but could be activated by extracellular ATP. ATP activation of the conductance could be mimicked by ionomycin and inhibited by BAPTA-AM, an agent that suppresses intracellular free Ca2+. This ATP-regulated cation conductance may be responsible for secreting K+ across the epididymal epithelium, resulting in an observed K+ concentration much higher in the lumen of the epididymis than in the blood.
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PMID:An ATP-activated cation conductance in human epididymal cells. 753

The regulatory pathways involved in the ATP-stimulated Cl- secretion across rat epididymal epithelium were investigated by the short-circuit current (ISC) technique. Biphasic characteristic was observed in the ISC responded to ATP (0.01-10 microM). Inhibitor of P1 receptor, 8-phenyltheophylline (up to 100 microM), did not have any effect on both phases of the ATP-stimulated ISC. The order of potency for stimulation of the two phases in ISC was ATP > ADP >> AMP, adenosine, consistent with the presence of P2-purinoceptors. Cl- channel blocker, disulfonic acid stilbene (DIDS, 300 microM), only inhibited the first peak of the ATP-stimulated ISC while diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced both, indicating the involvement of different conductance pathways. DIDS was found to have an inhibitory effect on Ca(2+)-activated ISC (induced by ionomycin, 10 microM) but not cAMP-activated ISC (induced by forskolin, 1 microM) which could only be blocked by DPC. Both peaks of the ATP-activated ISC could be significantly inhibited by pretreatment with a Ca(2+)-chelating agent, BAPTA-AM (50 microM). An increase in cellular cAMP content upon stimulation of ATP was measured by radioimmunoassay. No significant increase in cAMP production was observed in cells stimulated with adenosine. The ATP-induced cAMP increase was prevented by pretreatment with BAPTA-AM (100 microM) indicating that cAMP production upon ATP stimulation was secondary to an increase in intracellular Ca2+ concentration. These results indicate that the ATP-stimulated Cl- secretion could be mediated by Ca2+ and cAMP-dependent regulatory pathways giving rise to the biphasic nature of the ATP-induced ISC.
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PMID:Different regulatory pathways involved in ATP-stimulated chloride secretion in rat epididymal epithelium. 762 76

The effects of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), on Cl- secretion by rat cauda epididymal epithelium were studied through use of the short-circuit current (Isc) technique. PMA alone could stimulate the Isc in a dose-dependent manner. The PMA-induced Isc was blocked by the Cl channel blocker, diphenylamine-2-carboxylate, but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. PMA also exerted an inhibitory effect on the subsequent Ca(2+)-activated Isc. ATP or ionomycin-induced Isc was significantly reduced by treatment with PMA (5-10 min). Both stimulatory and inhibitory effects of PMA could be mimicked by a diacylglycerol analog, 1,2-dioctanoyl-sn-glycerol, but not an inactive analog of PMA, 4 alpha-phorbol 12,13-didecanote (4 alpha D). Down-regulation of protein PKC by prolonged treatment of epididymal cells with PMA (12 h) diminished both stimulatory and inhibitory effects of PMA on Isc. These results suggest that the dual effect of PMA on Isc was mediated by PKC. However, the PKC inhibitor, calphostin C, could block the inhibitory effect of PMA on ATP-induced Isc but not the stimulatory effect of PMA alone on Isc. The stimulatory effect of PMA was apparent only when PMA was applied to the apical aspect; in contrast, the inhibitory effect of PMA on ATP-induced Isc was readily seen with application of PMA to either side of the epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stimulatory and inhibitory effects of a phorbol ester on chloride secretion by rat epididymal epithelium. 775 57

A protein kinase that causes phosphorylation of serine and threonine residues of casein has been partially purified from goat cauda-epididymal sperm plasma membrane and characterized. The kinase, solubilized from the membrane with 1.0% Triton X-100, was purified to 480-fold by using DEAE-cellulose and casein-Sepharose affinity chromatographic techniques. The kinase is a strongly basic protein with pI of 9.5. The enzyme has a molecular mass of 310 kilodaltons as estimated by Sephacryl S-300 gel exclusion. The kinase showed affinity for protein substrates in the order membrane proteins > casein > phosvitin > histone > protamine. The apparent Km values of the kinase for casein and membrane proteins were 1 and 0.15 mg/mL, respectively. The synthetic peptides Kemptide and poly(Glu80Tyr20) did not serve as substrates of the enzyme. ATP, rather than GTP or PP(i), is the donor of phosphate for the phosphorylation reaction. Cyclic AMP and GMP, NaCl (0.25 M), KCl (0.25 M), Ca2+, calmodulin, phosphatidylserine, and muscle protein kinase inhibitor had no appreciable effect on the kinase activity. Heparin (0.5 microgram/mL) showed high affinity for inhibiting only 40% of the kinase activity, whereas polyamines at a relatively high concentration (5 mM) inhibited 40-50% of the enzymic activity. The kinase appears to be distinct from other protein kinases including casein kinases. The activity of the kinase derived from the purified sperm plasma membrane was markedly (approximately 90%) lost when the intact spermatozoa were pretreated with diazonium salt of sulfanilic acid, a membrane nonpenetrating surface probe.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and characterization of a protein kinase from goat sperm plasma membrane. 784 Sep 41

Bovine epididymal sperm resuspended in ionic buffers take up relatively large amounts of calcium. This uptake, which is almost entirely mitochondrial, apparently bypasses the sperm cytosol. The direct mitochondrial loading is an unusual aspect of sperm calcium uptake, which suggests that the plasma membrane region surrounding the mitochondria should be highly permeable to calcium, whereas the membrane domains surrounding the head and tail regions of sperm should be impermeable. This study was undertaken to determine the role of a plasma membrane calcium ATPase in sperm calcium homeostasis. Kinetics of calcium (45Ca2+) uptake into intact and permeabilized caudal epididymal sperm confirmed that mitochondrial calcium uptake occurs with virtually no resistance from the surrounding plasma membrane. Cytoplasmic calcium accumulation by sperm depleted of intracellular ATP, measured in the presence of mitochondrial calcium uptake inhibitors, showed no increase upon energy depletion as would be expected if an ATP-dependent calcium extrusion mechanism were present. Furthermore, lowering the incubation temperature to further reduce the activity of the calcium ATPase in these energy-depleted sperm was also without effect on calcium accumulation. The calcium ATPase inhibitor vanadate, even at high concentrations, failed to increase intracellular 45Ca2+ accumulation. However, vanadate was effective in inhibiting motility showing that the compound was accumulated into sperm to inhibit flagellar dyenin ATPase. Therefore, the lack of effect of vanadate on 45Ca2+ accumulation was not due to its inability to enter sperm. Other calcium ATPase inhibitors such as quercetin, thapsigargin, and cyclopiazonic acid, which readily demonstrate ATP-dependent calcium extrusion in other somatic cells, were also without effect on sperm calcium accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence against a functional ATP-dependent calcium extrusion mechanism in bovine epididymal sperm. 791 84

In common mammals, sperm leaving the testis are incapable of fertilizing a female gamete. Sperm have limited biosynthetic capability and need to minimize demand for ATP. Hence, modification of sperm to achieve their maturation requires pre-programmed cleavage of integral molecules (planned self-modification) and remodelling by action of molecules found in the suspending fluids. Most of these biocatalysts are secreted by a series of specialized regions in the epididymal epithelium, but some are provided in seminal plasma. The role of the epididymis in sperm maturation is postulated to be 'setting a series of triggers' each capable of initiating cellular changes either at emission or near or in the oocyte, and 'setting a safety' for each trigger to prevent premature occurrence of the event. The attributes required in a spermatozoon for in vitro fertilization and natural mating are different, and their expression is dependent on the site of sperm sampling. Some attributes needed for fertility are probably like an on-off switch, whereas others probably allow a gradually reduced probability of success before going to the off position (analogous to a conventional light switch and a dimmer-type light switch). All essential attributes of a spermatozoon must be expressed in a 'combined effective amount' for that cell to be fertile. Because of mixing, in any segment of the epididymal duct the population of sperm is heterogeneous in age and biological status. Thus, when assessing sperm maturation it is necessary to establish the proportion of sperm that has completed and retained all steps of maturation necessary to achieve fertilization of oocytes under the conditions imposed. In a normal animal, most sperm leaving the epididymis have a 'combined effective amount' of attributes, and the population has a high fertilizing potential.
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PMID:The epididymis and sperm maturation: a perspective. 815 87


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