Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Even in the presence of ATP, the motility of demembranated fowl spermatozoa was negligible at the avian body temperature of 40 degrees C. Motility could be restored by the addition of calyculin A, okadaic acid, specific inhibitors of phosphatase type 1 (PP1) and PP-2A, and inhibitor 1 or inhibitor 2, which are specific inhibitors of protein phosphatase type 1 (PP1). Demembranated spermatozoa, stimulated by calyculin A or okadaic acid, lost their motility following the addition of 1 mM CaCl2, but this was restored gradually by the stepwise addition of EGTA. Immunoblotting of sperm extract using an antibody to PP1 revealed a major cross-reacting protein of 36-37 kDa, which corresponded to the molecular weight of the known catalytic subunit of PP1. These results suggest that PP1 present in the fowl sperm axoneme may be involved in the inhibition of fowl sperm motility at 40 degrees C via Ca(2+)-dependent regulatory systems.
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PMID:Presence of protein phosphatase type 1 and its involvement in temperature-dependent flagellar movement of fowl spermatozoa. 806 11

The involvement of cAMP in the process of sperm capacitation has been the subject of several studies. In addition, the importance of protein-tyrosine phosphorylation in this process has been investigated, although only a few studies have been reported in the human. Since agents regulating the intracellular concentrations of cAMP affect sperm capacitation rates, the role of cAMP on the expression of phosphotyrosine-containing proteins was investigated during human sperm capacitation. Fetal cord serum ultrafiltrate, a known capacitation inducer in human spermatozoa, caused an increase in the phosphotyrosine content of 105- and 81-kDa proteins (p105 and p81), the two major phosphotyrosine-containing proteins of human spermatozoa. Similar effects were observed when spermatozoa were incubated with phosphodiesterase inhibitors or cell-permeant cAMP analogs, suggesting that cAMP is involved in these two processes. Forskolin, an adenylyl cyclase activator, also caused an increase in both sperm capacitation rates and tyrosine phosphorylation of p105 and p81, while 12-O-tetradecanoyl phorbol 13-acetate stimulated both capacitation and tyrosine phosphorylation of p105 and p81 only when spermatozoa were incubated in the presence of bicarbonate, in agreement with its reported effects on cAMP production and hamster sperm capacitation. The inhibition of these phenomena by cAMP-dependent protein kinase inhibitors, and the stimulation by protein phosphatase inhibitors, suggest that Ser/Thr protein phosphorylation plays an important role in the regulation of both sperm capacitation and protein-tyrosine phosphorylation pathways. However, observations that both calyculin A and okadaic acid stimulated sperm capacitation, whereas only calyculin A increased p105 and p81 phosphotyrosine content and sperm velocity, suggest that protein phosphatase PP1 is involved in the two latter phenomena while PP2A mediates sperm capacitation. These results suggest that divergent pathways might regulate tyrosine phosphorylation of p105 and p81 and sperm capacitation after cAMP-dependent phosphorylation of an intermediate protein.
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PMID:Cyclic adenosine 3',5'monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. 886 88

Spermatozoa undergoing capacitation, a necessary prerequisite event to successful fertilization that can be induced in vitro by reactive oxygen species (ROS), generate superoxide anion (O2.-). Because, in neutrophils, the generation of O2.- is associated with tyrosine phosphorylation of several proteins, the aim of the present study was to investigate the association between protein-tyrosine phosphorylation and ROS-induced human sperm capacitation. Human spermatozoa express two major phosphotyrosine-containing proteins of 105 and 81 kDa, the phosphotyrosine content of which is increased when spermatozoa are incubated under capacitating conditions. Superoxide dismutase and catalase abolish both sperm capacitation and tyrosine phosphorylation of p105 and p81, suggesting the involvement of O2.- and hydrogen peroxide in these two processes. Inhibitors of NADPH oxidase, the enzyme responsible for the neutrophil's respiratory burst, decrease both p105 and p81 tyrosine phosphorylation and sperm capacitation while hydrogen peroxide stimulates these two processes. Tyrosine phosphorylation of p105 and p81 occurs through a herbimycin A-sensitive tyrosine kinase, and sperm incubation with phosphotyrosine-protein phosphatase inhibitors results in an increase in phosphotyrosine content of these two proteins. Indirect immunocytochemical studies reveal phosphotyrosine-containing proteins mostly in the principal piece of the flagellum, in agreement with the localization of p105 and p81 in the human sperm fibrous sheath. Although tyrosine phosphorylation of p105 and p81 and sperm capacitation are related in a time-dependent fashion, some discrepancies are observed in the regulation of these two processes according to the redox status of the spermatozoa.
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PMID:Regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives. 901 27

The motility of demembranated fowl spermatozoa was vigorous at 30 degrees C in the presence of ATP, but decreased markedly following the addition of recombinant protein phosphatase type 1 (PP-1) supplemented with Mn2+. This inhibition was not restored by the addition of cAMP, within the range 1-1000 microM, but instantly restored by the addition of 50 ng/ml trypsin. Phosphorylation of demembranated fowl sperm proteins during incubation with [gamma-32P]ATP at 30 degrees C was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A marked difference in phosphorylation status was observed in approximately 116, 86, 79, 50 and 29-kDa proteins. These proteins were dephosphorylated in the presence of PP-1 and Mn2+ compared with those in control samples. These results suggest that PP-1-mediated dephosphorylation of some of these proteins of the axoneme and/or accessory cytoskeletal components of fowl spermatozoa may be involved in the inhibition of motility.
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PMID:Regulation of fowl sperm flagellar motility by protein phosphatase type 1 and its relationship with dephosphorylation of axonemal and/or accessory cytoskeletal proteins. 919 45

A rapid and gentle procedure for preparing demembranated, cytosol-free sperm models was applied to fowl spermatozoa. Intact spermatozoa were introduced to a Triton X-100-containing extraction medium layered on top of a discontinuous Percoll gradient in a 1.5 ml microfuge tube. After brief exposure to the extraction medium, spermatozoa were separated from the plasma membrane and detergent-soluble components by centrifugation through a 55% Percoll layer, finally collecting on top of a 90% Percoll cushion from where they were recovered. Optimum conditions consisted of a Triton X-100 concentration in the extraction medium of 0.15%, duration of demembranation time of 1.5 min and ATP concentration in the reactivation medium of 0.5 mM. Demembranated sperm models obtained by this procedure could be reactivated, and the motility at 30 degrees C was more than 60%, but negligible at 40 degrees C. These values were similar to those obtained from the conventional method, in which centrifugation is not carried out, and which results in some of the cytosolic components being transferred to the reactivation medium along with the spermatozoa. Inhibition of motility was observed following the addition of EGTA or myosin light chain kinase (MLCK) substrate peptide at 30 degrees C, whilst the presence of protein phosphatase inhibitors, such as calyculin A or okadaic acid, permitted the restoration of motility at 40 degrees C. These results demonstrate that the axoneme and/or accessory cytoskeletal components are directly involved in the temperature-dependent regulatory system of fowl sperm motility in the absence of plasma membrane and/or soluble components of cytoplasm.
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PMID:Temperature-dependent flagellar motility of demembranated, cytosol-free fowl spermatozoa. 988 71

Human and monkey ejaculated sperm contain protein phosphatase-1 (PP1), PP1 inhibitor 2 (12), and glycogen synthase kinase-3 (GSK-3). Inhibition of ejaculated human sperm protein phosphatase (PP) activity with calyculin-a (CL-A) significantly stimulates motility, implicating protein dephosphorylation in motility regulation. The present experiments were conducted to characterize and compare PP and GSK-3 activity in monkey caput and caudal epididymal sperm, to determine the cellular distribution of these enzymes, and to test the thesis that epididymal sperm PP activity is inversely related to motility. Caput epididymal sperm populations, (8.8% motile) contained levels of PP activity that were >3 times as high as those of caudal spermatozoa. This PP activity was further identified by inhibitor response profiles as PP1. In both caput and caudal sperm, the majority of this PP1 activity was localized in 100,000 x g soluble fractions. Western blot analysis indicated that a portion of this difference was the result of elevated amounts of PP1 in caput compared with caudal epididymal sperm. The presence of GSK-3 activity was undetectable in 100,000 x g insoluble fractions of epididymal sperm, whereas both caput and caudal sperm soluble fractions contained GSK-3 activity, which was approximately threefold higher in caput sperm compared with caudal populations. Treatment of caput epididymal sperm from the rhesus macaque with the PP inhibitor CL-A resulted in a significant, dose-dependent increase from 8 to 38% motile cells (without any effect on their path velocity). In contrast, CL-A had no significant influence on either percent motility or path velocity of caudal epididymal sperm. Cytosolic PP1 and GSK-3 activities appear to be inversely related to the motility of monkey epididymal sperm and may have a regulatory role in the development of the potential for motility in epididymal sperm.
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PMID:Motility potential of macaque epididymal sperm: the role of protein phosphatase and glycogen synthase kinase-3 activities. 1010 Apr 73

Plasma membranes of caput and cauda epididymal spermatozoa of hamster exhibited protein phosphatase activity. This membrane-associated protein phosphatase was identified as a protein tyrosine phosphatase based on its ability to hydrolyse a substrate specific for PTPase, by inhibition of its activity with a specific inhibitor of PTPase (sodium orthovanadate) and by the inability to inhibit its activity with calyculin, okadaic acid, trifluoperazine, calcium, EGTA, and EDTA, which are specific inhibitors of other protein phosphatases, namely PP-1, PP-2A, PP-2B, and PP-2C respectively. The specific activity of the protein tyrosine phosphatase both in the caput and cauda epididymal sperm plasma membranes was similar, implying that the enzyme may not be solely responsible for the differential phosphorylation of membrane proteins observed during maturation (Uma Devi et al. 1997. Mol Reprod Dev 47:341-350). Thus the significance of the PTPase activity in epididymal maturation still remains to be determined. The membrane-associated PTPase may not be essential for acquisition of motility. However, it appears that the activity is essential for the sustenance of motility since sodium orthovanadate, which specifically inhibits PTPase activity, also inhibits motility of spermatozoa and decreases the overall velocity of the spermatozoa by decreasing the average path velocity, straight line velocity, curvilinear velocity, and amplitude of lateral head displacement of the treated spermatozoa.
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PMID:Plasma membrane-associated protein tyrosine phosphatase activity in hamster spermatozoa. 1023 Aug 15

Both intact and demembranated fowl spermatozoa were incubated at 30 degrees C and 40 degrees C with adenosine, 3-deazaadenosine and homocysteine thiolactone. This combination of products is known to block intracellular protein-carboxyl methylation reaction. The motility of intact spermatozoa incubated at 30 degrees C was vigorous but decreased markedly after the addition of 100 microM adenosine+100 microM 3-deazaadenosine+100 microM homocysteine thiolactone. During this incubation period, the intracellular ATP concentrations of spermatozoa were maintained at approximately 40 nmol ATP/10(9) cells, in spite of the inhibition of motility. The motility of demembranated spermatozoa at 30 degrees C was not inhibited by the same concentrations of blocker. At 40 degrees C, the motility of intact spermatozoa without any effectors was almost negligible. The addition of blocker did not appreciably affect the motility of spermatozoa, which remained almost negligible. In contrast, motility became vigorous even at 40 degrees C when intact spermatozoa were suspended in fluid to which had been added 1 mM CaCl(2) or 100 nM calyculin A, a specific inhibitor of protein phosphatase-type 1 and -type 2. Stimulation of motility by Ca(2+) or calyculin A was inhibited by the presence of a blocker. Contrary to that of intact spermatozoa, the motility of demembranated spermatozoa stimulated by protein phosphatase inhibitor at 40 degrees C was not inhibited by the presence of a blocker. These results suggest that protein-carboxyl methylation may be involved in the regulation of fowl sperm motility. Furthermore, it appears that the methylating enzyme may be present in the cytoplasmic matrix and/or the plasma membrane but not retained in the axoneme and/or accessory cytoskeletal components.
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PMID:The possible role of protein-carboxyl methylation in the regulation of flagellar movement of fowl spermatozoa. 1070 Jun 51

Complementary DNAs encoding two subunits of scallop (Patinopecten yessoensis) testis calcineurin were cloned, and the nucleotide sequences of their coding regions were determined. The deduced amino acid sequences of the catalytic subunit, calcineurin A (486 amino acid residues, M(r) 55,005.91), and the regulatory subunit, calcineurin B (170 residues, M(r) 19,237.67), showed high similarity to those of mammalian calcineurins, especially to the brain-type ones rather than to the testis-specific isoforms. Northern blot analysis showed that only a single species for each subunit was expressed in testis and the expression of each subunit increased dramatically from January to March during the maturation stages of the one-year cycle. The period when the maximum amount of mRNAs for calcineurin was expressed corresponds to the one immediately after meiosis, that is, the maturation stage in which 20-80% of the average testis is occupied by spermatozoa. The result is consistent with the one as to the expression of the testis-specific isoform of calcineurin A in mouse, which occurs immediately after meiosis. This is the first report on the stage-specific expression of calcineurin in invertebrate testis and its sequence similarity to the mammalian brain-type isoforms may indicate that the mammalian testis-specific isoforms appeared in evolution after the divergence of mammals from the mollusks and then diverged rapidly for specific functions in testis.
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PMID:Molecular cloning of cDNA encoding two subunits of calcineurin from scallop testis: demonstration of stage-specific expression during maturation of the testis. 1078 81

A phosphoprotein phosphatase (PPase M-I) that dephosphorylates serine and threonine residues of histones was isolated from the goat cauda-epididymal sperm plasma membrane and partially characterized. The PPase was solubilized from the sperm membrane by treating it with 0.1 N NaOH at pH 11.4 and the solubilized enzyme was partially purified by concanavalin A-sepharose affinity chromatography and high-performance liquid chromatography (HPLC), revealing it to be a 520-kDa protein. The PPase gave a single protein band in native polyacrylamide gel electrophoresis (PAGE), but in the presence of SDS it resolved into multiple proteins (35-170 kDa) showing that the isolated enzyme contained a few contaminating proteins. The enzyme is a glycoprotein because it binds with high affinity to concanavalin A. It was maximally active at pH 8.0 and its activity was not dependent on bivalent metal ions. The enzyme is a specific phosphatase as it displayed higher affinity for dephosphorylation of large molecular weight phosphate esters. The PPase showed broad substrate specificity for the dephosphorylation of a variety of proteins. The membrane-associated PPase was strongly (70-80%) inhibited by detergents (0.5%) such as Nonidet P-40, Lubrol PX, Triton X-100 and Tween-20. Pyrophosphate (5 mm) and orthovanadate (400 microM) had no significant effect on the activity of the isolated PPase whereas polyamines such as spermine (10 mM) and spermidine (10 mM) slightly inhibited (20%) the enzymatic activity. Inorganic phosphate (10 mM) and NaF (10 mM), the well-known inhibitors of the cytosolic PPases, had no appreciable effect on the activity of PPase M-I, indicating that the membrane-bound PPase is distinct from the cytosolic PPases. The enzyme was radiolabelled when the intact spermatozoa were subjected to lactoperoxidase-mediated radioiodination reaction. The results show that the PPase M-I is an ecto-enzyme that may play an important role in sperm physiology by causing the dephosphorylation of the sperm outer surface phosphoproteins.
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PMID:Partial purification and characterization of a phosphoprotein phosphatase from sperm plasma membrane. 1097 6


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