Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Evidence for the occurrence of an ecto-(adenosine triphosphatase) in rat epididymal spermatozoa.

ABSTRACT: Intact spermatozoa from rat cauda epididymis possess a Mg2+-dependent ATPase activity that hydrolyses externally added [gamma-32P]ATP. The ATPase reaction was linear with time for approx. 6 min and there was no detectable uptake of ATP by these cells. The ATPase activity of the whole spermatozoa was not due to leakage of the intracellular enzymic activity, contamination of the broken cells or any possible cell damage during incubation and isolation of spermatozoa. The activity of the enzyme was strongly inhibited (approx. 85%) by p-chloromercuribenzenesulphonic acid (50 microM) or the diazonium salt of sulphanilic acid (50 microM), which are believed not to enter the cells, whereas ouabain (0.5 mM), NaF (10 mM), NaN3 (2.5 mM) and oligomycin (5 microM) had no appreciable effect on the activity of the spermatozoal APTase. There was little loss of ATPase activity from the cells when washed with 0.5 mM-EDTA and an iso-osmotic or hyperosmotic medium. These data are consistent with the view that the observed ATPase activity is located on the external surface of spermatozoa. The sperm ecto-ATPase activity is resistant to the action of proteinases (50 micrograms/ml), namely trypsin, chymotrypsin and Pronase. Studies with various unlabelled phosphate esters indicate that the sperm ecto-ATPase is not a non-specific phosphatase and it has high degree of substrate specificity for ATP.

SUBMITTER: Majumder GC

PROVIDER: S-EPMC1161656 | biostudies-other | 1979 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Enzymic characteristics of ecto-adenosine triphosphatase in rat epididymal intact spermatozoa.

Project description:Ecto-ATPase in rat cauda-epididymal intact spermatozoa has a high degree of substrate specificity for the hydrolysis of ATP and dATP rather than of ADP, AMP, GTP, dGTP, CTP, dCTP, TTP and UTP. The enzyme is activated by bivalent metal ions in the order Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+. The apparent Km values of the enzyme for Mg2+, Mn2+, Co2+ and Ca2+ are approx. 80, 100, 100 and 150 microM respectively. Addition of Ca2+ (0.1 or 1 mM) gives no further stimulation of the Mg2+-activated ecto-ATPase activity. The apparent Km value of the enzyme for ATP is 95 microM. Pi (16 mM) inhibits the enzymic activity (by 25%), whereas Na+ (50 mM) or K+ (10 mM) alone or in combination, polyamines (spermine and spermidine; 1--12.5mM) and nucleic acids (yeast RNA and calf thymus DNA; 0.12 or 0.62 mg/ml) had no significant effect on the activity of the enzyme. Orthovanadate at a relatively low concentration (20 microM) strongly inhibits (approx. 50%) the ecto-ATPase activity. Vanadate inhibition can be reversed by noradrenaline (2.5 mM). The vanadate-sensitivity of the enzyme increases markedly during spermatozoal maturation in the epididymis. However, the activity of the spermatozoal ecto-ATPase decreases progressively during the epididymal transit of the testicular spermatozoa.

| S-EPMC1162859 | biostudies-other

Enzymic characteristics of an ecto-cyclic AMP-dependent protein kinase in rat epididymal spermatozoa.

Project description:Intact spermatozoa from rat cauda epididymides possess an ecto-(cyclic AMP-dependent protein kinase) activity that causes the transfer of the terminal phosphate group of ATP to the serine residues of all the histone fractions. The enzyme showed a high degree of substrate specificity for the phosphorylation of histones rather than protamine, casein and phosvitin. The cell-external-surface protein kinase requires Mg2+ for activity, and other bivalent cations such as Mn2+ and Co2+ can substitute partially for Mg2+, whereas Ca2+ and Zn2+ are potent inhibitors of the enzyme. The enzyme has markedly higher affinity for cyclic AMP than for other cyclic nucleotides for its activation, with an apparent Km value for cyclic AmP of 80 nM. Spermatozoal ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. There was no loss of kinase activity from the cells when washed with 2 mM-EDTA, and the histones phosphorylated by intact spermatozoa were located outside the cells. Protein kinase activity of intact cells was strongly inhibited (approx. 90%) by p-chloromercuribenzenesulphonic acid (10 microM), which is believed not to enter the cells. These data provide further support for the localization of a protein kinase on the external surface of spermatozoa.

| S-EPMC1162860 | biostudies-other

Evidence that Mg2+- or Ca2+-activated adenosine triphosphatase in rat pancreas is a plasma-membrane ecto-enzyme.

Project description:Preparations of enzymically dispersed rat pancreatic cells hydrolyse externally added nucleoside triphosphates and diphosphates at high rates in the presence of Mg2+ or Ca2+. The lack of response to specific inhibitors and activators differentiates this hydrolytic activity from that of other well-characterized ion-transporting ATPases. Studies based on inactivation of this hydrolytic activity by the covalently reacting, slowly permeating probe diazotized sulphanilic acid indicated that this nucleoside tri- and di-phosphatase is primarily a plasma-membrane ecto-enzyme. It is the major ATPase activity associated with intact cells, homogenates and isolated plasma-membrane fractions. Concanavalin A stimulates this ATPase activity of intact cells and isolated plasma-membrane fractions. The insensitivity of this ATPase activity to univalent ions and inhibitors of pancreatic electrolyte secretion, taken together with the evidence that the active site is externally located, suggests that this enzyme is not directly involved in HCO3- secretion in the pancreas. Its actual function remains unknown.

| S-EPMC1152210 | biostudies-other

Freezability biomarkers in bull epididymal spermatozoa.

Project description:Sperm cryopreservation is an important tool for storing genetic traits and assisted reproduction techniques. Several studies have developed semen cryopreservation protocols. However, the sperm proteome is different between ejaculated and epididymal spermatozoa and little is known about cryopreservation effects on epididymal spermatozoa. Therefore, our study aimed to (i) investigate the differences of sperm parameters based on the freezing tolerance of spermatozoa and (ii) identify potential markers to predict the freezability of bull epididymal spermatozoa. Our preliminary study demonstrated that spermatozoa from individual bulls differ in cryopreservation freezability. We categorized spermatozoa into high freezing-tolerant spermatozoa and low freezing-tolerant spermatozoa group based on sperm motility after freezing/thawing. We evaluated several sperm functional parameters, including sperm motility/motion kinematics, sperm speed parameters, viability, mitochondrial activity, and capacitation status. Our results demonstrated that motility, sperm speed parameters, viability, and mitochondrial membrane potential had significant differences between the two groups but motion kinematics and capacitation status did not. In addition, the concentration of three proteins - glutathione s-transferase mu 5, voltage-dependent anion-selective channel protein 2, and ATP synthase subunit beta, differed between both groups. Thus, our research highlighted differences in bull epididymal spermatozoa freezability upon cryopreservation and these proteins might be useful markers to select high freezing-tolerant epididymal spermatozoa.

| S-EPMC6728342 | biostudies-literature

Data in support of peptidomic analysis of spermatozoa during epididymal maturation.

Project description:The final differentiation of the male germ cell occurs in the epididymal duct where the spermatozoa develop the ability to be motile and fertilize an ovum. Understanding of these biological processes is the key to understanding and controlling male fertility. Comparative studies between several epididymal maturation states could be an informative approach to finding sperm modifications related to maturation and fertility. Here we show the data from differential peptidomic/proteomic analyses on spermatozoa isolated from 4 epididymal regions (immature to mature stage) using a profiling approach based on MALDI-TOF mass spectrometry and, combined to top-down MS in order to identify peptidoforms and proteoforms. By this way, 172m/z peaks ranging between 2 and 20 kDa were found to be modified during maturation of sperm. A total of 62m/z were identified corresponding to 32 different molecular species. The interpretation of these data can be found in the research article published by Labas and colleagues in the Journal of Proteomics in 2014 [1].

| S-EPMC4459559 | biostudies-literature

Cryopreservation Differentially Alters the Proteome of Epididymal and Ejaculated Pig Spermatozoa.

Project description:Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How these proteome changes relate to the loss of sperm function during cryopreservation remains unsolved. The present study aimed to clarify this issue evaluating differential changes in the proteome of fresh and frozen-thawed pig spermatozoa retrieved from the cauda epididymis and the ejaculate of the same boars, with clear differences in cryotolerance. Spermatozoa were collected from 10 healthy, sexually mature, and fertile boars, and cryopreserved using a standard 0.5 mL-straw protocol. Total and progressive motility, viability, and mitochondria membrane potential were higher and membrane fluidity and reactive oxygen species generation lower in frozen-thawed (FT) epididymal than ejaculated spermatozoa. Quantitative proteomics of fresh and FT spermatozoa were analyzed using a LC-ESI-MS/MS-based Sequential Window Acquisition of All Theoretical Spectra approach. Cryopreservation quantitatively altered more proteins in ejaculated than cauda epididymal spermatozoa. Differential protein-protein networks highlighted a set of proteins quantitatively altered in ejaculated spermatozoa, directly involved in mitochondrial functionality which would explain why ejaculated spermatozoa deteriorate during cryopreservation.

| S-EPMC6479301 | biostudies-literature

Fibroblast growth factor 2 upregulates ecto-5'-nucleotidase and adenosine deaminase via MAPK pathways in cultured rat spinal cord astrocytes.

Project description:Adenosine triphosphate (ATP) and adenosine are neurotransmitters and neuromodulators in the central nervous system. Astrocytes regulate extracellular concentration of purines via ATP release and its metabolisms via ecto-enzymes. The expression and activity of purine metabolic enzymes in astrocytes are increased under pathological conditions. We previously showed that fibroblast growth factor 2 (FGF2) upregulates the expression and activity of the enzymes ecto-5'-nucleotidase (CD73) and adenosine deaminase (ADA). Here, we further demonstrate that this occurs in concentration- and time-dependent manners in cultured rat spinal cord astrocytes and is suppressed by inhibitors of the FGF receptor as well as the mitogen-activated protein kinases (MAPKs). We also found that FGF2 increased the phosphorylation of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK, leading to the increased expression and activity of CD73 and ADA. Our findings reveal the involvement of FGF2/MAPK pathways in the regulation of purine metabolic enzymes in astrocytes. These pathways may contribute to the control of extracellular purine concentrations under physiological and pathological conditions.

| S-EPMC7855126 | biostudies-literature

Thiol changes during epididymal maturation: a link to flagellar angulation in mouse spermatozoa?

Project description:Caput epididymal wild-type spermatozoa and cauda epididymal spermatozoa from mice null for the adenylyl cyclase Adcy10 gene are immotile unless stimulated by a membrane-permeant cyclic AMP analogue. Both types of spermatozoa exhibit flagellar angulation where the head folds back under these conditions. As sperm proteins undergo oxidation of sulfhydryl groups and the flagellum becomes more stable to external forces during epididymal transit, we hypothesized that ADCY10 is involved in a mechanism regulating flagellar stabilization. Although no differences were observed in global sulfhydryl status between caput and cauda epididymal spermatozoa from wild-type or Adcy10-null mice, two-dimensional fluorescence difference gel electrophoresis was performed to identify specific mouse sperm proteins containing sulfhydryl groups that became oxidized during epididymal maturation. A-kinase anchor protein 4, fatty acid-binding protein 9 (FABP9), glutathione S-transferase mu 5 and voltage-dependent anion channel 2 exhibited changes in thiol status between caput and cauda epididymal spermatozoa. The level and thiol status of each of these proteins were quantified in wild-type and Adcy10-null cauda epididymal spermatozoa. No differences in the abundance of any protein were observed; however, FABP9 in Adcy10-null cauda epididymal spermatozoa contained fewer disulfide bonds than wild-type sperm cells. In caput epididymal spermatozoa, FABP9 was detected in the cytoplasmic droplet, principal piece, midpiece, and non-acrosomal area of the head. However, in cauda epididymal spermatozoa, this protein localized to the perforatorium, post-acrosomal region and principal piece. Together, these results suggest that thiol changes during epididymal maturation have a role in the stabilization of the sperm flagellum.

| S-EPMC4253137 | biostudies-literature

Interaction of HSF1 and HSF2 with the Hspa1b promoter in mouse epididymal spermatozoa.

Project description:The Hspa1b gene is one of the first genes expressed after fertilization, with expression observed in the male pronucleus as early as the one-cell stage of embryogenesis. This expression can occur in the absence of stress and is initiated during the minor zygotic genome activation. There is a significant reduction in the number of embryos developing to the blastocyte stage when HSPA1B levels are depleted, which supports the importance of this protein for embryonic viability. However, the mechanism responsible for allowing expression of Hspa1b during the minor zygotic genome activation (ZGA) is unknown. In this report, we investigated the role of HSF1 and HSF2 in bookmarking Hspa1b during late spermatogenesis. Western blot results show that both HSF1 and HSF2 are present in epididymal spermatozoa, and immunofluorescence analysis revealed that some of the HSF1 and HSF2 proteins in these cells overlap the 4',6'-diamidino-2-phenylindole-stained DNA region. Results from chromatin immunoprecipitation assays showed that HSF1, HSF2, and SP1 are bound to the Hspa1b promoter in epididymal spermatozoa. Furthermore, we observed an increase in HSF2 binding to the Hspa1b promoter in late spermatids versus early spermatids, suggesting a likely period during spermatogenesis when transcription factor binding could occur. These results support a model in which the binding of HSF1, HSF2, and SP1 to the promoter of Hspa1b would allow the rapid formation of a transcription-competent state during the minor ZGA, thereby allowing Hspa1b expression.

| S-EPMC2574705 | biostudies-literature

Role of adenosine deaminase, ecto-(5'-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

Project description:1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.

| S-EPMC1161728 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data