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

1979 ◽  
Vol 183 (3) ◽  
pp. 737-743 ◽  
Author(s):  
G C Majumder ◽  
R Biswas
Keyword(s):  
Atpase Activity ◽  
External Surface ◽  
Diazonium Salt ◽  
Cell Damage ◽  
Enzymic Activity ◽  
Phosphate Esters ◽  
Appreciable Effect ◽  
Atpase Reaction ◽  
Epididymal Spermatozoa ◽  
High Degree

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.

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

1981 ◽  
Vol 195 (1) ◽  
pp. 103-110 ◽  
Author(s):  
G C Majumder
Keyword(s):  
Atpase Activity ◽  
Enzymic Activity ◽  
Testicular Spermatozoa ◽  
Calf Thymus ◽  
Km Value ◽  
Vanadate Inhibition ◽  
Bivalent Metal Ions ◽  
High Degree ◽  
Hydrolysis Of ◽  
Stimulation Of

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.

scholarly journals Decreased Erythrocyte NA+,K+-ATPase Activity and Increased Plasma TBARS in Prehypertensive Patients

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Carlos Ricardo Maneck Malfatti ◽  
Leandro Tibiriçá Burgos ◽  
Alexandre Rieger ◽  
Cássio Luiz Rüdger ◽  
Janaína Angela Túrmina ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Atpase Activity ◽  
Cell Damage ◽  
Venous Blood ◽  
Thiobarbituric Acid ◽  
Normal Blood Pressure ◽  
Thiobarbituric Acid Reactive Substances ◽  
Normotensive Subjects ◽  
The Relationship ◽  
Membrane Cell

The essential hypertension has been associated with membrane cell damage. The aim of the present study is investigate the relationship between erythrocyte Na+,K+-ATPase and lipoperoxidation in prehypertensive patients compared to normotensive status. The present study involved the prehypertensive patients (systolic:136±7 mmHg; diastolic:86.8±6.3 mmHg;n=8) and healthy men with normal blood pressure (systolic:110±6.4 mmHg; diastolic:76.1±4.2 mmHg;n=8) who were matched for age (35±4years old). The venous blood samples of antecubital vein (5 mL) were collected into a tube containing sodium heparin as anticoagulant (1000 UI), and erythrocyte ghosts were prepared for quantifying Na+,K+-ATPase activity. The extent of the thiobarbituric acid reactive substances (TBARS) was determined in plasma. The statistical analysis was carried out by Student’st-test and Pearson’s correlation coefficient. AP<0.05was considered significant. The Na+,K+-ATPase activity was lower in prehypertensive patients compared with normotensive subjects (4.9 versus 8.0 nmol Pi/mg protein/min;P<0.05). The Na+,K+-ATPase activity correlated negatively with TBARS content (r=-0.6;P<0.05) and diastolic blood pressure (r=-0.84;P<0.05). The present study suggests that Na+,K+-ATPase activity reduction and elevation of the TBARS content may underlie the pathophysiological aspects linked to the prehypertensive status.

scholarly journals Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles

1980 ◽  
Vol 188 (3) ◽  
pp. 807-815 ◽  
Author(s):  
E A Vasilyeva ◽  
A F Fitin ◽  
I B Minkov ◽  
A D Vinogradov
Keyword(s):  
Atpase Activity ◽  
Rate Constant ◽  
Adenosine Diphosphate ◽  
Mitochondrial Atpase ◽  
Dependent Manner ◽  
Submitochondrial Particles ◽  
Alternative Pathways ◽  
Atpase Reaction ◽  
Inhibitory Site ◽  
Km Value

The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.

STUDIES ON THE PRESERVATION OF BLOOD: VII. THE INFLUENCE OF INOSINE ON THE METABOLIC BEHAVIOR OF THE ERYTHROCYTE DURING THE PRESERVATION OF BLOOD IN THE COLD

1959 ◽  
Vol 37 (1) ◽  
pp. 69-79
Author(s):  
D. Rubinstein ◽  
S. Kashket ◽  
Rhoda Blostein ◽  
O. F. Denstedt
Keyword(s):  
Cold Storage ◽  
Organic Phosphate ◽  
Phosphate Esters ◽  
Blood Samples ◽  
The Third ◽  
Duration Of Storage ◽  
The Difference ◽  
Blood Specimens ◽  
Metabolic Behavior ◽  
High Degree

Inosine, like other purine nucleosides when added to preserved blood specimens, induces a resynthesis of organic phosphate esters in the erythrocytes. Apart from the difference in rates of reaction, the metabolic reconstitution of the cells is the same at 37° and 4 °C. The reconstitution of the esters occurs rapidly even at 4 °C, and the higher the concentration of inosine added, the more prolonged is the maintenance of the esters. When inosine was added repeatedly to blood samples during storage, a phase of synthesis was induced with each addition of nucleoside. The capacity of the erythrocytes to resynthesize 2,3-diphosphoglyceric acid (DPG) remained normal regardless of the storage age of the sample but the capacity to replenish ATP decreased with the duration of storage. Addition of inosine at the end of the third week of cold storage can effect a high degree of restoration of metabolic viability in the cells at 4 °C within 24 hours.

104 SURVIVAL AND APOPTOSIS RATES AFTER VITRIFICATION OF EARLY, EXPANDED, AND HATCHED CALF AND COW BLASTOCYSTS IN VITRO-PRODUCED USING CRYOTOP AS A DEVICE

2010 ◽  
Vol 22 (1) ◽  
pp. 211
Author(s):  
R. Morató ◽  
D. Izquierdo ◽  
M. T. Paramio ◽  
T. Mogas
Keyword(s):  
Cell Damage ◽  
Survival Rates ◽  
Developmental Competence ◽  
Staining Procedure ◽  
Tunel Staining ◽  
Dna Integrity ◽  
Blastocyst Development ◽  
Integrity Index ◽  
High Degree

Two experiments were designed to determine the ability of 7 and 8 day in vitro-produced blastocysts to survive to the vitrification procedure. Embryos were classified as early blastocysts, expanded, or hatching/hatched blastocysts. Vitrification was done using cryotop devices as described Du et al. (2007). After warming, blastocysts were incubated for 3 h in SOF medium. In the first experiment, we examined the developmental competence of early blastocysts, expanded blastocysts, and hatching/hatched blastocysts after vitrification using the Cryotop method. In the second experiment, warmed blastocysts that had been vitrified on cryotops were fixed in 4% formaldehyde and incubated with TUNEL staining for detecting DNA damaged nuclei. The percentage of TUNEL positive and negative blastomeres was assessed by confocal microscopy. In all experiments cow and calf blastocysts were compared. When the results according to the developmental stage were analyzed, no differences in the survival rates after vitrification of expanded and hatched blastocysts were observed at Day 8 from cow and calf blastocysts. After warming, survival rates of 52.4 and 50% were noted in the groups of expanded and hatched blastocysts respectively from Day 8 cow blastocysts. Similar results were observed in the groups of expanded (54.5%) and hatched (59.4%) blastocysts from Day 8 calf blastocysts. When embryos were vitrified at Day 7, survival rates of 78.4 and 66.7% were observed after warming expanded and hatched blastocysts from cows. In calves, a significant increase in the survival up to 83.3 and 80% was observed after warming expanded and hatched blastocysts. The lowest survival rates were observed in early blastocysts (from 26 to 51%), particularly in those vitrified at Day 8 (≤40%). Following vitrification, cell death was monitored in blastocysts 3 h after warming by TUNEL labelling of cells with damaged DNA. The TUNEL staining procedure was undertaken on Day 7 calf (n = 23) and cow (n = 25) blastocysts, as well as on Day 8 calf (n = 22) and cow (n = 30) blastocysts. When taking into account the stage of blastocyst development, there was a trend toward higher DNA integrity index after warming of expanded and hatched blastocysts compared with early blastocysts in calf and cow groups. So, cell damage was minimal in those blastocysts vitrified at expanded and hatched stage and rates were comparable with those from control fresh blastocysts. These findings suggest that the Cryotop technique seems to be particularly useful for blastocysts presenting a high degree of expansion (expanded and hatched blastocysts), mainly those blastocysts vitrified and warmed at Day 7.

Enzymic characteristics of ecto-phosphoprotein phosphatase in goat epididymal intact spermatozoa

1987 ◽  
Vol 65 (7) ◽  
pp. 602-609 ◽  
Author(s):  
Madhabi Barua ◽  
Gopal C. Majumder
Keyword(s):  
Specific Activity ◽  
Enzymic Activity ◽  
Significant Inhibition ◽  
Appreciable Effect ◽  
Flagellar Motility ◽  
Extracellular Medium ◽  
Intact Cells ◽  
Phosphoprotein Phosphatase ◽  
Nitrophenyl Phosphate ◽  
Enzyme Markers

Intact washed spermatozoa from goat cauda epididymis possess an ecto-phosphoprotein phosphatase that causes dephosphorylation of phosphoserine and phosphothreonine residues of exogenous 32P-labelled histones. The cell-bound ecto-enzyme has high affinity for proteins (histones, casein, phosvitin, and protamine) rather than phosphate esters, such as p-nitrophenyl phosphate, β-glycerophosphate, AMP, and ATP. The activity of the enzyme is inhibited by 4 mM Mg2+, Ca2+, Mn2+, or Co2+. Pi (10 mM), NaF (10 mM), and Zn2+ (1 mM) inhibit the enzyme by approximately 50, 35, and 100%, respectively. Polyamines such as spermine and spermidine at 10 mM each caused significant inhibition (60 and 30%, respectively) of the cell-bound phosphoprotein phosphatase activity, whereas cAMP, orthovanadate, and calmodulin (with or without Ca2+) had no appreciable effect. Under the standard assay conditions, spermatozoa remain intact as evidenced by assay of cytosolic enzyme markers. Both the washed and "native" intact spermatozoa showed nearly the same specific activity of the ectoenzyme. The product of the reaction (Pi) was found in the extracellular medium. Sonication doubled the enzymic activity of the intact cells. The specific activity of the enzyme was nearly fourfold higher in the intact forwardly motile cells than the "composite" spermatozoa. These data provide further support for the localization of a phosphoprotein phosphatase on the external surface of spermatozoa and that the ectoenzyme may have a role in the regulation of flagellar motility.

scholarly journals REACTIONS OF VALONIA AND OF HALICYSTIS TO COLLOIDS

1933 ◽  
Vol 16 (6) ◽  
pp. 925-935 ◽  
Author(s):  
E. M. East ◽  
Benjamin White
Keyword(s):  
Diphtheria Toxin ◽  
Sea Water ◽  
Animal Origin ◽  
Appreciable Effect ◽  
High Tolerance ◽  
High Concentration ◽  
Probable Case ◽  
Egg Albumen ◽  
High Degree ◽  
Very High

From the results of these tests it is clear that both Halicystis and Valonia have a high degree of tolerance for animal peptone, and a very high degree of tolerance for animal proteose and for egg albumen. The products of bacterial growths fostered by these proteins have a deleterious effect upon both species of algae; but, if it were possible to prevent bacterial growth entirely and at the same time supply proper food, it is probable that Halicystis and Valonia would show normal growth indefinitely in the presence of these three colloids. This is not true where exposure is made to yeast nucleic acid dissolved in sea water containing 0.00093 gm. per cc. of NaOH. Valonia is markedly less tolerant of this medium (perhaps of NaOH rather than the colloid used) than Halicystis. Such differential effects, however, reach a high point in the case of the solutions of diphtheria toxin and of edestin. Halicystis has a very high tolerance for diphtheria toxin, and Valonia a very low tolerance. In the case of edestin, the relationship is reversed. Here Halicystis has a very low tolerance, and Valonia a very high tolerance. In fact, it may be said that diphtheria toxin has no appreciable effect upon Halicystis, and edestin a very slight effect upon Valonia; while diphtheria toxin is extremely toxic to Valonia, and edestin is extremely toxic to Halicystis. We can offer no suggestions, at present, as to the way in which these effects are produced. It is probable that the very thin protoplasmic layer of these species, which is certainly no thicker than 8µ, is sufficient to obstruct the passage of proteins having large molecules, like egg albumen, with a degree of efficiency that is extraordinary. In the tests we have reported, areas of from 20 sq. cm. to 40 sq. cm. have been submitted to the action of a relatively high concentration of egg albumen for several days without permitting the passage of sufficient amounts to give definable tests either with Spiegler's or with Tanret's method,— presumably less than 1 part in 250,000. In the tests of the proteins having much smaller molecules (though the size may not be the explanation), there is some probability that the membranes exhibit a little permeability. The peptone and the proteose of animal origin, or biuret-positive substances derived from them, apparently pass the protoplasmic membranes occasionally in quantities sufficient to give biuret tests. The most probable case of protein passage, however, was that of the proteose of the scarlet runner bean, where specific detection of less than 1 part per 80,000 was possible. In this instance the proteose appeared to pass membranes that were healthy and were functioning normally. But since the cells of the algae had to be destroyed in making the tests, one cannot maintain this point. All one can say is that protein passage was indicated in carefully examined cells of both species, where no breaks in the protoplasmic membrane were discernible, and where samples of the treated cells behaved normally after treatment.

Utilization of positional isotope exchange experiments to evaluate reversibility of ATP hydrolysis catalyzed by Escherichia coli Lon proteaseThis paper is one of a selection of papers published in this special issue entitled 8th International Conference on AAA Proteins and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Jennifer Thomas ◽  
Jennifer Fishovitz ◽  
Irene Lee
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Isotope Exchange ◽  
Atp Hydrolysis ◽  
Peer Review Process ◽  
Lon Protease ◽  
Peptidase Activity ◽  
E Coli ◽  
Atpase Reaction ◽  
Half Site

Lon protease, also known as protease La, is an ATP-dependent serine protease. Despite the presence of a proteolytic Ser–Lys dyad, the enzyme only catalyzes protein degradation in the presence of ATP. Lon possesses an intrinsic ATPase activity that is stimulated by protein and certain peptide substrates. Through sequence alignment and analysis, it is concluded that Lon belongs to the AAA+ protein family. Previous kinetic characterization of the ATPase domain of Escherichia coli Lon protease implicates a half-site reactivity model in which only 50% of the ATP bound to Lon are hydrolyzed to yield ADP; the remaining ATPase sites remain bound with ATP and are considered non-catalytic. In this model, it is implied that ATP hydrolysis is irreversible. To further evaluate the proposed half-site reactivity model, the reversibility of the ATPase activity of E. coli Lon was evaluated by positional isotope exchange experiments. The ATPase reactions were conducted in the 18O-enriched buffer such that the extent of 18O incorporation into inorganic phosphate generated from ATP hydrolysis could be used to evaluate the extent of reversibility in ATP hydrolysis. Collectively, our experimental data reveal that the ATPase reaction catalyzed by E. coli Lon in the presence and absence of peptide substrate that stimulated the enzyme’s ATPase activity is irreversible. Therefore, the half-site ATPase reactivity of E. coli Lon is validated, and can be used to account for the kinetic mechanism of the ATP-dependent peptidase activity of the enzyme.

scholarly journals INVESTIGATIONS ON THE MITOCHONDRIA OF THE HOUSEFLY, MUSCA DOMESTICA L

1954 ◽  
Vol 37 (3) ◽  
pp. 343-359 ◽  
Author(s):  
Bertram Sacktor
Keyword(s):  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Musca Domestica ◽  
Flight Muscle ◽  
Phosphate Uptake ◽  
Systematic Investigation ◽  
Appreciable Effect ◽  
Considerable Influence ◽  
Isolation Medium ◽  
Normal Morphology

It has been found that mitochondria isolated from the flight muscle of the housefly, Musca domestica, are capable of effecting oxidative phosphorylation. A systematic investigation of the factors which regulate this coupling was undertaken. It was found: 1. The molarity of the isolation medium had considerable influence on the morphology of the mitochondria. These physical alterations were associated with changes in oxidation, phosphorylation, and ATPase activity. 2. In addition to an optimum isolation medium, the normal morphology of the mitochondria needed to be further stabilized by serum albumin. 3. A "latent" ATPase activity in insect mitochondria was demonstrated. An inverse relationship was found between oxidative phosphorylation and ATPase activity. 4. Oxygen consumption and the uptake of phosphate were linear with respect to time. 5. A respiratory substrate was necessary for phosphorylation and for maintenance of spatially organized mitochondria. 6. No differences in oxygen uptake were found in the presence or absence of inorganic phosphate. 7. Magnesium was required for optimal oxidative phosphorylation. Calcium and manganese inhibited both respiration and phosphorylation. 8. The addition of cytochrome c had no effect on either oxygen or phosphate uptake. 9. ATP, ADP, or AMP were capable of participating in oxidative phosphorylation, but the glucose-hexokinase trapping system was necessary. 10. Fluoride inhibited the phosphorylation of AMP, but increased P/O when ATP was used. This stimulation was not due to the inhibition of ATPase. 11. Neither arginine nor creatine was phosphorylated. 12. The addition of other isolated fractions of flight muscle to the mitochondrial system had no appreciable effect on respiration or phosphorylation.

Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin

2002 ◽  
Vol 363 (3) ◽  
pp. 697-705 ◽  
Author(s):  
Christiane ROSE ◽  
Stéphanie VOISIN ◽  
Claude GROS ◽  
Jean-Charles SCHWARTZ ◽  
Tanja OUIMET
Keyword(s):  
Transition State ◽  
Plasma Membranes ◽  
Bioactive Peptides ◽  
Specific Activity ◽  
Chinese Hamster ◽  
Enzymic Activity ◽  
Amide Bond ◽  
Cell Trafficking ◽  
Sequence Identity ◽  
High Degree

Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity.

Sign in / Sign up

Export Citation Format

Share Document