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.

Inhibitory effect of soil humic compounds on the proteolytic enzyme pronase

Soil Research ◽  
1969 ◽  
Vol 7 (3) ◽  
pp. 241
Author(s):  
JN Ladd ◽  
JHA Butler
Keyword(s):  
Humic Acids ◽  
Maximum Velocity ◽  
Kinetic Studies ◽  
Inhibitory Effect ◽  
Enzymic Activity ◽  
Humic Compounds ◽  
Km Value ◽  
Group Basis ◽  
Inhibitory Power ◽  
Hydrolysis Of

Neutralized solutions of soil humic acids inhibit the proteolytic activity of the enzyme pronase when tested against a variety of substrates. Protein hydrolysis was less sensitive than hydrolysis of dipeptide derivatives; 50% inhibition of benzyloxycarbonylglycylleucine hydrolysis was achieved with concentrations of humic acids as low as 1-2 �g/ml or less than 10-5M, on a carboxyl group basis. Humic acids, extracted from soils with different crop histories, showed only slight differences in their effectiveness as inhibitors of pronase activity. Their inhibitory power was comparable with that of other high molecular weight polyanions, e.g. polyacrylic acid and polycondensates derived from p-benzoquinone and catechol. Alginic acid was a relatively poor inhibitor. Preincubation of humic acids for various periods with either pronase or substrate (albumin or benzyloxycarbonylglycylleucine) had little or no effect on the subsequent inhibition of enzymic activity. However, inhibition is decreased by increasing substrate concentrations, following preincubation of humic acids and pronase. Both observations are consistent with a reversible inhibitory mechanism. Kinetic studies demonstrate that humic acids inhibit pronase activity towards albumin and N-benzyloxycarbonyl dipeptides by effectively reducing the affinity of pronase for the substrate, i.e. by increasing the Km value for the reaction. With benzoylarginine amide and benzoylarginine ethyl ester as substrates, the reaction velocity is lowered due to a reduction of the maximum velocity of the system. Both effects may possibly be explained by a conformational change in the enzyme structure due to combination with the humic acid molecules.

Corrigenda - Inhibitory effect of soil humic compounds on the proteolytic enzyme pronase

Soil Research ◽  
1969 ◽  
Vol 7 (3) ◽  
pp. 241
Author(s):  
JN Ladd ◽  
JHA Butler
Keyword(s):  
Humic Acids ◽  
Maximum Velocity ◽  
Kinetic Studies ◽  
Inhibitory Effect ◽  
Enzymic Activity ◽  
Humic Compounds ◽  
Km Value ◽  
Group Basis ◽  
Inhibitory Power ◽  
Hydrolysis Of

Neutralized solutions of soil humic acids inhibit the proteolytic activity of the enzyme pronase when tested against a variety of substrates. Protein hydrolysis was less sensitive than hydrolysis of dipeptide derivatives; 50% inhibition of benzyloxycarbonylglycylleucine hydrolysis was achieved with concentrations of humic acids as low as 1-2 �g/ml or less than 10-5M, on a carboxyl group basis. Humic acids, extracted from soils with different crop histories, showed only slight differences in their effectiveness as inhibitors of pronase activity. Their inhibitory power was comparable with that of other high molecular weight polyanions, e.g. polyacrylic acid and polycondensates derived from p-benzoquinone and catechol. Alginic acid was a relatively poor inhibitor. Preincubation of humic acids for various periods with either pronase or substrate (albumin or benzyloxycarbonylglycylleucine) had little or no effect on the subsequent inhibition of enzymic activity. However, inhibition is decreased by increasing substrate concentrations, following preincubation of humic acids and pronase. Both observations are consistent with a reversible inhibitory mechanism. Kinetic studies demonstrate that humic acids inhibit pronase activity towards albumin and N-benzyloxycarbonyl dipeptides by effectively reducing the affinity of pronase for the substrate, i.e. by increasing the Km value for the reaction. With benzoylarginine amide and benzoylarginine ethyl ester as substrates, the reaction velocity is lowered due to a reduction of the maximum velocity of the system. Both effects may possibly be explained by a conformational change in the enzyme structure due to combination with the humic acid molecules.

scholarly journals A kinetic study of the soluble 5′-nucleotidase of rat liver

1977 ◽  
Vol 162 (3) ◽  
pp. 611-616 ◽  
Author(s):  
G van den Berghe ◽  
C van Pottelsberghe ◽  
H G Hers
Keyword(s):  
Uric Acid ◽  
Rat Liver ◽  
Liver Cell ◽  
Adenine Nucleotides ◽  
Enzymic Activity ◽  
Kinetic Properties ◽  
Stimulatory Action ◽  
Physiological Conditions ◽  
Hydrolysis Of ◽  
Stimulation Of

1. The kinetic properties of the 5′-nucleotidase (EC 3.1.3.5) present in the cytosol of rat liver were investigated in relation to the conversion of adenine nucleotides into uric acid, with particular reference to the stimulation of this process by fructose. The enzyme was assayed by the release of Pi and by a new and more sensitive radiochemical procedure. 2. When IMP was used as substrate, the partially purified enzyme displayed almost hyperbolic kinetics (h = 1.1) with S0.5 = 1.2 mM. Similar kinetics were observed with GMP and other nucleoside 5′-monophosphates, except AMP. 3. Vmax. of the enzyme for AMP was about the same as for IMP, but the kinetics were sigmoidal (h = 1.6) with S 0.5 = 10 mM. 4. The hydrolysis of IMP was inhibited competitively by GMP. IMP, at concentrations up to 0.5 mM, had a paradoxical stimulatory action on the hydrolysis of 2-5 mM-AMP and was inhibitory at higher concentrations. 5. The activity of the enzyme towards AMP and IMP was stimulated by ATP and GTP, and inhibited by Pi. Activators and inhibitor approximately cancelled each others' effects. At pH 7.4, the enzymic activity with 0.2 mM-AMP was undetectable under physiological conditions. 6. It is concluded that, in the liver cell, AMP is not hydrolysed by the soluble 5′-nucleotidase, but that its degradation requires prior deamination to IMP.

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 Renal sodium-potassium adenosine triphosphatase. Optical localization and x-ray microanalysis.

1975 ◽  
Vol 23 (11) ◽  
pp. 828-839 ◽  
Author(s):  
R Beeuwkes ◽  
S Rosen
Keyword(s):  
Atpase Activity ◽  
Electron Probe ◽  
Sequential Analysis ◽  
Relative Sensitivity ◽  
Initial Reaction ◽  
Initial Product ◽  
Sodium Potassium ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of ◽  
Convoluted Tubules

The distribution of sodium-potassium adenosine triposphatase (Na-K-ATPase) activity in kidney sections has been studied by a method based on the hydrolysis of p-nitrophenyl phosphate in alkaline medium containing dimethyl sulfoxide. The products at each stage in the reaction sequence have been subjected to electron probe microanalysis. The initial product was identified as a mixture of KMgPO4 and Mg(PO4)2, and sequential analysis demonstrated the linearity of conversion of this product to a visible form. In human, rabbit and rat kidneys the distribution of activity was found to be essentially identical, with highest levels located in thick ascending limbs and distal convoluted tubules. The initial reaction was completely potassium dependent and was inhibited by ouabain in concentrations reflecting the relative sensitivity of microsomal Na-K-ATPase in each species. Measurement of initial product phosphorus by means of the electron probe is presented as a practical technique for direct quantitation of Na-K-ATPase activity in identified tubule segments.

TRIGEMINAL PROJECTIONS IN THE RETICULAR FORMATION OF THE PONS IN THE CAT

1962 ◽  
Vol 40 (1) ◽  
pp. 7-12
Author(s):  
J. M. Langlois ◽  
Guy Lamarche
Keyword(s):  
Reticular Formation ◽  
Trigeminal Nerve ◽  
Unit Activity ◽  
Functional Organization ◽  
Pontine Reticular Formation ◽  
Recording Unit ◽  
Spatial Convergence ◽  
The Face ◽  
High Degree ◽  
Stimulation Of

The projections of the trigeminal nerve in the pontine reticular formation of the cat have been investigated by recording unit activity, after physiological stimulation of the face, in 30 "encéphales isolés" preparations. No somatotopical arrangement was found but a high degree of spatial convergence onto pontine reticular units exists and a certain degree of functional organization was observed.

The Appearance Of PF1 And PF3 In Activated Human Platelets

1981 ◽  
Author(s):  
H Sandberg ◽  
A P Bodet ◽  
F A Dombrosei ◽  
L O Andersson ◽  
B R Lentz
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Void Volume ◽  
Factor V ◽  
Dense Granules ◽  
Protein Particles ◽  
Hydrolysis Of ◽  
Platelet Pellet ◽  
Stimulation Of

Collagen and thrombin induced platelet activation were examined, in vitro, with regard to the appearance of surface-associated Factor V-like activity (PF1) and catalytic phospholipid-like surface activity (PF3). Two test systems were used: a clotting assay (a modified KAPTT) and a chromogenic substrate assay (maximum hydrolysis of S-2238). Following stimulation of normal platelets, both PF1 and PF3 appeared simultaneously in the supernatant and platelet pellet. When normal platelets were collected and carefully washed in a buffer containing adenosine, PGE1, and theophylline, the appearance of both PF1 and PF3 was blocked, as was the release of ATP from dense granules, the release of β-TG and PF4 from α-granules, and the occurrence of aggregation. When platelets were collected in this same inhibitor-containing buffer, and then gel filtered/centrifuge-washed in an inhibitor-free buffer, the appearance of PF1 and PF3 was still blocked. This occurred even though release of ATP, β-TG and PF4 as well as aggregation followed a pattern equivalent to platelets never exposed to these inhibitors. When the release supernatant from normal platelets isolated in the absence of inhibitors was gel filtered on Sepharose CL-4B in the presence of EDTA, the carbohydrate-free, lipid- protein particles (70-170nm diam.) that provide PF3 appeared in the void volume. When the release supernatant from normal platelets was gel filtered in the presence of Ca2+, both, PF1 and PF3 eluted in the void volume. With platelets isolated from severe F.V-deficient donors, only PF3 was found in the void volume, in the presence or absence of Ca2+. It seems that the appearance of PF1 and PF3 as coagulant activities is completely separate from both the release of dense granule and α-granule contents as well as platelet aggregation and that the appearance of PF1 requires the presence of Ca2+.

Stimulation of β-(l → 6)-glucanase production by oxidized pustulan

1972 ◽  
Vol 18 (10) ◽  
pp. 1543-1550 ◽  
Author(s):  
Robert G. Brown
Keyword(s):  
Enzyme Activity ◽  
Isoelectric Focusing ◽  
Gel Filtration ◽  
Tween 80 ◽  
Sole Source ◽  
Cellulase Production ◽  
Low Degree ◽  
Degree Of Oxidation ◽  
High Degree ◽  
Stimulation Of

A strain of Penicillium lilacinum, isolated from soil, produced pustulanase, β-(1 → 3)-glucanase, (EC. 3.2.1.6) and cellulase (EC.3.2.1.4) when cultivated on a medium containing pustulan as the sole source of carbon. If pustulan was replaced by ketopustulan, the production of pustulanase was stimulated about 10-fold although the amount of stimulation was dependent on the degree of oxidation of pustulan. β-(1 → 3)-Glucanase production was stimulated slightly by ketopustulan; however, the degree of oxidation did not affect significantly the yield of this enzyme. Cellulase production was either unaffected by the oxidized polymer, or at higher degrees of oxidation, decreased. Tween 80 stimulated the production of the three enzymes in media containing ketopustulan with a low degree of oxidation but was inhibitory to pustulanase and cellulase production in media containing ketopustulan with a high degree of oxidation. A combination of gel filtration and isoelectric focusing revealed that each enzyme activity was attributable to at least two proteins.

scholarly journals Purinergic stimulation of rat cardiomyocytes induces tyrosine phosphorylation and membrane association of phospholipase Cγ: a major mechanism for InsP3 generation

1996 ◽  
Vol 318 (2) ◽  
pp. 723-728 ◽  
Author(s):  
Michel PUCEAT ◽  
Guy VASSORT
Keyword(s):  
Tyrosine Kinase ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Extracellular Atp ◽  
Rat Cardiomyocytes ◽  
Major Mechanism ◽  
Adult Rat ◽  
Hydrolysis Of ◽  
Purinergic Stimulation ◽  
Stimulation Of

Phospholipase Cγ (PLCγ) expression and activation by a purinergic agonist were investigated in adult rat cardiomyocytes. PLCγ is expressed in isolated cardiomyocytes. Stimulation of cells with extracellular ATP induces a rapid increase in membrane-associated PLCγ immunoreactivity most probably due to redistribution of the lipase from the cytosol to the membrane. The purine triggers a significant phosphorylation on tyrosine residues of a cytosolic pool of PLCγ with a time course that correlates with that of translocation. Extracellular ATP also increases intracellular Ins(1,4,5)P3 content. All these events (translocation and phosphorylation of PLCγ, InsP3 formation) are blocked by genistein, a tyrosine kinase inhibitor. The purinergic effect on both PLCγ translocation and phosphorylation are Ca-sensitive. We thus propose that the purinergic stimulation activates a non-receptor tyrosine kinase that phosphorylates PLCγ in the presence of an increased Ca level and induces PLCγ redistribution to the membrane. There, PLCγ becomes activated leading to the hydrolysis of phosphatidylinositol diphosphate and in turn Ins(1,4,5)P3 formation. This cascade of events may play a significant role in the induction of arrhythmogenesis by purinergic agonists.

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