scholarly journals The basal Mg2+-dependent ATPase activity is not part of the (H++K+)-transporting ATPase reaction cycle

1990 ◽  
Vol 267 (3) ◽  
pp. 565-572 ◽  
Author(s):  
H T W M Van der Hijden ◽  
S Kramer-Schmitt ◽  
E Grell ◽  
J J H H M de Pont
Keyword(s):  
Gastric Mucosa ◽  
Atpase Activity ◽  
Hydrolytic Activity ◽  
Reaction Cycle ◽  
Dependent Atpase ◽  
Atpase Reaction ◽  
Phosphorylation Reaction ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

Purified gastric (H(+)+K+)-transporting ATPase [(H(+)+K+)-ATPase] from the parietal cells always contains a certain amount of basal Mg2(+)-dependent ATPase (Mg2(+)-ATPase) activity. lin-Benzo-ATP (the prefix lin refers to the linear disposition of the pyrimidine, benzene and imidazole rings in the ‘stretched-out’ version of the adenine nucleus), an ATP analogue with a benzene ring formally inserted between the two rings composing the adenosine moiety, is an interesting substrate not only because of its fluorescent behaviour, but also because of its geometric properties. lin-Benzo-ATP was used in the present study to elucidate the possible role of the basal Mg2(+)-ATPase activity in the gastric (H(+)+K+)-ATPase preparation. With lin-benzo-ATP the enzyme can be phosphorylated such that a conventional phosphoenzyme intermediate is formed. The rate of the phosphorylation reaction, however, is so low that this reaction with subsequent dephosphorylation cannot account for the much higher rate of hydrolysis of lin-benzo-ATP by the enzyme. This apparent kinetic discrepancy indicates that lin-benzo-ATP is not a substrate for the (H(+)+K+)-ATPase reaction cycle. This idea was further supported by the finding that lin-benzo-ATP was unable to catalyse H+ uptake by gastric-mucosa vesicles. The breakdown of lin-benzo-ATP by the (H(+)+K+)-ATPase preparation must be due to a hydrolytic activity which is not involved in the ion-transporting reaction cycle of the (H(+)+K+)-ATPase itself. Comparison of the basal Mg2(+)-ATPase activity (with ATP as substrate) with the hydrolytic activity of (H(+)+K+)-ATPase using lin-benzo-ATP as substrate and the effect of the inhibitors omeprazole and SCH 28080 support the notion that lin-benzo-ATP is not hydrolysed by the (H(+)+K+)-ATPase, but by the basal Mg2(+)-ATPase, and that the activity of the latter enzyme is not involved in the (H(+)+K+)-transporting reaction cycle (according to the Albers-Post formalism) of (H(+)+K+)-ATPase.

scholarly journals Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase.

1984 ◽  
Vol 99 (2) ◽  
pp. 734-741 ◽  
Author(s):  
W A Braell ◽  
D M Schlossman ◽  
S L Schmid ◽  
J E Rothman
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Low Ph ◽  
Coated Vesicles ◽  
Magnesium Concentration ◽  
Cross Linking ◽  
Dependent Atpase ◽  
Event Triggering ◽  
Hydrolysis Of

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.

Functional role of ecto-ATPase activity in goldfish hepatocytes

1998 ◽  
Vol 274 (4) ◽  
pp. R1031-R1038 ◽  
Author(s):  
Pablo J. Schwarzbaum ◽  
Michael E. Frischmann ◽  
Gerhard Krumschnabel ◽  
Rolando C. Rossi ◽  
Wolfgang Wieser
Keyword(s):  
Atpase Activity ◽  
Functional Role ◽  
Maximal Activity ◽  
Hydrolysis Rate ◽  
Hyperbolic Function ◽  
Extracellular Release ◽  
Transport Atpases ◽  
Hydrolysis Of ◽  
Hepatocyte Suspension

Extracellular [γ-32P]ATP added to a suspension of goldfish hepatocytes can be hydrolyzed to ADP plus γ-32Pidue to the presence of an ecto-ATPase located in the plasma membrane. Ecto-ATPase activity was a hyperbolic function of ATP concentration ([ATP]), with apparent maximal activity of 8.3 ± 0.4 nmol Pi ⋅ (106cells)−1 ⋅ min−1and substrate concentration at which a half-maximal hydrolysis rate is obtained of 667 ± 123 μM. Ecto-ATPase activity was inhibited 70% by suramin but was insensitive to inhibitors of transport ATPases. Addition of 5 μM [α-32P]ATP to the hepatocyte suspension induced the extracellular release of α-32Pi[8.2 pmol ⋅ (106cells)−1 ⋅ min−1] and adenosine, suggesting the presence of other ectonucleotidase(s). Exposure of cell suspensions to 5 μM [2,8-3H]ATP resulted in uptake of [2,8-3H]adenosine at 7.9 pmol ⋅ (106cells)−1 ⋅ min−1. Addition of low micromolar [ATP] strongly increased cytosolic free Ca2+([Formula: see text]). This effect could be partially mimicked by adenosine 5′- O-(3-thiotriphosphate), a nonhydrolyzable analog of ATP. The blockage of both glycolysis and oxidative phosphorylation led to a sixfold increase of[Formula: see text] and an 80% decrease of intracellular ATP, but ecto-ATPase activity was insensitive to these metabolic changes. Ecto-ATPase activity represents the first step leading to the complete hydrolysis of extracellular ATP, which allows 1) termination of the action of ATP on specific purinoceptors and 2) the resulting adenosine to be taken up by the cells.

An ATP-inhibited soluble 5'-nucleotidase of rat kidney

1988 ◽  
Vol 254 (2) ◽  
pp. F191-F195
Author(s):  
M. Le Hir ◽  
U. C. Dubach
Keyword(s):  
High Speed ◽  
Catalytic Properties ◽  
Divalent Cations ◽  
Rat Kidney ◽  
Maximal Activity ◽  
Rate Of Hydrolysis ◽  
Membrane Bound ◽  
Ph Range ◽  
Hydrolysis Of

Hydrolysis of 5'-AMP by 5'-nucleotidase is a possible source of adenosine in the kidney. A renal membrane-bound ecto-5'-nucleotidase has been previously described. The present study deals with the catalytic properties of a 5'-AMP phosphohydrolase partially purified from high-speed supernatants of rat kidney homogenates. It exhibits phosphatase activity toward 5'-AMP, 5'-IMP, and 5'-GMP, but not toward 2'- and 3'-AMP and corresponds therefore to a 5'-nucleotidase. The hydrolysis of 5'-AMP by the soluble 5'-nucleotidase requires divalent cations. Maximal activity is reached with 10 microM of either Mn2+ or Co2+, whereas half-maximal activity is obtained with approximately 400 microM Mg2+. The soluble 5'-nucleotidase exhibits Michaelis-Menten kinetics with a Km of 9.5 microM for 5'-AMP. In the presence of 1 mM of free Mg2+, physiological concentrations of ATP provoke an increase of the Km for 5'-AMP and a decrease of Vmax. An increase of the pH of 0.4 units in the pH range 6.4-7.4 roughly doubles the rate of hydrolysis of 5'-AMP. The effects of ATP and of the pH are compatible with a role of the renal soluble 5'-nucleotidase in the hydrolysis of 5'-AMP and in the production of adenosine during hypoxia.

scholarly journals Molecular and Physiological Role of the Trehalose-Hydrolyzing α-Glucosidase from Thermus thermophilus HB27

2008 ◽  
Vol 190 (7) ◽  
pp. 2298-2305 ◽  
Author(s):  
Susana Alarico ◽  
Milton S. da Costa ◽  
Nuno Empadinhas
Keyword(s):  
Amino Acids ◽  
Minimal Medium ◽  
Thermus Thermophilus ◽  
Physiological Role ◽  
Hydrolytic Activity ◽  
Recombinant Enzyme ◽  
New Feature ◽  
Hydrolysis Of ◽  
Reciprocal Replacement

ABSTRACT Trehalose supports the growth of Thermus thermophilus strain HB27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. We expressed a putative α-glucosidase gene (TTC0107), characterized the recombinant enzyme, and found that the preferred substrate was α,α-1,1-trehalose, a new feature among α-glucosidases. The enzyme could also hydrolyze the disaccharides kojibiose and sucrose (α-1,2 linkage), nigerose and turanose (α-1,3), leucrose (α-1,5), isomaltose and palatinose (α-1,6), and maltose (α-1,4) to a lesser extent. Trehalose was not, however, a substrate for the highly homologous α-glucosidase from T. thermophilus strain GK24. The reciprocal replacement of a peptide containing eight amino acids in the α-glucosidases from strains HB27 (LGEHNLPP) and GK24 (EPTAYHTL) reduced the ability of the former to hydrolyze trehalose and provided trehalose-hydrolytic activity to the latter, showing that LGEHNLPP is necessary for trehalose recognition. Furthermore, disruption of the α-glucosidase gene significantly affected the growth of T. thermophilus HB27 in minimal medium supplemented with trehalose, isomaltose, sucrose, or palatinose, to a lesser extent with maltose, but not with cellobiose (not a substrate for the α-glucosidase), indicating that the α-glucosidase is important for the assimilation of those four disaccharides but that it is also implicated in maltose catabolism.

Determination of Na-K-ATPase activity in single segments of the mammalian nephron

1979 ◽  
Vol 237 (2) ◽  
pp. F105-F113 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz ◽  
F. Morel
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Inorganic Phosphate ◽  
Rapid Freezing ◽  
Phosphate Release ◽  
Large Numbers ◽  
Hydrolysis Of ◽  
Single Tubules

A micromethod for the determination of Na-K-ATPase in discrete segments of nephrons from rabbit, rat, and mouse kidneys is described. To facilitate tubule microdissection, the kidneys were perfused with collagenase after it had been verified that collagenase had no effect on ATPase activity. Individual tubule segments were dissected under stereomicroscopic observation, exposed to a hypotonic environment followed by rapid freezing, and incubated in 1 microliter assay medium. Enzyme activity was determined by direct measurement of labeled inorganic phosphate release by the hydrolysis of [gamma-32P]ATP and was expressed as a function of tubule length. This method is technically simple enough to permit simultaneous measurement of the enzyme in large numbers of tubules and sufficiently sensitive to determine its activity in each region of the nephron. Correlation of Na-K-ATPase activity in single tubules with functional measurements obtained in the corresponding segment of the nephron with the perfused tubule or micropuncture techniques should help define the role of this enzyme in tubular ion transport.

scholarly journals Adsorption of soluble proteins to rumen bacteria and the role of adsorption in proteolysis

1985 ◽  
Vol 53 (2) ◽  
pp. 399-408 ◽  
Author(s):  
R. J. Wallace
Keyword(s):  
Adsorption Mechanism ◽  
Binding Capacity ◽  
Rumen Bacteria ◽  
Maximum Binding Capacity ◽  
Rate Of Hydrolysis ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Capsular Material ◽  
Hydrolysis Of ◽  
Relative Adsorption

1. Following the addition ofI4C-labelled casein to mixed rumen bacteria at 39°, some radioactivity was adsorbed to the bacteria before the casein was hydrolysed. At O°, the rate of hydrolysis was greatly diminished but adsorption still occurred, and this enabled a study of the adsorption mechanism to be made.2. The adsorption of14C-labelled casein to rumen bacteria was a saturable process. The maximum binding capacity was about 10 μI4C-labelled casein/mg bacterial protein.3. The ability of bacteria to adsorb14C-labelled casein was abolished when they had been boiled for 5 min. Boiling caused the release of material from the bacteria which rendered some undigested protein soluble in 50 g trichloroacetic acid/l.4. Adsorbed14C-labelled casein could be partly displaced by the addition of Triton XI00 or an excess of unlabelled casein, or by boiling, or by removal of capsular material by blending. Adsorbed14C-labelled haemoglobin could similarly be displaced by an excess of cold casein.5. When an excess of casein was added to bacteria to which glucose-6-phosphate dehydrogenase (EC I. I.I.49) and glucosephosphate isomerase (EC 5.3. I.9) had been adsorbed, little active enzyme was displaced.6. The susceptibility of different14C-labelled proteins to hydrolysis corresponded to their relative adsorption affinities.7. The pattern of sensitivity to inhibitors of the adsorption mechanism was the same as that for the inhibition of the bacterial hydrolysis of 14C-labelled casein, and the synthetic substrates leucine p-nitroanilide and benzoyl arginine p-nitroanilide.8. It was concluded that the adsorption site and the catalytic site for proteolysis by rumen bacteria are probably identical and so not likely to be subject to independent manipulation.

Role of Insulin in Regulation of Na+-/K+-Dependent ATPase Activity and Pump Function in Corneal Endothelial Cells

2010 ◽  
Vol 51 (8) ◽  
pp. 3935 ◽  
Author(s):  
Shin Hatou ◽  
Masakazu Yamada ◽  
Yoko Akune ◽  
Hiroshi Mochizuki ◽  
Atsushi Shiraishi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Atpase Activity ◽  
Corneal Endothelial Cells ◽  
Pump Function ◽  
Dependent Atpase

scholarly journals Reaction mechanism of the gastric H+ + K+-dependent ATPase. Effects of inhibitor and pH

1987 ◽  
Vol 241 (1) ◽  
pp. 175-181 ◽  
Author(s):  
J Nandi ◽  
T K Ray
Keyword(s):  
Reaction Mechanism ◽  
Atp Hydrolysis ◽  
Dependent Effect ◽  
Dramatic Effect ◽  
Ph Values ◽  
Dependent Atpase ◽  
P Concentration ◽  
Atpase Reaction ◽  
Enzyme Turnover ◽  
Hydrolysis Of

The effect of nolinium bromide [2-(3,4-dichlorophenylamino)quinolizium bromide], which acts as a K+ antagonist in the gastric H+ +K+-dependent ATPase reaction, was investigated at the level of 32P-labelled intermediates of the gastric ATPase reaction. A concentration-dependent effect of nolinium bromide was observed on the concentrations of phosphorylated intermediates. At low (up to 50 microM) concentrations the drug did not interfere with the concentrations of intermediates but exhibited a competition with K+ at the level of both 32P-labelled intermediates and hydrolysis of ATP at pH 7.0. Similar competition was noted in the H+ +K+-dependent ATPase reaction. Low nolinium bromide concentrations also drastically slowed the enzyme turnover. The concentrations of the intermediates were lowered appreciably between 50 microM- and 100 microM-nolinium bromide without affecting the ATP hydrolysis, and the effects were independent of pH. Similar to the effects at pH 7.0, the drug also exhibited competition with K+ in lowering the E approximately P concentration at pH 5.0. A dramatic effect of pH on the K+-sensitivity as well as on turnover of the 32P-labelled intermediates was observed. Although the concentrations of intermediates remained nearly unaltered at various pH values, the K+-stimulated hydrolysis of ATP showed an optimum at pH 7.0 with sharp declines at pH 5 and 8. The data suggest a critical involvement of H+ in the conversion of the K+-insensitive E1 approximately P into the K+-sensitive E2 approximately P form of the enzyme. Nolinium bromide appears to function as a K+ analogue and seems to block the entry of K+ at the K X E2 step, thereby interfering with the enzyme turnover.

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