scholarly journals A HISTOCHEMICAL STUDY OF ADENOSINE TRIPHOSPHATASE IN THE TOAD (BUFO SPINULOSUS) GASTRIC MUCOSA

1970 ◽  
Vol 18 (5) ◽  
pp. 340-353 ◽  
Author(s):  
CECILIA KOENIG S. ◽  
JUAN D. VIAL C.
Keyword(s):  
Gastric Mucosa ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Atp Hydrolysis ◽  
Lead Ion ◽  
Outer Side ◽  
Reaction Products ◽  
Fresh Tissue ◽  
Toad Bufo ◽  
Hydrolysis Of

Adenosine triphosphatase (ATPase) activity was studied by histochemical methods in the gastric mucosa of Bufo spinulosus. Two types of activity were established. One is activated by Mg++, and is localized mainly at the intercellular boundaries and the basal zone of the oxyntic-peptic cells; the reaction products are found only on the outer side of the cells. The other is activated by HCO3– and is mainly localized at the microvilli at the apical zone of the oxyntic-peptic cells. The intensity and/or distribution of the reactions are influenced by histamine stimulation. Control experiments demonstrated that: the microsomal fraction of gastric mucosa contained a Mg++-requiring ATPase activity which was enhanced by addition of HCO3–; 25% of the ATPase activity of fresh tissue was maintained after fixation and incubation in presence of lead ion; the medium employed in the histochemical studies did not enhance the lead-catalyzed, nonenzymatic hydrolysis of ATP by more than 20% when compared with spontaneous ATP hydrolysis; and incubation in media with different ATP-Pb ratios did not significantly alter the location of the staining.

scholarly journals LEAD ION AND PHOSPHATASE HISTOCHEMISTRY II. EFFECT OF ADENOSINE TRIPHOSPHATE HYDROLYSIS BY LEAD ION ON THE HISTOCHEMICAL LOCALIZATION OF ADENOSINE TRIPHOSPHATASE ACTIVITY

1966 ◽  
Vol 14 (10) ◽  
pp. 702-710 ◽  
Author(s):  
HAROLD L. MOSES ◽  
ALAN S. ROSENTHAL ◽  
DAVID L. BEAVER ◽  
SHIRLEY S. SCHUFFMAN
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Lead Nitrate ◽  
Histochemical Localization ◽  
Lead Ion ◽  
Membrane Localization ◽  
Fixed Tissue ◽  
Plasma Membrane Localization ◽  
Hydrolysis Of

The lead method of Wachstein and Meisel for the histochemical localization of adenosine triphosphatase (ATPase) involves the incubation of sections of fixed tissue in reaction mixtures containing ATP, lead nitrate, magnesium sulfate and a Tris-maleate buffer, pH 7.2. Both fixation and the presence of lead ion were shown to inhibit tissue ATPase activity markedly and to inactivate the sodium- plus potassium-dependent membrane ATPase. In addition, recent studies have demonstrated that lead ion, in the concentration used in the Wachstein-Meisel system, will catalyze the hydrolysis of ATP. Studies on the effect of this nonenzymatic reaction on the histochemical localization of ATPases demonstrated that plasma membrane localization occurred only with lead and ATP concentrations which gave significant nonenzymatic hydrolysis of ATP by lead. In addition, nuclear and mitochondrial localization without accompanying plasma membrane localization could be obtained in formalin-fixed tissue with decreased concentrations of lead or with increased concentrations of ATP in the reaction mixture. The amount of lead-catalyzed hydrolysis was in the same order of magnitude as fixed tissue ATPase activity and could quantitatively account for the amount of phosphate needed to give recognizable localization of lead salt deposits in sections of fixed tissue.

scholarly journals The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1970 ◽  
Vol 120 (1) ◽  
pp. 15-24 ◽  
Author(s):  
P. S. G. Goldfarb ◽  
R. Rodnight
Keyword(s):  
Potassium Chloride ◽  
Magnesium Chloride ◽  
Adenosine Triphosphatase ◽  
Time Course ◽  
Atp Hydrolysis ◽  
Protein Ratio ◽  
Low Concentrations ◽  
Hydrolysis Of ◽  
Emission Flame

1. The intrinsic Na+, K+, Mg2+ and Ca2+ contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na+ from 90±20 to 24±12, the bound K+ from 27±3 to 7±2, the bound Mg2+ from 20±2 to 3±1 and the bound calcium from 8±1 to <1nmol/mg of protein. 3. The activities of the Na++K++Mg2+-stimulated adenosine triphosphatase and the Na+-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5μm (ATP/protein ratio 12.5pmol/μg). 4. The Na+-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5μm-magnesium chloride and 2μm-potassium chloride. Addition of 2.5μm-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na+-dependent ATP hydrolysis was partly restored with 2.5μm-magnesium chloride; addition of K+ in the range 2–10μm-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0°C with 0.5nmol of K+/mg of protein so that the final added K+ in the reaction mixture was 0.1μm restored the Na+-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [42K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K+/mg of protein was linear over a period of 20min and was inhibited by Na+. Half-maximal inhibition of 42K+-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na+-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K+ and Mg2+ of the Na++K++Mg2+-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K+ from a solution of 0.5μm-potassium chloride.

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.

scholarly journals THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES

1967 ◽  
Vol 35 (2) ◽  
pp. 385-404 ◽  
Author(s):  
Vincent T. Marchesi ◽  
George E. Palade
Keyword(s):  
Atpase Activity ◽  
Outer Surface ◽  
Adenosine Triphosphatase ◽  
Atp Hydrolysis ◽  
Biochemical Assay ◽  
Lead Salt ◽  
Red Cell ◽  
Cell Ghost ◽  
Marked Inhibition ◽  
Cytochemical Demonstration

The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO3)2 resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb2+ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of Pi on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis Pi is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase.

THE HYDROLYSIS OF ADENOSINE TRIPHOSPHATE BY BRAIN SUSPENSIONS

1954 ◽  
Vol 32 (1) ◽  
pp. 471-483 ◽  
Author(s):  
Hanna M. Pappius ◽  
K. A. C. Elliott
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphate ◽  
Atp Hydrolysis ◽  
Standard Procedure ◽  
High Energy ◽  
Unit Weight ◽  
Bicarbonate Buffer ◽  
Low Magnesium ◽  
Optimum Ph ◽  
Hydrolysis Of

Adenosine triphosphate (ATP) hydrolysis by unfractionated brain suspensions has been measured manometrically and chemically in bicarbonate buffer. Both magnesium and, less strongly, calcium accelerate ATP hydrolysis by brain suspensions. The effectiveness of low magnesium or calcium concentrations is decreased by relatively high ATP concentrations. The optimum pH for phosphate liberation is about 8. The ratio of carbon dioxide evolved to inorganic phosphate liberated depends on the pH. Evidence is given that the suspensions contain a Mg-activated ATPase which is strongly inhibited by calcium, a Ca-activated ATPase, and a Mg-activated PPase which is strongly inhibited by calcium but is different from the Mg-ATPase. These enzymes were not obtained in "latent" form nor activated by glutathione. Ethylenediamine tetraacetate can inhibit Ca-ATPase completely but is less effective on the Mg-ATPase and can release the latter from calcium inhibition. Other inhibitors have been tested.The ATPase activity of cerebral cortex of mouse, rat, cat, beef, and man has been measured by a standard procedure. The activity per unit weight decreases with increasing size of the animal parallel to the decrease in the oxygen uptake rate. Calculations show that the ATPase activity is high enough to release all high energy phosphate which could be produced by esterification coupled with respiration.

scholarly journals A BIOCHEMICAL AND CYTOCHEMICAL STUDY OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE PHLOEM OF NICOTIAN A TABACUM

1974 ◽  
Vol 60 (1) ◽  
pp. 221-235 ◽  
Author(s):  
Jamison Gilder ◽  
James Cronshaw
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Phloem Transport ◽  
Cytochemical Localization ◽  
Gtp Hydrolysis ◽  
Cytochemical Study ◽  
P Protein ◽  
Competitive Inhibitors ◽  
Hydrolysis Of

A biochemical and cytochemical study has been made of the distribution of ATPase in mature and differentiating phloem cells of Nicotiana tabacum and of the substrate specificity and effects of fixation on enzyme activity. Homogenates of unfixed leaf midveins and midveins fixed in formaldehyde-glutaraldehyde were assayed for enzyme activity by determining the amount of Pi, liberated per milligram of protein from various substrates in a 30 min period at pH 7.2. In fresh homogenates, hydrolysis of ATP was not significantly different from that of ITP, CTP, and UTP. Hydrolysis of GTP was slightly higher than that of ATP. ATP hydrolysis by fresh homogenates was 17% more extensive than that of ADP, 76% more extensive than that of 5'-AMP, and was inhibited by fluoride and p-chloromercuribenzoate (PCMB). There was little or no hydrolysis of the competitive inhibitors 2'- and 3'-AMP nor with the alternate substrates p-nitrophenylphosphate (PNP) or ß-glycerophosphate (ß-GP). In homogenates of material fixed in formaldehyde-glutaraldehyde for 1¼ h, ATPase activity was 13% preserved. Hydrolysis of ATP by fixed homogenates was not significantly different from that of ADP, 5'-AMP, ITP, CTP, and GTP. Hydrolysis of UTP was lower. Fluoride and PCMB inhibited fixed ATPase activity. The results of cytochemical localization experiments using a lead phosphate precipitation technique were in agreement with the biochemical results. Similar localization patterns were obtained with the nucleoside triphosphates ATP, CTP, GTP, ITP, and UTP. Activity was also localized with ADP and 5'-AMP but not with the competitive inhibitors 2'- and 3'-AMP, nor with PNP or ß-GP. Little or no reaction product was deposited in other controls incubated without substrate or with substrate plus fluoride, PCMB, or N-ethylmaleimide. ATPase activity was demonstrated chiefly at the plasma membrane of mature and differentiating phloem cells and was associated with the P-protein of mature sieve elements. It is suggested that the phloem transport system derives its energy from the demonstrated nucleoside triphosphatase activity.

Localization of adenosine triphosphatase activity in motile bacteria

1973 ◽  
Vol 19 (10) ◽  
pp. 1265-1267 ◽  
Author(s):  
Z. Vaituzis
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Uniform Distribution ◽  
Adenosine Triphosphatase ◽  
Periplasmic Space ◽  
Reaction Products ◽  
Adenosine Triphosphatase Activity ◽  
Atpase Reaction ◽  
Motile Bacteria

Cytochemical studies on motile bacteria revealed magnesium-dependent adenosine triphosphatase (ATPase) activity at the membranous sites of flagellar origin. The studies were done on bacteria representing three types of flagellation, namely, peritrichate, lophotrichate, and monotrichate. Escherichia coli and S. serpens showed a uniform distribution of ATPase reaction products throughout the periplasmic space. In B. licheniformis and V. metchnikovii the reaction products were found in the cytoplasm accumulated in areas where flagella originate.

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 Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1974 ◽  
Vol 143 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Hans Vilhardt ◽  
Derek B. Hope
Keyword(s):  
Atpase Activity ◽  
Adenosine Triphosphatase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Subcellular Fractions ◽  
Neural Lobe ◽  
Bivalent Cations ◽  
Pituitary Glands ◽  
Membrane Bound ◽  
Triton X ◽  
Hydrolysis Of

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg2+-dependent, (b) Ca2+-dependent and (c) Mg2++Na++K+-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg2++Na++K+-dependent enzyme, whereas the Mg2+- and Ca2+-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg2+- and Ca2+-ATPases; however, the activity of the Mg2++Na++K+-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg2+- and Ca2+-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg2+or Ca2+and the other activated only by Ca2+. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.

scholarly journals Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM

2020 ◽  
Author(s):  
Andrés López-Perrote ◽  
Nele Hug ◽  
Ana González-Corpas ◽  
Carlos F. Rodríguez ◽  
Marina Serna ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Mrna Decay ◽  
Atp Hydrolysis ◽  
Unknown Mechanism ◽  
Macromolecular Complexes ◽  
Wide Range ◽  
Nonsense Mediated Mrna Decay ◽  
Aaa Atpases ◽  
Hydrolysis Of

AbstractNonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1 and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.

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