Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An endogenous protein activator (AF) responsible for the activation of the gastric H+,K+-ATPase system, identified recently as the biochemical mechanism for the transport of H+, has been purified to homogeneity and partially characterized. The purification procedure (at 0-4 degrees C) involves simultaneous concentration and dialysis of the cytosolic fraction from dog fundic cells under negative pressure, pH 4.8 precipitation and two consecutive gel filtration steps on sephacryl S-200 columns. The highly purified and active AF is a protein of 80 Kd consisting of two identical subunits of 40 Kd each. The AF not only stimulates the gastric H+,K+-ATPase activity but also greatly enhances the rate of ATPase dependent proton pumping inside gastric microsomal vesicles. The data clearly suggest an important regulatory role of the cytosolic AF in the gastric HCl secretory process.
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PMID:Purification and characterization of a cytosolic activator protein for the gastric H+,K+-ATPase system from dog fundic mucosa. 301 Feb 73

The accessibility of the tryptophans in dog kidney Na,K-ATPase was studied with the technique of quenching by acrylamide. By use of a modified Stern-Volmer equation, fa, the effective fraction of tryptophans most exposed to quencher, and Ka, the effective quenching constant, were calculated. The direct Stern-Volmer plots are nonlinear under nondenaturing conditions, indicating that the tryptophan residues are unequally accessible to quencher. Modified Stern-Volmer plots revealed marked differences in the exposure of tryptophans in the E1 and E2 states. In the presence of Na or ADP, ligands that stabilize E1, these plots curve downward, indicating that the in addition to buried (unquenched) tryptophans, there is a heterogeneous class of tryptophans. In the presence of K or ouabain, conditions that favor E2, the modified Stern-Volmer plots are linear, consistent with a homogeneous population of tryptophans. Treatment with chymotrypsin to block the E1 to E2 transition results in a new set of quenching parameters which are unchanged with Na or K. Even after detergent denaturation (1% sodium dodecyl sulfate for 30 min), Stern-Volmer plots are nonlinear, and a significant fraction of tryptophan residues remain inaccessible to quencher. Denaturation with urea or guanidine HCl plus dithiothreitol increases the fraction of quenchable fluorescence even more, but still a small fraction, about 7-13%, is buried. The observed changes in exposure of the tryptophan residues would seem to account for the differences in intrinsic fluorescence seen on adding K and Na to Na,K-ATPase. The present results provide new evidence that a significant rearrangement of amino acid residues results from the E1 to E2 transition. Furthermore, a region of the molecule is inaccessible even after denaturation; this may correspond to highly hydrophobic stretches that are normally buried in the membrane.
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PMID:Accessibility of tryptophan residues in Na,K-ATPase. 303 Oct 29

The objective of this work is to establish a procedure to study the stimulation-dependent membrane redistribution and properties of H+-K+-ATPase in an in vitro model system, rabbit isolated gastric glands. Stimulated (10(-4) M histamine plus 10(-5) M forskolin) and resting (10(-4) M metiamide) glands were homogenized and fractionated into PO (40 g, 5 min), P1 (400 g, 10 min), P2 (14,500 g, 10 min), P3 (48,200 g, 90 min), and supernatant, S3. Significant changes occurred in the distribution of our marker for H+-K+-ATPase (K+-p-nitrophenyl phosphatase) activity: a reduction in activity of P3 and a compensatory increment in P1. P3 showed valinomycin (Val)-dependent vesicular H+ uptake, while H+ uptake in P1 was Val independent. Direct measurements of ATPase revealed that H+-K+-ATPase activity of P3 was Val dependent and decreased by stimulation; H+-K+-ATPase activity of P1 was Val independent and increased by stimulation. Further density gradient purification of P1 showed that membranes lighter than 17% Ficoll contained higher specific H+-K+-ATPase activity, and the observed increase in H+-K+-ATPase associated with stimulation was more pronounced. Also, the lighter fractions from stimulated P1 had much latent H+-K+-ATPase activity that was unmasked by n-octylglucoside. The properties of membrane fractions from isolated glands were consistent with results obtained in vivo: high H+-K+-ATPase activity of P3 from resting glands corresponds to cytoplasmic tubulovesicles lacking KCl transport pathways; high activity of P1 from stimulated glands corresponds to apical plasma membrane vesicles containing KCl transport in addition to the H+-K+-ATPase, and full competency for the generation of HCl.
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PMID:Stimulation-associated redistribution of H+-K+-ATPase activity in isolated gastric glands. 303 93

Purified epithelial basolateral membrane vesicles were prepared from lobster hepatopancreas by sorbitol gradient centrifugation. Na+-K+-adenosinetriphosphatase, alkaline phosphatase, and cytochrome-c oxidase enzyme activities in the final membrane preparation were enriched 9.6-, 1.4-, and 0.4-fold, respectively, compared with their activities in the original tissue homogenate. Vesicle osmotic reactivity was demonstrated using 60-min equilibrium 36Cl uptake experiments at a variety of transmembrane osmotic gradients. 36Cl uptake into vesicles preloaded with HCO3 was significantly greater than into vesicles lacking HCO3. This exchange process was stimulated by a transmembrane proton gradient (internal pH greater than external pH). Proton-gradient-dependent Cl-HCO3 exchange was potential sensitive and stimulated by an electrically negative vesicle interior. 36Cl influx (4-s exposures) into HCO3-loaded vesicles occurred by the combination of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid sensitive, carrier-mediated transfer and "apparent diffusion." 36Cl influx was a hyperbolic function of both internal [HCO3] and internal [Cl]. The two internal anions displayed a 100-fold difference in apparent affinity constants with HCO3 being strongly preferred. 36Cl influx was stimulated more by preloaded monovalent than by divalent anions. Na was an inhibitor of proton-dependent anion antiport, whereas K had no effect. A model for HCl-HCO3 antiport is suggested that employs combined transmembrane concentration gradients of Cl and HCO3 to power anion exchange and transfer protons against a concentration gradient.
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PMID:Proton-stimulated Cl-HCO3 antiport by basolateral membrane vesicles of lobster hepatopancreas. 303 81

The parietal cells possess the unique capacity to produce large quantities of acid at a high concentration, and this is reflected in unique properties at the cellular level. The cells are comparatively large, and they are equipped with secretory canaliculi, a multitude of mitochondria, and cytoplasmic tubulovesicles. During secretion many of the tubulovesicles merge with the secretory canaliculi, which then expand. In the process H+, K+-ATPase is transferred from the tubulovesicular membrane to the secretory membrane. This enzyme catalyses the final step in the production of HCl. Parietal cell activity is regulated through receptors on the basolateral cell surfaces. In the isolated gland and in the isolated parietal-cell fractions, stimulation of receptors for histamine evokes higher secretion than receptor stimulation with cholinergic compounds or with gastrin. In these experimental models, specific inhibitors are required to block acid secretion; for example histamine H2-receptor antagonists will block histamine-induced secretion but will be inactive when secretion is evoked by gastrin or by cholinergic stimulation. These stimuli cause a more or less marked increase in the intracellular levels of Ca2+, which acts as a second messenger, leading to the activation of phosphokinases and, ultimately, to morphological transformation of the parietal cells and acid secretion. Another such intracellular messenger is cAMP, which is formed in response to histamine stimulation only; prostaglandins may prevent this process and block acid secretion. The final step in the production of acid requires K+ and Cl- channels in the secretory membrane and the H+, K+-ATPase-catalysed exchange of K+ for H+ across this membrane. This reaction consumes large amounts of energy and depends on the aerobic production of ATP by the parietal cells. Substituted benzimidazoles, such as omeprazole, accumulate in the acid compartments of the parietal cells and inhibit the H+, K+-ATPase, thereby blocking acid production.
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PMID:Physiology and pharmacology of the parietal cell. 304 49

Effects of respiratory and metabolic acidosis (pH approximately 6.8) on myocardial function were studied in the newborn and adult rabbits. Mechanical function was studied in the isolated arterially perfused heart preparation. Acidosis was induced either by increase of the perfusate PCO2 or by decrease of the bicarbonate content. During respiratory acidosis, developed tension (DT) decreased to 43 +/- 3% of control (n = 18) in the adult and this depression was significantly greater than in the newborn (DT = 92 +/- 4%, n = 6). Depression of DT by respiratory acidosis was observed even at high extracellular Ca. During metabolic acidosis, mechanical function decreased gradually and DT at 30 min into acidosis in the adult was 78 +/- 3% of control (n = 6). This depression of DT in the adult was significantly greater than in the newborn (DT at 30 min = 96 +/- 1% of control, n = 6). Statistical analysis using paired t test showed that respiratory acidosis, but not metabolic acidosis, caused significant negative inotropism in the newborn. Myofibrils were isolated and the ATPase was measured at 10(-8) to 10(-4) M Ca and at pH of 7.1 (control), 6.5, and 6.0. Reducing pH depressed the ATPase activity similarly in the newborn and adult. Intracellular buffer capacity was determined by titrating muscle homogenate with HCI. Although the initial pH was not different, addition of HCl to the homogenate caused less decrease in pH in the newborn. These data indicate that contractile function in the newborn heart is more resistant to acidosis and this may be due partly to the greater intracellular buffer capacity.
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PMID:Effect of acidosis on contractile function in the newborn rabbit heart. 315 69

The membranes of Sulfolobus, a thermoacidophilic archaebacterium showed two types of ATP hydrolyzing activity. One was that of a neutral ATPase at an optimum pH around 6.5. This enzyme was activated by 10 mM sulfate with a shift of optimum pH to 5. In these respects, the enzyme was similar to membrane-bound ATPase of Thermoplasma, another thermoacidophilic archaebacterium, reported by Searcy and Whatley [1982) Zbl. Bakt. Hyg., I. Abt. Orig. C3, 245-257). The enzyme hydrolyzed ATP and other NTPs, but not ADP or AMP. It was highly thermostable, but irreversibly inactivated in 0.1 M HCl. The other activity was that of an acidic apyrase at an optimum pH around 2.5. This enzyme was extremely stable toward high temperature and acid and inhibited by sulfate. Both of these ATP hydrolyzing enzymes were resistant to N,N'-dicyclohexylcarbodiimide (DCCD), azide, oligomycin, N'-ethylmaleimide, p-chloromercuribenzoate, orthovanadate, or ouabain. Sulfolobus ATPases differ from F1 and other transport ATPases so far described.
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PMID:Membrane-bound ATPase of a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius. 315 31

Effects of topical application of omeprazole on transmucosal potential difference (PD), luminal pH and histamine-stimulated acid secretion were examined in anesthetized rat stomachs, and they were compared with those of systemic administration. Omeprazole was suspended in 1% CMC with NaHCO3 (pH 9.0) or dissolved in 0.1 N HCl (pH 1.0). Both omeprazole (30 mg/kg, pH 9.0) and cimetidine (100 mg/kg), given i.d., increased the pH and inhibited acid secretion induced by histamine (8 mg/kg/hr, i.v.), while basal gastric PD was markedly elevated only by the former. Similar responses in PD, pH and acid output were obtained dose-dependently after brief exposure of the stomach (10 min) to omeprazole (0.3-30 mg/kg), even in acidic conditions, but the effects of acidified omeprazole disappeared depending upon the latency period in 0.1 N HCl; there was no effect when applied at more than 30 min after dissolution. Of interest, subsequent exposure of the stomach to a mercaptane compound (cysteine, 100 mg/kg) for 30 min significantly reversed the antisecretory effect of omeprazole (both i.d. and i.g.) but not of cimetidine. These results suggest that omeprazole has a local antisecretory action even in acidic stomachs, probably through an inhibition of the H+/K+ATPase activity, and the increase of PD caused by omeprazole may be a characteristic phenomenon seen after the blockade of H+/K+ ATPase, but is not associated with acid inhibition itself.
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PMID:Effects of topical application of acidified omeprazole on acid secretion and transmucosal potential difference in anesthetized rat stomachs. 318 68

Two modifications to Western blots which enhance immunochemical recognition have been developed. The first is transfer in carbonate buffer at pH 9.9, rather than the more commonly used Tris-glycine buffer at pH 8.3. This alteration improved the recognition of four of the five subunits of Escherichia coli F1-ATPase by monoclonal antibodies, the smaller subunits showing the greatest effects. Recognition of dinitrophenyl groups attached to the subunits by polyclonal antibodies was improved by the carbonate buffer only for the smallest ATPase subunit, epsilon. The second modification was incubation of the gel in mild buffers, designed to promote the renaturation of proteins, before the electrophoretic transfer step. The most effective buffer was 20% glycerol in 50 mM Tris-HCl, pH 7.4. Improvements in the signal obtained with monoclonal antibodies to all the subunits of ATPase were obtained by this procedure. As the subunits vary markedly in size, isoelectric point, and other properties, this method should be useful for most proteins. The fate of the 15,000-Da epsilon subunit, labeled with 125I, was followed through a blotting experiment. As long as no sodium dodecyl sulfate was added to the transfer buffer, epsilon was bound to nitrocellulose efficiently in either Tris-glycine or carbonate buffer. However, the epsilon was retained much more strongly during the subsequent incubation steps if the transfer was done in the carbonate buffer. The binding of epsilon to the nitrocellulose was even more stable when the gel had been treated with the buffered glycerol solution before transfer. These results indicate that the conditions under which epsilon subunit first encounters the nitrocellulose markedly affect the stability of binding during subsequent steps. The F1-ATPase was partially fragmented by treatment with proteases and then run on a gel and either transferred immediately in Tris-glycine buffer or else treated with the buffered glycerol solution and transferred in the carbonate buffer. The second blot gave stronger recognition of residual alpha subunit and fragments by an anti-alpha monoclonal antibody, with the largest improvement for the smaller fragments. This result suggests that the modified procedure may be particularly useful in enhancing the detection of small proteins.
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PMID:Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. 353 63

It was shown that substoichiometric concentrations of chaetoglobosin J, one of the fungal metabolites belonging to cytochalasins, inhibited the elongation at the barbed end of an actin filament. Stoichiometric concentrations of chaetoglobosin J decreased both the rate and the extent of actin polymerization in the presence of 75 mM KCl, 0.2 mM ATP and 10 mM Tris-HCl buffer at pH 8.0 and 25 degrees C. In contrast, stoichiometric concentrations of cytochalasin D accelerated actin polymerization. Chaetoglobosin J slowly depolymerized F-actin to G-actin until an equilibrium was reached. Analyses by a number of different methods showed the increase of monomer concentration at equilibrium to depend on chaetoglobosin J concentrations. F-actin under the influence of stoichiometric concentrations of chaetoglobosin J only slightly activated the Mg2+-enhanced ATPase activity of myosin at low ionic strength. It is suggested that when the structure of the chaetoglobosin-affected actin filaments is modified, the equilibrium is shifted to the monomer side, and the interaction with myosin is weakened.
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PMID:Effects of chaetoglobosin J on the G-F transformation of actin. 377 15


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