Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

The activity of calcium-stimulated ATPase (E.C. 3.6.1.3) in homogenates of the secretory enamel organ of rat incisors was studied biochemically. ATP hydrolysis was estimated from the amount of inorganic phosphate liberated. An analysis of the total degradation of ATP was initially performed to ensure that the enzyme assays pertained to the original substrate, ATP, and were not influenced by reaction products formed. Standard incubations were run in tris-maleate buffer, pH 8.2, with 3 mM ATP, 3 mM Ca2+ and 0.5 mM R 8231 at 37 degrees C. The presence of R 8231 was necessary to inhibit nonspecific alkaline phosphatase. The calcium-stimulated ATPase was completely inhibited when heated at 55-60 degrees C for 5 min. The pH optimum was found to be 8.2. The hydrolysis was substantially dependent on Ca2+ and was fastest when the ATP:Ca2+ ratio was 1:1. High substrate concentrations inhibited the hydrolysis. The addition of 1 mM Zn2+ and Ni2+ to the incubation medium markedly inhibited the hydrolysis as did, though less strongly, p-hydroxymercuribenzoate, oligomycin, EDTA and ruthenium red. l-Cysteine, mercaptoethanol, iodoacetic acid and sodium azide were without effect. F- was without effect unless added to a final concentration above 15 mM to media where Ca2+ had first been allowed to react with ATP.
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PMID:Calcium-stimulated ATPase activity in homogenates of the secretory enamel organ in the rat. 2 89

The uptake of the siderophore-iron complex ferrienterochelin was found to be strongly dependent upon an energized membrane state, as demonstrated by its sensitivity to dinitrophenol, azide, and cyanide. Ferrienterochelin uptake may also be dependent upon phosphate bond energy, as indicated by sensitivity to arsenate and iodoacetic acid. Although the adenosine triphosphatase does not appear to be involved in this energy coupling mechanism, ferrienterochelin uptake was shown to be less dependent upon phosphate bond energy than was glutamine uptake. Sensitivity of ferrienterochelin uptake to osmotic shock was shown to be due to the release of a ferrienterochelin binding compound located in the outer membrane of the cells and probably identical to the colicin B receptor protein.
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PMID:Uptake of ferrienterochelin by Escherichia coli: energy dependent stage of uptake. 14 Jan 61

In the present work the uptake of foreign materials by macrophages has been studied in order to elucidate its possible energy-dependent mechanisms. We used monolayer cultures of macrophages from human peripheral venous blood, treated with the following metabolic inhibitors: iodoacetic acid, fluoroacetic acid, sodium fluoride, sodium malonate, sodium azide, 2-4-dinitrophenol, cycloheximide, and ouabain. The test assay was performed by using a zymosan particles suspension in Mc Coy 5 A medium supplemented as follows. The quantitation of phagocytosis was obtained by direct count of intracellular zymosan particles by oil 100X microscopy and the results were submitted to a statistical evaluation. The most effective inhibitor we found was iodoacetate, an inhibitor of anaerobic glycolysis, but fluoride, which acts on the same metabolic pathway at a different site, was quite ineffective. The same ineffectiveness we found for fluoracetate and malonate which act on the Krebs cycle. On the contrary, dinitrophenol (uncoupler of oxidative phosphorylation), azide (inhibitor of cytochrome linked-phosphorylation), ouabain (inhibitor of membrane ATPase activity) and cycloheximide (inhibitor of protein synthesis) give a remarkable decrease of index of phagocytosis after a 3h incubation. In conclusion, we can suppose that the energy-dependent phagocytosis is first depending on transport across the cell membrane (ATPase activity and protein synthesis) and second both on anaerobic glycolysis and oxidative phosphorylation.
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PMID:The influence of some metabolic inhibitors on in vitro phagocytizing macrophages. I. The behaviour of human macrophages. 91 75

1. The biochemical basis of the slowing of relaxation seen in fatigue has been examined using an isolated mouse soleus preparation. 2. Slowing of relaxation occurred during prolonged tetani under anaerobic conditions when ATP and PC fell and lactate accumulated. 3. Slowing of relaxation was also demonstrated with muscles poisoned with cyanide and iodoacetic acid when there was a fall in ATP and PC but no accumulation of lactate. During a period of anaerobic recovery following a fatiguing tetanus, relaxation became faster at a time when lactate was accumulating in the muscle. 4. It is concluded that the slowing of relaxation in fatigue is not a consequence of lactate accumulation, and a relationship is demonstrated between the ATP content of the muscle and the rate of relaxation in muscles fatigued by prolonged stimulation, 5. Rates of ATP turn-over in fresh muscle, and at intervals throughout a tetanus are consistent with the suggestion that the rate limiting step for myofibrillar ATPase may be directly related to the rate limiting step for the decay of tension during relaxation.
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PMID:Metabolic changes associated with the slowing of relaxation in fatigued mouse muscle. 118 65

An assay for the Ca pump ATPase of intact human red blood cells (RBCs) was developed. The assay utilized a small volume (typically 10 microliters) of packed RBCs in 1 ml of a buffer of known composition. The assay was based on the exposure of intact RBCs to the ionophore, A23187, in the presence of Ca. Such exposure caused a rapid degradation of ATP in RBCs. This degradation process is modeled in a numerical simulation in a companion paper (Vincenzi, F. F. and Hinds, T. R. (1992) Biochim. Biophys. Acta 1105, 63-70). The loss of ATP followed pseudo-first-order kinetics, and the rate constants for ATP degradation was taken as a measure of the capacity of the Ca pump ATPase. A number of variables were examined to optimize the activity of the ATPase. These variables included the concentrations of Ca and A23187. Because A23187 can promote loss of cellular Mg, it was necessary to include MgCl2 in the incubation medium to optimize ATPase activity. Likewise, it was determined that inclusion of iodoacetic acid optimized the rate of ATP loss, presumably by preventing the resynthesis of ATP from ADP and inorganic phosphate. Cobalt inhibited the ionophore-dependent loss of ATP by apparent competition with Ca for binding to A23187. Results of many assays demonstrated substantial differences in the rate constant for ATP loss in RBCs from different individuals. RBCs were selected according to density. Density associated loss of Ca pump ATPase activity was observed both by the intact RBC assay, and by assay of Ca pump ATPase activity in saponin lysates of RBCs. The correlation coefficient between the two assays was 0.93. It is suggested that the rate constant for ATP loss in intact RBCs exposed to A23187 and Ca can be taken as a measure of the Ca pump ATPase activity. This may be useful when isolated membrane ATPase assays fail (e.g., dog RBCs). The intact cell assay can also be carried out on very small volumes of cells and may be of particular value when RBC volumes are limited.
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PMID:Assay of the Ca pump ATPase activity of intact red blood cells. 131 64

We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.
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PMID:A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin. 142 Apr 39

The objective of this study was to determine the relationship between cytosolic pH and vesicular pH during ATP depletion. Using digitized video microscopy and single, cultured rat hepatocytes, cytosolic pH and vesicular pH were quantitated by ratio imaging of BCECF (2', 7' biscarboxyethyl-5,6-carboxyfluorescein) fluorescence and fluorescein-dextran fluorescence, respectively. Basal value for cytosolic pH was 7.26 and basal value for vesicular pH was 4.86. During ATP depletion by metabolic inhibition with KCN plus iodoacetic acid or antimycin A, cytosolic pH decreased 0.71 units to 6.55. In separate experiments under identical conditions, vesicular pH increased 1.59 units to 6.45, suggesting that protons were leaking from acidic vesicles during ATP depletion. Fluorescein-dextran fluorescence remained punctate, indicating that the rise in vesicular pH was due to an efflux of protons from vesicles and not loss of vesicle integrity. To determine whether efflux of protons from acidic vesicles can acidify cytosolic pH, we used two maneuvers that result in leakage of protons from acidic vesicles without significantly decreasing cellular ATP: (a) hypotonic stress in K(+)-free media and (b) exposure of the cells to the H(+)-ATPase inhibitor NBD-Cl. Both hypotonic stress and NBD-Cl decreased cytosolic pH 0.4 units to 6.86 and increased vesicular pH 2.0 units to 6.76, resulting in near-equilibration of cytosolic pH and vesicular pH. Thus an efflux of protons from intracellular compartments will acidify cytosolic pH of hepatocytes (pH 6.86), but not to the same degree as ATP depletion (pH 6.55).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Efflux of protons from acidic vesicles contributes to cytosolic acidification of hepatocytes during ATP depletion. 171 57

We evaluated the contributions of calcium loading and impaired energy production to metabolic and ultrastructural manifestations of cell injury in a cultured neonatal rat ventriculocyte model. Direct calcium loading was produced by incubation in K(+)-free medium to inhibit the Na+,K(+)-ATPase and promote Na(+)-Ca2+ exchange, and inhibition of energy metabolism was produced by incubation with 30 microM iodoacetic acid (IAA). Measurements were made of total cell calcium, [3H] arachidonic acid (AA) release (an index of membrane phospholipid degradation), ATP, and ultrastructural features of cell damage. Inhibition of the Na(+),K(+) pump resulted in the rapid onset of cellular calcium loading, increased [3H]AA release, and moderate ATP reduction. After return to control medium for 24 hours, myocytes previously exposed to K(+)-free medium for 1 hour showed recovery of ATP level and little additional [3H]AA release. However, after 2 to 3 hours of calcium loading, the ATP level remained moderately depressed, residual [3H]AA release was greater, and a mixed population of relatively normal and severely damaged myocytes was observed by electron microscopy. IAA treatment for 1 hour resulted in moderate ATP reduction without calcium accumulation or [3H]AA release, whereas IAA treatment for 3 hours resulted in marked ATP reduction associated with calcium accumulation and [3H]AA release. Reversal experiments showed substantial recovery of ATP level after 1 hour of IAA exposure, and marked ATP depression and [3H]AA release associated with widespread irreversible injury after 3 hours. Thus, the data indicate that increased calcium accumulation itself can initiate accelerated membrane phospholipid degradation, but that progression to irreversible injury is influenced by other factors, including the magnitude of ATP depression associated with calcium loading.
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PMID:Effects of calcium loading and impaired energy production on metabolic and ultrastructural features of cell injury in cultured neonatal rat cardiac myocytes. 216 2

The relationship between the oxygen uptake and the release of amylase and sialic acid induced by pilocarpine was investigated in dog submandibular glands. Pilocarpine dose-dependently stimulated the oxygen uptake. The dose required for the maximal response was 10 microM. The release of amylase and sialic acid induced by pilocarpine was inhibited by the addition of iodoacetic acid, malonic acid, 2, 4-dinitrophenol, antimycin A or sodium azide. The oxygen uptake induced by pilocarpine was significantly inhibited by iodoacetic acid, malonic acid, antimycin A or sodium azide. On the other hand, 2, 4-dinitrophenol further stimulated the oxygen uptake by pilocarpine. The increase in the oxygen uptake or the release of amylase and sialic acid induced by pilocarpine was significantly inhibited by ouabain. The Na+, K+-ATPase activity ratio in the microsomal fraction of dog submandibular glands was dose-dependently increased by pilocarpine. The Na+, K+-ATPase activity ratio induced by pilocarpine was significantly inhibited by ouabain, antimycin A, oligomycin or 2, 4-dinitrophenol. The pilocarpine-induced Na+, K+-ATPase activity ratio was significantly inhibited by the removal Ca2+ from the medium or the addition of 2 mM EGTA. These results suggest that the increase in the oxygen uptake by pilocarpine is profoundly involved in the energy supply for the process of amylase and sialic acid release. In particular, the energy supply demanded for the activation of Na+ pump may play a role in the mechanism by which pilocarpine induces the oxygen uptake.
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PMID:[Studies on the relationship between the oxygen uptake and the release of amylase and sialic acid]. 241

Pancreatic ductal cell secretion has not been well characterized due to the difficulty in obtaining sufficient quantities of purified ductal cells. To determine if the MIA PaCa-2 cell line would provide a useful model for in vitro studies of pancreatic ductal cell secretion, the present study was designed to characterize these cells in greater detail. In this investigation, the human pancreatic undifferentiated cell line, MIA PaCa-2, was compared with PANC-1 cells (a human ductal cell line previously characterized), isolated rat and human ducts, acinar cells, and nonpancreatic cell lines. The results indicate that while the morphology of the MIA PaCa-2 cell line is nonpolarized and generally atypical of either ductal or acinar cells, the cell line has retained certain biochemical similarities to ductal cells. Additional morphological studies indicated (a) the presence of intermediate filaments characteristic of epithelial cells, (b) the absence of zymogen granules, and (c) an apparent basolateral plasma membrane localization of Na+, K+-ATPase. Similar to ductal cells, biochemical analyses indicated (a) the presence of Na+, K+-ATPase based on [3H]-ouabain binding assays, (b) high levels of carbonic anhydrase, (c) low levels of gamma-glutamyl transpeptidase, (d) nondetectable levels of amylase, and (e) protein composition and protein synthetic patterns comparable to PANC-1 cells. Finally, as with PANC-1 cells and isolated rat and human ducts, the major sulfated secretory product of MIA PaCa-2 cells was a protein with a molecular weight of approximately 660,000 to 1 million.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparative analysis of a human pancreatic undifferentiated cell line (MIA PaCa-2) to acinar and ductal cells. 247 96


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