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 binding of N-acetyl-L-glutamate, the physiological allosteric activator, to rat liver carbamoyl-phosphate synthetase (ammonia) was studied by techniques of rate of dialysis and of ultracentrifugation in the Airfuge. There is one binding site for acetylglutamate per enzyme monomer (Mr 165 000). K+, Mg2+ (free) and ATP were required to demonstrate binding. The concentrations of ATP required indicate that binding of ATPA (the ATP molecule that yields Pi) is needed. HCO-3 was not essential, but it enhanced binding of acetylglutamate. Glycerol also favored binding. Plots of Kd values versus the reciprocal of free Mg2+ and ATP concentrations are linear and indicate that ATPA, K+ and Mg2+ bind before acetylglutamate. In the presence of these ligands and HCO-3, ammonia increased drastically the Kd value for acetylglutamate, whereas in absence of HCO-3 ammonia had little effect. This suggests that acetylglutamate dissociates with the products and explains the higher Km for acetylglutamate in the synthetase (overall) reaction than in the ATPase (partial) reaction. In the absence of ATP acetylglutamate was bound with high affinity if ADP and carbamoyl phosphate were present. ADP or carbamoyl phosphate alone did not promote substantial binding. Binding of acetylglutamate at low concentration was slow; it was accelerated at higher concentrations of the activator. Exchange of bound acetylglutamate with acetylglutamate in solution was fast. A scheme proposed earlier for allosteric activation of the enzyme [Rubio, V., Britton, H. G. and Grisolia, S. (1983) Eur. J. Biochem. (in preparation)] is refined to incorporate the new information. Binding of ATPA, K+ and Mg2+ and formation of 'active CO2' (the central complex) are greatly favored by acetylglutamate.
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PMID:Binding of N-acetyl-L-glutamate to rat liver carbamoyl phosphate synthetase (ammonia). 688 68

The influence of ouabain and physostigmine on 42K and 86Rb uptake in isolated from sartorii with normal [Na]i(12-14 mmol.kg -1 wet weight) and low [Na]i (6 mmol.kg-1 wet weight) was compared. Both in normal sodium and in low sodium muscles application of 10-3 M physostigmine reduces potassium influx by about 70%. About one forth of potassium-uptake in normal-sodium muscles is inhibited by ouabain (10-4 M) and only a very slight fraction of potassium uptake is ouabain-sensitive in low-sodium muscle. The effects of ouabain and physostigmine on 42K influx are additive. The greater parts of the Rb-fluxes are through the ouabain-sensitive pathway. Glycerol treatment has no effect on ouabain-sensitive channels although it inhibits markedly the K-flux through the physostigmine-sensitive pathway. The results suggest that the Na-K-ATPase is located in the surface membrane while most of the physostigmine-sensitive K-exchange is within the tubules.
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PMID:The subcellular location of potassium flux pathways in frog skeletal muscle. 697 36

Recent work on the structure and function of cholinergic synaptic vesicles isolated from the electrometer nerve terminals of Torpedo marmorata, in which lipid and protein analysis, water space and density measurements, proton and 31P NMR spectroscopy and uptake were used, is reviewed. A consistent model emerges of a vesicle with a highly hydrated core, containing acetylcholine in free solution at an approximate concentration of 0.5 M, ATP and Ca2+ at an approximate concentration of 0.2 M, and a pH of about 6.7 enclosed within a lipoprotein membrane with a fairly high (approximately 30%) water content and about five specific protein constituents. One of these is actin; another, resembling the ADP/ATP exchange carrier of mitochondria, is thought to be the ATP carrier; and a third may be a Ca2+,Mg2+-activated ATPase known to be associated with these vesicles. The vesicular ATP carrier is less specific than the mitochondrial carrier. Small amounts of glucosaminoglycan are present, the negative charges of which may have a role in binding Ca2+ and acetylcholine. Actin is the only common constituent of the vesicle and presynaptic plasma membrane, which shows that the former retains its identity through one or more cycles of exo- and endocytosis. Glycerol water space measurements show that the small, dense synaptic vesicles generated by stimulation-induced cycles of exo- and endocytosis contain less water than vesicles from unstimulated tissue. It is suggested that these changes are secondary to osmotic pressure changes in the vesicle core accompanying discharge and repletion of transmitter and ATP.
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PMID:Biophysical and biochemical studies of isolated cholinergic vesicles from Torpedo marmorata. 711 51

We have examined the temperature-dependent effects of several organic compounds on the activity of the purified Ca(2+)-ATPase of erythrocytes. The monomeric enzyme was activated either by interaction with calmodulin or by oligomerization in the absence of calmodulin. Of the four homologous solute series studied including polyols, alkanols, aprotic solvents, and N-methyl derivatives of formamide and acetamide only polyols stabilized the enzyme over a broad range of concentration and temperature. Similarity of Ca(2+)-ATPase activity patterns at 25 and 37 degrees C and in the presence of glycerol is in agreement with indirect, stabilizing interactions. Glycerol also protected the Ca(2+)-ATPase from thermal denaturation at 45 degrees C. Within each homologous series, inhibitory effects increased with increasing solute concentration and with increasing structural similarity to detergents, indicating that direct destabilizing interactions are responsible for the observed inhibition. These were comparable to the destabilizing effect of urea. Oligomers were more resistant to all inhibitory solutes as compared to calmodulin-activated monomers suggesting that the nonpolar patches of the oligomerized enzyme are less accessible to solutes.
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PMID:Stabilizing and destabilizing effects on plasma membrane Ca(2+)-ATPase activity. 785 37

Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae. Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0. 4-0.7 micromol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated. By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone. Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp. Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K(MgATP)(M), V(max), and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies.
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PMID:Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: high-yield expression and purification of human P-glycoprotein. 1072 88

The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146-6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H(+) ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.
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PMID:Regulation of carbon and electron flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol mixtures. 1116 Jan 7

Loss of skeletal muscle is a major factor in the poor survival of patients with cancer cachexia. This study examines the mechanism of catabolism of skeletal muscle by a tumour product, proteolysis-inducing factor (PIF). Intravenous administration of PIF to normal mice produced a rapid decrease in body weight (1.55 +/- 0.12 g in 24 h) that was accompanied by increased mRNA levels for ubiquitin, the Mr 14 000 ubiquitin carrier-protein, E2, and the C9 proteasome subunit in gastrocnemius muscle. There was also increased protein levels of the 20S proteasome core and 19S regulatory subunit, detectable by immunoblotting, suggesting activation of the ATP-ubiquitin-dependent proteolytic pathway. An increased protein catabolism was also seen in C(2)C(12)myoblasts within 24 h of PIF addition with a bell-shaped dose-response curve and a maximal effect at 2-4 nM. The enhanced protein degradation was attenuated by anti-PIF antibody and by the proteasome inhibitors MG115 and lactacystin. Glycerol gradient analysis of proteasomes from PIF-treated cells showed an elevation in chymotrypsin-like activity, while Western analysis showed a dose-related increase in expression of MSSI, an ATPase that is a regulatory subunit of the proteasome, with a dose-response curve similar to that for protein degradation. These results confirm that PIF acts directly to stimulate the proteasome pathway in muscle cells and may play a pivotal role in protein catabolism in cancer cachexia.
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PMID:Activation of ATP-ubiquitin-dependent proteolysis in skeletal muscle in vivo and murine myoblasts in vitro by a proteolysis-inducing factor (PIF). 1146 Oct 93

Candida halophila CBS 4019 (syn. C. versatilis) is an extremely salt-tolerant yeast. It was chosen to study the physiology of long-term resistance to salt stress in cells cultivated at increasing NaCl concentrations up to 4 or 5 M. Growth under stress was slow, severely affected not by salt, but rather by initial external pH. Growing on glucose, glycerol and mannitol were produced. Glycerol is the osmolyte and is transported by H(+)/symport. Transport-driven accumulation was though not affected by salt. The role of mannitol is unknown. Internal pH and intracellular volume were constant during growth at all initial pH/salt combinations. H(+)-ATPase activity was not affected by salt.
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PMID:Physiological studies on long-term adaptation to salt stress in the extremely halotolerant yeast Candida versatilis CBS 4019 (syn. C. halophila). 1268 33

Dorfin, a RING-IBR type ubiquitin ligase (E3), can ubiquitylate mutant superoxide dismutase 1, the causative gene of familial amyotrophic lateral sclerosis (ALS). Dorfin is located in ubiquitylated inclusions (UBIs) in various neurodegenerative disorders, such as ALS and Parkinson's disease (PD). Here we report that Valosin-containing protein (VCP) directly binds to Dorfin and that VCP ATPase activity profoundly contributes to the E3 activity of Dorfin. High through-put analysis using mass spectrometry identified VCP as a candidate of Dorfin-associated protein. Glycerol gradient centrifugation analysis showed that endogenous Dorfin consisted of a 400-600-kDa complex and was co-immunoprecipitated with endogenous VCP. In vitro experiments showed that Dorfin interacted directly with VCP through its C-terminal region. These two proteins were colocalized in aggresomes in HEK293 cells and UBIs in the affected neurons of ALS and PD. VCP(K524A), a dominant negative form of VCP, reduced the E3 activity of Dorfin against mutant superoxide dismutase 1, whereas it had no effect on the autoubiquitylation of Parkin. Our results indicate that VCPs functionally regulate Dorfin through direct interaction and that their functional interplay may be related to the process of UBI formation in neurodegenerative disorders, such as ALS or PD.
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PMID:Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders. 1545 87

The isolation of the chloroplast ATP synthase complex (CF(0)-CF(1)) and of CF(1) from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil alpha subunit migrates ahead of the beta subunit and epsilon-migrates ahead of subunit II of CF(0) when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF(1) isolated from D. bardawil resembles the CF(1) isolated from Chladmydomonas reinhardi in that a reversible, Mg(2+)-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-P(i) exchange catalyzed by D. bardawil CF(0)-CF(1) proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg(2+)-ATPase of the D. bardawil CF(1) is more resistant to glycerol inhibition than the octylglucoside-activated, Mg(2+)-ATPase of spinach CF(1) or the ethanol-activated, Mg(2+)-dependent ATPase of the C. reinhardi CF(1). Both cyclic photophosphorylation and ATP-P(i) exchange catalyzed by D. bardawil CF(0)-CF(1) are more sensitive to high concentrations of NaCl than is the spinach complex.
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PMID:Purification and Characterization of a Glycerol-Resistant CF(0)-CF(1) and CF(1)-ATPase from the Halotolerant Alga Dunaliella bardawil. 1666 7


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