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Query: UNIPROT:P20020 (adenosine triphosphatase)
 3,299 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The chemical properties of two highly purified preparations of (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) and their subunits have been compared. One preparation is derived from the rectal gland of the spiny dogfish shark, Squalus acanthias and the other preparation is derived from the electric organ of the electric eel, Electrophorus electricus. Ouabain binding and phosphorylation from [gamma-32-P]ATP for both enzymes ranged from 4000 to 4300 pmol per mg of protein. This gives a stoichiometry for ouabain binding and phosphorylation of 1:1 for both enzymes. The molar ratios of catalytic subunit to glycoprotein was 2:1 for both enzymes, suggesting a minimum molecular weight of 250, 000, which agrees with the molecular weight obtained by radiation inactivation. Assuming that only one of the two catalytic subunits is phosphorylated and binds ouabain per (sodium + potassium)-activated adenosine triphosphatase molecule the data on phosphorylation and ouabain binding also give a molecular weight of 250, 000. The data on phosphorylatiion, ouabain binding, subunit composition, and molecular weight based on radiaion inactivation are thus all internally consistent. A technique has been developed for isolation of pure catalytic subunit and glycoprotein in good yields by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of chemical studies have been carried out with the purified subunits. The amino acid composition of the catalytic subunit was different from that of the glycoprotein, but the amino acid composition of each of the two subunits was essentially the same for both species. However, the NH2-terminal amino acid for the catalytic subunit was alanine for the rectal gland enzyme and serine for the electric organ enzyme, suggesting some differencesin amino acid sequences for the two species. The NH2-terminal amino acid for the glycoprotein was alanine for the two species. The glycoproteins from both species contained the same carbohydrates but in quite differing amounts. The carbohydrates were glucosamine, sialic acid, fucose, galactose, mannose, and glucose. The release of all the sialic acid from the electric organ enzyme and the release of 40% of the sialic acid from the rectal gland enzyme did not affect (sodium + potassium)-activated adenosine triphosphatase activity. Both enzymes contained the following phospholipids, which accounted for 98 to 100% of the total phospholipid phosphorus: sphingomyelin, lecithin, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylserine, the amount of any phospholipid per mg of enzyme as well as the total phospholipid content were quite different for the two enzymes.
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PMID:Molecular properties of purified (sodium + potassium)-activated adenosine triphosphatases and their subunits from the rectal gland of Squalus acanthias and the electric organ of Electrophorus electricus. 12 22

Sodium- and potassium-activated adenosine triphosphatase (NaK-ATPase) was purified from nasal salt glands of the duck (Anas platyrhynchos). Enzyme of specific activity 2,000 to 2,300 mumol of Pi/mg/hour was routinely obtained by sodium dodecyl sulfate treatment of a microsomal fraction of gland homogenate in the presence of 3 mM ATP followed by pelleting of the enzyme through a sucrose density gradient. Purified NaK-ATPase was stable for over 3 months at -20 degree. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography purified NaK-ATPase was shown to contain two polypeptide chains of molecular weight 94,000 and 60,000, the smaller of which was a glycoprotein. Purified enzyme of activity 2,300 mumol of Pi/mg/hour bound 3,600 pmol of ouabain/mg of enzyme protein. Reaction with [gamma-32P]ATP in the presence of Mg2+ and Na+ gave 7,025 pmol of acyl phosphate/mg of enzyme protein. The turnover number calculated from phosphorylation data was 5,460 min-1. Amino acid analysis of the polypeptide components of duck salt gland enzyme after separation by gel filtration chromatography in sodium dodecyl sulfate demonstrated strong compositional homology with highly purified NaK-ATPase preparations from other organs and species. The NH2-terminal amino acid of the 94,000-dalton component was glycine and of the 60,000-dalton component, alanine. With a combination of manual sequencing and automated Edman degradation, the NH2-terminal amino acid sequence of the 94,00-dalton catalytic subunit was found to be Gly-Arg-Asn-Lys-Tyr-Glu-Thr-Thr-Ala-()-Ser-Glu.
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PMID:Sodium- and potassium-activated adenosine triphosphatase of the nasal salt gland of the duck (Anas platyrhynchos). Purification, characterization, and NH2-terminal amino acid sequence of the phosphorylating polypeptide. 13 47

When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited.
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PMID:Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae. 13 54

1. The properties of membrane vesicles from the extreme thermophile Bacillus caldolyticus were investigated. 2. Vesicles prepared by exposure of spheroplasts to ultrasound contained cytochromes a, b and c, and at 50 degrees C they rapidly oxidized NADH and ascorbate in the presence of tetramethyl-p-phenylenediamine. Succinate and l-malate were oxidized more slowly, and dl-lactate, l-alanine and glycerol 1-phosphate were not oxidized. 3. In the absence of proton-conducting uncouplers the oxidation of NADH was accompanied by a net translocation of H(+) into the vesicles. Hydrolysis of ATP by a dicyclohexylcarbodi-imide-sensitive adenosine triphosphatase was accompanied by a similarly directed net translocation of H(+). 4. Uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone or valinomycin plus NH(4) (+)) prevented net H(+) translocation but stimulated ATP hydrolysis, NADH oxidation and ascorbate oxidation. The last result suggested an energy-conserving site in the respiratory chain between cytochrome c and oxygen. 5. Under anaerobic conditions the reduction of cytochrome b by ascorbate (with tetramethyl-p-phenylenediamine) was stimulated by ATP hydrolysis, indicating an energy-conserving site between cytochrome b and cytochrome c. However, no reduction of NAD(+) supported by oxidation of succinate, malate or ascorbate occurred, neither did it with these substrates in the presence of ATP under anaerobic conditions, suggesting that there was no energy-conserving site between NADH and cytochrome b. 6. Succinate oxidation, in contrast with that of NADH and ascorbate, was strongly inhibited by uncouplers and stimulated by ATP hydrolysis. These effects were not observed when phenazine methosulphate, which transfers electrons from succinate dehydrogenase directly to oxygen, was present. It was concluded that in these vesicles the oxidation of succinate was energy-dependent and that the reoxidation of reduced succinate dehydrogenase was dependent on the outward movement of H(+) by the protonmotive force. 7. In support of the foregoing conclusion it was shown that the reduction of fumarate by NADH was an energy-conserving process. 8. If the activities of vesicles accurately represent those of the intact organism it appears that in B. caldolyticus the reduction of fumarate to succinate at the expense of reducing equivalents from NADH is energetically favoured over succinate oxidation even under aerobic conditions. This may be related to the need for an ample supply of succinate for haem synthesis in order to provide cytochromes for the organism.
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PMID:The oxidative activities of membrane vesicles from Bacillus caldolyticus. Energy-dependence of succinate oxidation. 20 11

Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase.
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PMID:The isolation and partial characterization of the plasma membrane from Trypanosoma brucei. 48 94

The ATP-energy transducing system in membranes of Escherichia coli is inhibited by dicyclohexylcarbodiimide. The protein component of this complex with which carbodiimides covalently react to inhibit function was previously identified by labeling wild type and dicyclohexylcarbodiimide-resistant mutants with dicyclohexyl[14C]carbodiimide (Fillingame, R. H. (1975) J. Bacteriol. 124, 870-883). This specific carbodiimide-reactive protein has now been purified. The protein was extracted from the membrane with chloroform:methanol and chromatographed on DEAE-cellulose and hydroxypropyl Spehadex G-50 in this sulvent mixture. The resultant 700-fold purification yielded a protein that was homogeneous on dodecyl sulfate-acrylamide gel electrophoresis and virtually free of phospholipid. It remained soluble in neutral chloroform:methanol throughout the purification procedure. The amino acid composition of the purified protein was extraordinary in that only 16% of the amino acids present could be considered polar. Histidine, serine, cysteine, and tryptophan were not found. Abnormally high contents of methionine, glycine, alanine, and leucine were present. One mole of lysine and threonine were found/mole of dicyclohexyl[14C]carbodiimide bound. The minimum molecular weight based on the amino acid composition was 8400. The specific carbodiimide-reactive protein has also been purified without prior modification by dicyclohexylcarbodiimide. The unmodified protein eluted from DEAE-cellulose at a higher salt concentration than the dicyclohexylcarbodiimide-modified form, which suggested that the reaction with the carbodiimide neutralized the negative charge. Only one-third of the total carbodiimide-reactive protein in the membrane was modified by dicyclohexylcarbodiimide under conditions which maximally inhibited adenosine triphosphatase activity. These results rais the possibility that the carbodiimide-reactive protein may be present as an oligomer in the energy-transducing complex. The purification of the unmodified carbodiimide-reactive protein should permit assessment of tis biological function, particularly its role in the protein-translocation process that is catalyzed by this energy-transducing complex.
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PMID:Purification of the carbodiimide-reactive protein component of the ATP energy-transducing system of Escherichia coli. 78 71

The role of the mammalian mesonephric kidney is not completely understood. It has been established that outpouchings of the mesonephric excretory ducts give origin to parts of the urogenital system of the adult. It is also known that mammalian mesonephric urine is formed as an ultrafiltrate. The mesonephric renal tubules have Na+/K+ adenosine triphosphatase (Na+/K+ pump), secrete phenol red, and reabsorb protein. Prior to this work, the possibility of epithelial transport of ions and metabolic substrates across mammalian mesonephric tubules had not been directly evaluated. Proximal mesonephric tubules obtained from 17 to 18-days-old rabbit embryos were isolated and perfused in vitro. Continuous intracellular electrical recordings were obtained with Ling-Gerard-type microelectrodes and a high input impedance electrometer. In tubules perfused and bathed in standard mammalian Ringer's solutions, the average transmembrane electrical cell potential difference (PD) was -43 +/- 0.5 mV (76 cells). The cellular PD decreased by 30 percent when the temperature of the bath was cooled from 37 degrees C to 30 degrees C. The cells also depolarized by 25 percent in the first five minutes of exposure to 0.1 mM ouabain. In addition, the cell PD decreased by 40 and 60 percent when the extracellular potassium concentration was raised from five to 25 and 50 mM, respectively. The uptake of glucose and alanine was similarly electrogenic (delta:1 mV/mM). The cell PD, the K+ conductance, and the electrogenicity induced by luminal exposure to 5 mM glucose or alanine are significantly lower in the mesonephric as compared to the metanephric proximal tubules of the rabbit. These observations suggest that sodium-coupled transepithelial transport mechanisms, driven by the Na+/K+ pump, are already present in the mammalian mesonephric proximal tubule. Increases in the number of Na+/K+ pumps, conductive K+ channels, and sodium-substrate cotransporters seem to be at the core of proximal tubular ontogeny.
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PMID:Renal ontogeny: epithelial transport in the mammalian mesonephric proximal tubule. 164 30

Brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) were simultaneously prepared from surgically resected pieces of morphologically intact human duodenum with a modified Percoll gradient centrifugation method. Alkaline phosphatase was enriched 20-fold in BBMV, whereas (Na+ + K+)-stimulated adenosine triphosphatase was enriched 15-fold in BLMV. BBMV and BLMV were preincubated with 3 microM synthetic somatostatin-14 or 3 microM SMS 201-995 for 10 min at 5 degrees C. In BBMV calcium, sodium, D-glucose, L-alanine, and D-mannitol uptake was unaffected by somatostatin-14 and SMS 201-995. In BLMV we found two Ca++ transport systems: Na+/Ca++ exchange and ATP-driven Ca++ transport. Somatostatin-14 had no effect on either of the two transport mechanisms. SMS 201-995 had no effect on Na+/Ca++ exchange but significantly inhibited basolateral ATP-dependent Ca++ transport (-40% +/- 5%, p less than 0.005).
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PMID:Effect of the somatostatin analogue SMS 201-995 on ATP-dependent calcium transport of basolateral vesicles from human duodenum. 289 47

Amino acid transport was studied in C1 cells which contain amplified levels of sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase), in C4 cells which are ouabain-sensitive revertants, and in parental HeLa S3. Sodium-dependent uptake of aminoisobutyric acid and alanine was increased 2-fold in the amplified C1 cells. After a 6 h amino acid starvation period, the rate of sodium-dependent uptake of methylaminoisobutyric acid was 70-90% greater for C1 than for C4 and HeLa. This uptake was inhibitable by ouabain and the apparent Km values for high affinity uptake were similar in all three lines. Overall, neutral amino acid uptake through Systems A, ASC, and L was 2-fold higher in the Na,K-ATPase amplified C1 cells relative to C4 or HeLa. The induction of System A uptake of methylaminoisobutyric acid after starvation was more rapid in both the amplified C1 cells and the revertant C4 when compared to HeLa, which suggests that the selection for amplification of the Na,K-ATPase produced membrane alterations affecting the adaptive regulation of System A.
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PMID:Alterations in amino acid transport in Na,K-ATPase amplified HeLa cells. 300 Oct 56

1. The alanyl-s-RNA synthetase of tomato roots has been purified by ammonium sulphate precipitation, adsorption on calcium phosphate gel and DEAE-cellulose chromatography and its properties have been investigated. 2. Enzyme activity was measured by using the hydroxamate assay, the [(32)P]pyrophosphate-ATP-exchange assay and the [(14)C]alanyl-s-RNA assay. The purified enzyme was specific for l-alanine and was activated by Mg(2+) ions and to a smaller extent by Co(2+) and Mn(2+) ions. It was free from adenosine triphosphatase, pyrophosphatase and ribonuclease, and possessed a specific activity comparable with that of the most highly purified aminoacyl-s-RNA synthetases from animal and microbial systems. 3. The properties of the purified enzyme were similar in many respects to most other highly purified aminoacyl-s-RNA synthetases. It differed, however, in that the pH optimum of the hydroxamate assay was almost the same as that of the pyrophosphate-ATP-exchange assay and in requiring a high concentration of l-alanine for maximum activity (100mumoles/ml.). 4. The purified enzyme was not absolutely specific for tomato-root s-RNA; slight activity was also observed with yeast s-RNA. 5. The properties of this enzyme are fully consistent with the suggestion that the enzymic formation of alanyl-s-RNA proceeds via the intermediate formation of alanyl acyl-adenylate with the elimination of pyrophosphate from ATP. It remains to be shown the extent to which alanyl-s-RNA participates further in subsequent stages of protein synthesis in plants.
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PMID:The purification and properties of the alanyl-transfer ribonucleic acid synthetase of tomato roots. 428 91


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