Assuntos
ATPase Trocadora de Hidrogênio-Potássio/química , Animais , Fluoresceína-5-Isotiocianato , ATPase Trocadora de Hidrogênio-Potássio/fisiologia , ATPase Trocadora de Hidrogênio-Potássio/ultraestrutura , Humanos , Transporte de Íons , Ligantes , Microscopia de Fluorescência/métodos , Polietilenoglicóis , Estrutura Quaternária de Proteína , SolubilidadeRESUMO
Pig kidney Na/K-ATPase preparations showed a positive cooperative effect for pNPP in Na-pNPPase activity. Measurements of the Na-pNPPase activity, Na-ATPase activity and the accumulation of phosphoenzyme (EP) under conditions of pNPP saturation showed several different ATP affinities. The presence of pNPP reduced both the maximum amount of EP and Na-ATPase activity to half showing a value of 4 and a 3,700-fold reduced ATP affinity for EP formation, and a 7 and 1,300-fold reduced affinity for Na-ATPase activity. The presence of low concentrations of ATP in the phosphorylation induced a 2-fold enhancement in Na-pNPPase activity despite a reduction in available pNPP sites. However, higher concentrations of ATP inhibited the Na-pNPPase activity and a much higher concentration of ATP increased both the phosphorylation and Na-ATPase activity to the maximum levels. The maximum Na-pNPPase activity was 1.7 and 3.4-fold higher without and with ATP, respectively, than the maximum Na-ATPase activity. These data and the pNPP dependent reduction in both Na-ATPase activity and the amount of enzyme bound ATP provide new evidence to show that ATP, pNPP and ATP with pNPP, respectively, induce different subunit interactions resulting a difference in the maximum Na(+)-dependent catalytic activity in tetraprotomeric Na/K-ATPase.
Assuntos
4-Nitrofenilfosfatase/química , Trifosfato de Adenosina/química , Modelos Biológicos , ATPase Trocadora de Sódio-Potássio/química , Animais , Domínio Catalítico , Ativação Enzimática , Rim/enzimologia , Nitrofenóis/química , Compostos Organofosforados/química , Fosforilação , SuínosRESUMO
BACKGROUND/AIMS: Anti-parietal cell antibody (APCA) is used for diagnosis of pernicious anemia and type A gastritis. APCA is present in patients with Helicobacter pylori (H. pylori)-positive gastritis and is related to atrophic gastritis and gastric carcinoma. The aim of the study was to determine whether an enzyme-linked immunosorbent assay (ELISA) can be used to detect APCA. METHODOLOGY: Study subjects were 134 patients (74 men, 60 women; mean age, 63 years; range, 18-88 years) with gastric carcinoma, gastric ulcer, duodenal ulcer, gastritis, type A gastritis or pernicious anemia and H. pylori-negative normal mucosa subjects in the stomach. APCA was measured in all subjects by our ELISA method. Results of ELISA were compared with results of indirect immunofluorescence (IIF). RESULTS: The percentage of samples that were APCA-positives by IIF at a 1/20 or greater serum dilutions was similar to that by ELISA (86.9%). Agreement between ELISA and IIF for presence of APCA was 78.3% in patients with gastric carcinoma, 80.1% (gastric ulcer), 59.1% (duodenal ulcer), 51.7% (gastritis), 75.0% (type A gastritis), 100% (pernicious anemia) and 80.0% (normal). CONCLUSIONS: ELISA is a useful method for detection of APCA for diagnosis of PA and type A gastritis.
Assuntos
Autoanticorpos/sangue , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Células Parietais Gástricas/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Activity-oligomeric assembly relationships using octaethylene glycol dodecyl ether (C12E8) solubilized pig gastric H/K-ATPase (unmodified H/K-ATPase) or H/K-ATPase modified with Fluorescein 5'-isothiocyanate (FITC-H/K-ATPase) were examined. The amount of oligomeric species in FITC-H/K-ATPase, which retained little H/K-ATPase activity was estimated by a single-molecule detection technique using total internal reflection fluorescence microscopy. Solubilization of the FITC-H/K-ATPase reduced the potassium-dependent p-nitrophenyl phosphatase (K-pNPPase) activity to around 5% of the level of the membrane-bound enzyme with the formation of 50% protomer and 40% diprotomer. The solubilization of unmodified H/K-ATPase also reduced both the K-pNPPase and H/K-ATPase activities to around 5%. However, solubilization with increasing concentrations of potassium acetate induced significant and similar increases in K-pNPPase activity (K0.5 = 35 mM) with an increase in the amount of the tetraprotomer of FITC-H/K-ATPase, and the K-pNPPase (K0.5 = 28 mM) and H/K-ATPase (K0.5 = 40 mM) activities of the unmodified H/K-ATPase. The correlation coefficient between the proportion of tetraprotomer and the proportion of the K-pNPPase activity for the same FITC-H/K-ATPase preparation was estimated to be 0.93. Similar coefficients were also obtained between the proportion of tetraprotomer in the FITC-H/K-ATPase and the proportion of K-pNPPase and H/K-ATPase activities in the unmodified H/K-ATPase, with value of 0.85 and 0.86, respectively. Such positive correlations were not obtained between these activities and other oligomeric species. These data, the first direct comparison of oligomeric assembly and enzyme activity both stabilized by K+ in C12E8-solubilized gastric H/K-ATPase, provide strong evidence that the catalytic unit of C12E8-solubilized gastric H/K-ATPase is a tetraprotomer.
Assuntos
Mucosa Gástrica/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Estrutura Quaternária de Proteína , 4-Nitrofenilfosfatase/química , 4-Nitrofenilfosfatase/metabolismo , Animais , Éteres/química , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Microscopia de Fluorescência , Acetato de Potássio , SuínosRESUMO
The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in the transmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 and Glu-954 in M9, the side chains of which appear to be in close proximity, were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K, V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistant cell lines were obtained from T774A, V920E, E953A, and E954A, whereas Q923N/E/D/L, E953K, and E954K could only be transiently expressed as fusion proteins with an enhanced green fluorescent protein. The apparent K0.5 values for Na+, as estimated by the Na+-dependent phosphoenzyme formation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), were increased by around 2 approximately 8-fold in the case of T774A, V920E, and E954A. The apparent K0.5 values for K+, as estimated by the Na,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity (K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, except that V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparent K0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a high affinity ATP effect), were increased by 1.6 approximately 2.6-fold in the case of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity (K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPase activity (low affinity ATP effects) were, respectively, increased by 1.8-fold and unchanged in the case of T774A but decreased by 2- and 4.8-fold in the case of V920E and were slightly changed and increased by 1.7-fold in the case of E954A. The E953A showed little significant change in the apparent affinities. These results suggest that Gln-923 in M8 is crucial for the active transport of Na+ and/or K+ across membranes and that the side chain oxygen atom of Thr-774 in M5, the methyl group(s) of Val-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly play some role in the transport of Na+ and also in the high and low affinity ATP effects rather than the transport of K+.
Assuntos
Ácido Glutâmico/química , Glutamina/química , ATPase Trocadora de Sódio-Potássio/química , Treonina/química , Valina/química , 4-Nitrofenilfosfatase/farmacologia , Trifosfato de Adenosina/química , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cátions , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Cinética , Bicamadas Lipídicas/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ouabaína/farmacologia , Oxigênio/química , Fosforilação , Potássio/farmacologia , Conformação Proteica , Ratos , Proteínas Recombinantes de Fusão/química , Homologia de Sequência de Aminoácidos , Sódio/farmacologia , Cloreto de Sódio/farmacologia , TransfecçãoRESUMO
Membrane-bound H/K-ATPase was solubilized by octaethylene glycol dodecyl ether (C(12)E(8)) or n-octyl glucoside (nOG). H/K-ATPase activity and the distribution of protomeric and oligomeric components were evaluated by high-performance gel chromatography (HPGC) and by single-molecule detection using total internal reflection fluorescence microscopy (TIRFM). As evidenced by HPGC of the C(12)E(8)-solubilized enzyme, the distribution of oligomers was 12% higher oligomeric, 44% diprotomeric, and 44% protomeric species, although solubilization by C(12)E(8) reduced the H/K-ATPase activity to 1.8% of that of the membrane-bound enzyme. The electron microscopic images of the C(12)E(8)-solubilized enzyme showed the presence of protomers and a combination of two and more protomers. While the nOG-solubilized H/K-ATPase retained the same turnover number and 71% of the specific activity as that of the membrane-bound enzyme, 56% higher oligomeric, 34% diprotomeric, and 10% protomeric species were detected. TIRFM analysis of solubilized fluorescein 5'-isothiocyanate (FITC)-modified H/K-ATPase at Lys-518 of the alpha-chain showed a quantized photobleaching of the FITC fluorescence intensity. For the C(12)E(8)-solubilized FITC-enzyme, the fraction of each of the initial relative fluorescence intensity units of 4, 2, and 1 was, respectively, 5%, 44% and 51%. In the case of the nOG-solubilized FITC-enzyme, each fraction of 4 and 2 units was, respectively, 54% and 46% with no detectable 1 unit fraction. This represents the first direct observation of H/K-ATPase in aqueous solution. The correlation between the enzymatic activities and distribution of oligomeric forms of H/K-ATPase by HPGC and the observation of a single molecule of H/K-ATPase and others suggests that the tetraprotomeric form of H/K-ATPase molecules represents the functional species in the membrane.
Assuntos
ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Animais , Fracionamento Químico , Cromatografia em Gel , Detergentes/química , Ativação Enzimática , Mucosa Gástrica/enzimologia , Glucosídeos/química , ATPase Trocadora de Hidrogênio-Potássio/ultraestrutura , Isoenzimas/química , Isoenzimas/metabolismo , Isoenzimas/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Peso Molecular , Solubilidade , Soluções , Relação Estrutura-Atividade , SuínosRESUMO
A highly conserved amino acid sequence 442GDASE446 in the ATP binding pocket of rat Na/K-ATPase was mutated, and the resulting proteins, G442A, G442P, D443A, S445A, and E446A, were expressed in HeLa cells to investigate the effect of individual ligands on Na/K-ATPase. The apparent Km for the high and low affinity ATP effects was estimated by ATP concentration dependence for the formation of the Na-dependent phosphoenzyme (Kmh) and Na/K-ATPase activity (Kml). The apparent Km for p-nitrophenylphosphate (pNPP) for K-dependent-pNPPase (KmP) and its inhibition by ATP (Ki,0.5) and the apparent Km for Mg2+, Na+, K+, and vanadate in Na/K-ATPase were also estimated. For all the mutants, the value for ATP was approximately 2-10-fold larger than that of the wild type. While the turnover number for Na/K-ATPase activity were unaffected or reduced by 20 approximately 50% in mutants G442(A/P) and D443A. Although both affinities for ATP effects were reduced as a result of the mutations, the ratio, Kml Kmh, for each mutant was 1.3 approximately 3.7, indicating that these mutations had a greater impact on the low affinity ATP effect than on the high affinity effect. Each KmP value with the turnover number suggests that these mutations favor the binding of pNPP over that of ATP. These data and others indicate that the sequence 442GDASE446 in the ATP binding pocket is an important motif that it is involved in both the high and low affinity ATP effects rather than in free Mg2+, Na+, and K+ effects.
Assuntos
Trifosfato de Adenosina/metabolismo , ATPase Trocadora de Sódio-Potássio/química , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Compostos de Anilina/farmacologia , Animais , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Cinética , Ligantes , Magnésio/química , Magnésio/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Compostos Organofosforados/farmacologia , Potássio/química , Cloreto de Potássio/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Sódio/química , Cloreto de Sódio/farmacologia , Vanadatos/químicaAssuntos
Trifosfato de Adenosina/metabolismo , Mucosa Gástrica/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Fosfatos/metabolismo , Potássio/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Animais , Cinética , SuínosAssuntos
ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Sódio-Potássio/química , Animais , Mucosa Gástrica/enzimologia , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Rim/enzimologia , Cinética , Substâncias Macromoleculares , Microscopia de Fluorescência/métodos , ATPase Trocadora de Sódio-Potássio/metabolismo , SuínosRESUMO
It has been well established that phosphorylation is an important reaction for the regulation of protein functions. In the N-terminal domain of the alpha-chain of pig gastric H(+)/K(+)-ATPase, reversible sequential phosphorylation occurs at Tyr 10 and Tyr 7. In this study, we determined the structure of the peptide involving the residues from Gly 2 to Gly 34 of pig gastric H(+)/K(+)-ATPase and investigated the tyrosine phosphorylation-induced conformational change using CD and NMR experiments. The solution structure showed that the N-terminal fragment has a helical conformation, and the peptide adopted two alpha-helices in 50% trifluoroethanol (TFE) solvent, suggesting that the peptide has a high helical propensity under hydrophobic conditions. Furthermore, the CD and NMR data suggested that the structure of the N-terminal fragment becomes more disordered as a result of phosphorylation of Tyr 10. This conformational change induced by the phosphorylation of Tyr 10 might be an advantageous reaction for sequential phosphorylation and may be important for regulating the function of H(+)/K(+)-ATPase.
Assuntos
ATPase Trocadora de Hidrogênio-Potássio/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , ATPase Trocadora de Hidrogênio-Potássio/genética , Técnicas In Vitro , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosforilação , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Estômago/enzimologia , Suínos , Tirosina/químicaRESUMO
To investigate the relationship between the high and the low affinity ATP-binding site, which appears during the Na(+)/K(+)-ATPase reaction, four amino acids were mutated, the side chains of which are exposed to inside of the ATP-binding pocket. Six mutants, F475Y, K480A, K480E, K501A, K501E, and R544A, where the numbers correspond to the pig Na(+)/K(+)-ATPase alpha-chain, were expressed in HeLa cells. The apparent affinities were determined by high affinity ATP-dependent phosphorylation and by the low affinity activation of Na(+)/K(+)-ATPase or low affinity ATP inhibition of K(+)-para-nitrophenylphosphatase (pNPPase). For the mutants K480A and K501A, little affinity change was detected for either the high affinity or the low affinity effect. In contrast, the other four mutants reduced both apparent affinities. Strikingly, R544A had a 30-fold greater effect on the high affinity ATP site than the low affinity site. For the F475Y mutant, it is likely that there was a greater effect on the low affinity site than the high affinity site, but for both F475Y and K480E the affinity for the low affinity ATP effect was reduced so much that it was not possible to estimate a K(0.5). However, both the affinities for the K480E were reduced to approximately 1/20. The turnover number of the Na(+)/K(+)-ATPase and the apparent affinity for Na(+) and pNPP was reduced slightly or not at all for these mutants, but the turnover number of K(+)-pNPPase and the apparent affinity for K(+) were increased. These and other data suggest the presence of only one ATP-binding site, which can change its conformation to accept ATP with a high and low affinity. The requirement of Arg-544 and possibly Lys-501 is more important in forming a high affinity ATP binding conformation, and Phe-475 and possibly Lys-480 are more important in forming the low affinity ATP binding conformation.
Assuntos
Trifosfato de Adenosina/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Membrana Celular/enzimologia , Primers do DNA , Células HeLa , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Subunidades Proteicas , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , TransfecçãoRESUMO
BACKGROUND: Atrophic gastritis is more common in Japan than in Germany. The expression of anti-parietal cell antibody has been implicated in the genesis of atrophic gastritis associated with Helicobacter pylori infection. OBJECTIVE: We investigated the difference in serum levels of pepsinogens and in anti-parietal cell antibody expression between Japanese and German patients. METHODS: We recruited 102 Japanese and 46 German patients with dyspepsia. Endoscopic examination detected no localized lesions in the upper gastrointestinal tract of any patients. Anti-parietal cell antibody was investigated by enzyme-linked immunosorbent assay with the purified porcine H+,K+-ATPase fraction and immunohistochemistry. H. pylori infection was diagnosed by the presence of anti-H. pylori antibody, by using the urease test and by histological examination. Serum levels of pepsinogen I and II and of gastrin were measured by a modified radioimmunoassay. RESULTS: Seventy-one Japanese (70%) and 17 Germans (37%) were positive for H. pylori. Serum levels of anti-parietal cell antibody were not significantly different between Japanese and Germans in both H. pylori negative and positive groups. The serum pepsinogen I/II ratio and gastrin levels were altered by H. pylori infection in both populations. Moreover, anti-parietal cell antibody levels were higher in H. pylori-positive patients with low pepsinogen levels than in those with high pepsinogen levels in both populations. CONCLUSIONS: The levels of anti-parietal cell antibody do not differ statistically between Japanese and Germans. Anti-parietal cell antibody might play a role in the progression of atrophic gastritis in both Japanese and German patients.
Assuntos
Anticorpos Anti-Idiotípicos/análise , Gastrite Atrófica/imunologia , Células Parietais Gástricas/imunologia , Pepsinogênio A/sangue , Pepsinogênio C/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Gastrite Atrófica/epidemiologia , Alemanha/epidemiologia , Infecções por Helicobacter/imunologia , Helicobacter pylori , Humanos , Imuno-Histoquímica , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Radioimunoensaio , Estudos SoroepidemiológicosRESUMO
We investigated serum levels of anti-parietal cell antibody (APCA) in relation to various gastric diseases. Subjects were 224 Japanese patients including 58 with gastric cancer. All patients underwent gastroscopy, and APCA was investigated by enzyme-linked immunosorbent assay. Unexpectedly, there was no difference in APCA levels between patients with gastric cancer and those with gastritis. Among H. pylori-positive patients, APCA levels were closely correlated with grades of atrophy when no gastric cancer was present, but no correlation was found when gastric cancer was present. APCA-negative gastric cancer was found mainly in males and was characterized by massive infiltration of neutrophils in the background mucosa. The 24 patients with gastric cancer were APCA-negative and showed low pepsinogen levels. The odds ratio for the incidence of gastric cancer in these patients was 7.90 (95% CI 3.4-18.4). This suggests APCA-negative gastric cancer is the predominant form of gastric cancer in Japan.
Assuntos
Autoanticorpos/sangue , Gastroenteropatias/imunologia , Células Parietais Gástricas/imunologia , Neoplasias Gástricas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Mucosa Gástrica/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/patologia , Pepsinogênio A/sangue , Fatores SexuaisRESUMO
The maximum amount of acid-stable phosphoenzyme (E32P)/mol of alpha chain of pig gastric H/K-ATPase from [gamma-32P]ATP (K(1/2) = 0.5 microM) was found to be approximately 0.5, which was half of that formed from 32P(i) (K(1/2) = 0.22 mM). The maximum 32P binding for the enzyme during turnover in the presence of [gamma-32P]ATP or [alpha-32P]ATP was due to 0.5 mol of E32P + 0.5 mol of an acid-labile enzyme-bound [gamma-32P]ATP (EATP) or 0.5 mol of an acid-labile enzyme-bound [alpha-32P]ATP, respectively. The K(1/2) for EATP formation in both cases was 0.12 approximately 0.14 mM. The turnover number of the enzyme (i.e., the H+-ATPase activity/(EP + EATP)) was very close to the apparent rate constants for EP breakdown and P(i) liberation, both of which decreased with increasing concentrations of ATP. The ratio of the amount of P(i) liberated to that of EP that disappeared increased from 1 to approximately 2 with increasing concentrations of ATP (i.e., equal amounts of EP and EATP exist, both of which release phosphate in the presence of high concentrations of ATP). This represents the first direct evidence, for the case of a P-type ATPase, in which 2 mol of P(i) liberation occurs simultaneously from 1 mol of EP for half of the enzyme molecules and 1 mol of EATP for the other half during ATP hydrolysis. Each catalytic alpha chain is involved in cross-talk, thus maintaining half-site phosphorylation and half-site ATP binding which are induced by high- and low-affinity ATP binding, respectively, in the presence of Mg2+.