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)

Systems for overexpression and purification of active alpha, beta and gamma subunits of Escherichia coli H(+)-ATPase were established. The alpha and beta subunits recovered as soluble form were purified by hydroxyapatite column chromatography. Since the gamma subunit was overexpressed as the insoluble form, this subunit was purified by polyacrylamide gel-electrophoresis containing sodium dodecyl sulfate. By subsequent denaturation of this subunit with guanidine hydrochloride and renaturation, the active gamma subunit for reconstitution of the F1-ATPase activity with the purified alpha and beta subunit was obtained. The delta and epsilon subunits which were fused to the carboxy terminus of glutathione S-transferase (GST) were overproduced and purified by affinity chromatography. These fused proteins (delta-GST and epsilon-GST) were incubated with the purified alpha, beta and gamma subunits and applied to affinity chromatography. The alpha beta gamma delta-GST and alpha beta gamma epsilon-GST complex were eluted specifically by addition of glutathione and exhibited high and low ATPase activity, respectively, with a subunit stoichiometry similar to that in the native F1-ATPase, indicating that active complexes could be reconstituted with the fused proteins. These results suggested that the amino-terminal ends of the delta and epsilon subunits are not involved in formation of the active complex. The fused epsilon-GST bound the gamma subunit strongly, and the alpha subunit weakly. The delta-GST bound the gamma subunit significantly, and the alpha and beta subunits very weakly.
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PMID:Reconstitution of the F1-ATPase activity from purified alpha, beta, gamma and delta or epsilon subunits with glutathione S-transferase fused at their amino termini. 857 96

As demonstrated previously, liver acini draining the blood from intraportally transplanted pancreatic islets in streptozotocin-diabetic rats are altered in various respects. The hepatocytes in these acini store glycogen and/or fat, and they show an increase in proliferation as well as in apoptotic activity. Thus, they are phenotypically similar to carcinogen-induced preneoplastic liver foci (glycogen-storing foci and sometimes also mixed cell foci). By means of catalytic enzyme histochemistry or immunohistochemistry, we investigated the activity of key enzymes of alternative pathways of carbohydrate metabolism and some additional marker enzymes (well known from studies on preneoplastic hepatic foci) in the altered liver acini surrounding the islet isografts. In addition, the expression of glucose transporter proteins 1 and 2 (GLUT-1 and GLUT-2) were investigated immunohistochemically. The activities of hexokinase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and glucose-6-phosphate dehydrogenase were increased, whereas the activities of glycogen phosphorylase, adenylate cyclase, glucose-6-phosphatase, and membrane-bound adenosine triphosphatase were decreased in the altered liver acini. The expression of GLUT-2 was also decreased. GLUT-1 and glutathione S-transferase placental form were not expressed, and the activities of glycogen synthase and gamma-glutamyl-transferase remained unchanged. All changes of the enzyme activities were in line with the well known effects of insulin and resembled alterations characteristic of preneoplastic liver foci observed in different models of hepatocarcinogenesis. It remains to be clarified in long-term experiments whether or not these foci represent preneoplastic lesions and may proceed to neoplasia.
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PMID:Altered liver acini induced in diabetic rats by portal vein islet isografts resemble preneoplastic hepatic foci in their enzymic pattern. 864 65

Hepatic sinusoidal uptake of bile acids is mediated by defined carrier proteins against unfavourable concentration and electrical gradients. Putative carrier proteins have been identified using bile acid photoaffinity labels and more recently using immunological probes, such as monoclonal antibodies. At the sinusoidal domain, proteins with molecular weights of 49 and 54 kDa have been shown to be carriers for bile acid transport. The 49 kDa protein has been associated with the Na(+)-dependent uptake of conjugated bile acids, while the 54 kDa carrier has been involved in the Na(+)-independent bile acid uptake process. Within the hepatocyte, cytosolic proteins, such as the glutathione S-transferase (also designated the Y protein), the Y binders and the fatty acid binding proteins, are able to bind bile acids and possibly facilitate their movement to the canalicular domain. At the canalicular domain a 100 kDa carrier protein has been isolated and it has been shown by several laboratories that this particular protein is concerned with canalicular bile acid transport. The system is ATP-dependent and follows Michaelis-Menten kinetics. Interference with bile acid transport has been demonstrated by several chemicals. The mechanisms by which these chemicals inhibit bile acid transport may explain the apparent cholestatic properties observed in patients and experimental animals treated with these agents. Several studies have shown that Na+/K(+)-ATPase activity is markedly decreased in cholestasis induced by ethinyloestradiol, taurolithocholate and chlorpromazine. However, other types of interference have been described and the cholestatic effects may be the result of several mechanisms. Cholestasis is associated with several adaptive changes that may be responsible for the accumulation of bile acids and other cholephilic compounds in the blood of these patients. It may be speculated that the nature of these changes is to protect liver parenchymal cells from an accumulation of bile acids to toxic levels. However, more detailed quantitative experiments are necessary to answer questions with regard to the significance of these changes and the effect of various hepatobiliary disorders in modifying these mechanisms. It is expected that the mechanisms by which bile acid transport is regulated and efforts to understand the molecular basis for these processes will be among the areas of future research.
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PMID:Current concepts of hepatic uptake, intracellular transport and biliary secretion of bile acids: physiological basis and pathophysiological changes in cholestatic liver dysfunction. 871 9

Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.
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PMID:Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium. 879 66

One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells, is sec independent and dedicated to proteins lacking an N-terminal signal peptide. It depends on ABC protein-mediated exporters, which consist of three cell envelope proteins, two inner membrane proteins, an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide. Erwinia chrysanthemi metalloproteases B and C and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein. Using as protein substrates HasA and GST-PrtC, a chimeric protein which has a glutathione S-transferase moiety fused to a large C-terminal domain of protease C, we developed a simple system to identify proteins bound to the substrate based on substrate affinity-chromatography using heme- or glutathione-agarose. We show an ordered association between the protein substrates and the three exporter components: the substrate recognizes the ABC protein which interacts with the MFP which in turn binds the outer membrane component. Substrate binding is required for assembly of the three components.
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PMID:Protein secretion in gram-negative bacteria: assembly of the three components of ABC protein-mediated exporters is ordered and promoted by substrate binding. 891 58

We examined the effect of selective thromboxane A2 (TXA2) receptor antagonists, calcium 5(Z)-1R, 2S, 3S, 4S-7-[3-phenylsulphonylaminobicyclo [2.2.1] hept-2-yl]-5-heptonoate hydrate (S-1452) and +/- -7-(3,5,6,-trimethyl-1,4-benzoquinon-2-yl)-7-phenylhaptanoic acid (AA-2414), on sensitivity to cis-diamminedichloroplatinum (II) (CDDP) in non-small-cell lung cancer cell lines. IC50 values to CDDP using MTT assay were decreased 2.1- and 4.6-fold respectively by treatment with 250 or 500 microM S-1452, for a 2 h simultaneous drug exposure, and those of PC-9/CDDP, a CDDP-resistant cell line, were decreased 3.1- and 6.1-fold. Sensitivity to carboplatin was also enhanced by the treatment with S-1452. IC50 values to CDDP and carboplatin were decreased by treatment with AA-2414 in a dose-dependent manner. Isobologram analysis showed that the combination of CDDP with S-1452 or AA-2414 produced supra-additive or additive effects in each cell line. Neither glutathione content nor glutathione S-transferase activity was changed in either cell line by treatment with 500 microM S-1452. Accumulation of platinum into PC-9 and PC-9/CDDP was increased by the treatment in a dose-dependent manner. Na+, K+-ATPase activity of PC-9 and PC-9/CDDP was enhanced by the treatment of S-1452 in a dose-dependent manner. These data show that the TXA2 receptor antagonists may enhance the sensitivity of non-small-cell lung cancer cell lines to platinum agents. Increase in Na+, K+-ATPase activity induced by S-1452 may be the mechanism of its sensitising effect through increase in platinum accumulation.
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PMID:Modulation of sensitivity to cis-diamminedichloroplatinum (II) by thromboxane A2 receptor antagonists in non-small-cell lung cancer cell lines. 893 34

Tissue transglutaminase (tTG) exhibits a magnesium-dependent GTP/ATPase activity that is involved in the regulation of the cell cycle and cell receptor signaling. The portion of the molecule involved in GTP/ATP hydrolysis is unknown. We expressed and purified a series of C-terminal truncation mutants of human tTG as glutathione S-transferase fusion proteins (DeltaS538, DeltaE447, DeltaP345, DeltaC290, DeltaV228, and DeltaF185) to determine the effect on GTP/ATPase activity. The truncation of the C terminus did not change significantly the apparent Km value for either GTP or ATP. In contrast, the Kcat value for GTP was increased by 4.6- and 3-fold for the DeltaS538 and DeltaE447 mutants, respectively. The DeltaP345 mutant had the highest hydrolysis activity with a 34-fold increase. The hydrolysis activity then declined to 8.1-, 8.7-, and 1. 9-fold for the DeltaC290, DeltaV228, and DeltaF185 mutants, respectively. The Kcat for ATP changed in parallel with the GTPase results. Thin layer chromatography analysis of the hydrolysis reaction products revealed that ATP was rapidly converted to ADP followed by a much slower conversion of ADP to AMP when incubated with wild type tTG or the DeltaP345 mutant. There was a substantial decrease in the calcium-dependent TGase activity when the last 149 amino acid residues were deleted from the C terminus. Less than 5% of the TGase activity was detected for the DeltaS538 and DeltaE447 mutants. In conclusion, we have located the ATP and GTP hydrolytic domain to amino acid residues 1-185. The C terminus functions to inhibit the expression of endogenous GTP/ATPase activity of tTG, and the potential role of the C terminus in modulating this activity is discussed.
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PMID:C-terminal deletion of human tissue transglutaminase enhances magnesium-dependent GTP/ATPase activity. 894 Jan 19

Phospholamban (PLB) is a small hydrophobic protein that regulates contractility in the heart. This membrane protein expressed in bacterial cells is resistant to purification by conventional strategies that have been used to isolate expressed soluble proteins. Therefore, in order to obtain both wild-type and mutant PLB proteins, we have amplified the PLB gene by the polymerase chain reaction from genomic DNA of porcine heart and inserted it into the pGEX-2T plasmid expression vector. In this vector, the gene product fused to glutathione S-transferase has been expressed in JM109 Escherichia coli cells. The expressed fusion protein was found associated predominantly with insoluble cellular constituents. However, most of the fusion protein was readily extracted with SDS. PLB was subsequently purified by a simple procedure consisting of isolation of the fusion protein by preparative SDS-gel electrophoresis, followed by a second electrophoretic separation of PLB after its cleavage from the fusion protein by thrombin. This isolation method yields 3-4 mg of PLB per liter of cells, in a form which is capable of functional interaction with the Ca-ATPase in reconstituted proteoliposomes.
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PMID:Purification of porcine phospholamban expressed in Escherichia coli. 895 94

Monoclonal antibody alpha110 recognizes Leu-456 in the alpha subunit of the Escherichia coli F1-ATPase. Binding of this antibody to the alpha subunit or mutation of this residue to Pro caused enhancement of the ATPase activity, suggesting that this residue is involved in the catalytic mechanism of this molecule (H. Kanazawa et al. (1995) Arch. Biochem. Biophys. 317, 348-356). Leu-456 together with Gly-454 and Tyr-455 are the only residues in the carboxy-terminal 75 amino acids conserved among various species, suggesting that these three residues play important roles in catalysis by the ATPase. Here, we introduced site-directed mutations into these residues. Not only L456P but also G454L, Y455K, Y455L, and L456N mutations caused enhancement of the ATPase activity. Surprisingly, Y455V, L456H, and L456S caused assembly defects of F1 subunits on the membrane. Reconstitution of the alpha betagamma complex from the wild-type beta and gamma subunits with the mutant alpha subunit (L4gamma6P) exhibited enhanced ATPase activity. Addition of delta or epsilon fused to glutathione S-transferase which are functionally similar to the delta and epsilon subunits, respectively, to the reconstituted F1-ATPase did not cause significant enhancement of its activity. Decreased interaction between alpha and beta subunits with the L456P mutation was detected by the yeast two-hybrid system. According to the deduced three-dimensional structure of the bovine a subunit, Leu-456, Gly-454, and Tyr-455 are included in a small alpha helix. These results suggest that this alpha helix affects interaction of the alpha subunit with the beta subunit but not with delta or epsilon, which may be important for the catalytic mechanism and F1 assembly.
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PMID:Catalytic and structural importance of Gly-454, Tyr-455, and Leu-456 in the carboxy-terminal region of Escherichia coli F1-ATPase alpha subunit. 901 94

Elongation factor 3 (EF-3) is an essential requirement of the fungi for translational elongation. EF-3 is an ATPase, and the hydrolytic activity is stimulated 2 orders of magnitude by yeast ribosomes. Limited trypsinolysis of EF-3 results in the cleavage of a single peptide bond between residues 774 (Arg) and 775 (Gln), generating polypeptides of approximate molecular mass 90 and 30 kDa. The 90-kDa fragment is relatively resistant to proteolysis and retains ribosome-independent ATPase activity. The 30-kDa fragment is further proteolyzed into smaller fragments and retains the specificity for binding to yeast ribosomes. Both the intact EF-3 and the 30-kDa fragment are protected from proteolysis by yeast ribosomes. EF-3 is NH2 terminally blocked, and so is the 90-kDa fragment. The COOH terminally derived 30-kDa fragment contains glutamine (residue 775) at the NH2-terminal end. A construct was designed representing the COOH-terminal domain of EF-3 (30-kDa fragment), subcloned, and expressed as a glutathione S-transferase fusion in yeast. The glutathione S-transferase-30-kDa peptide remains stringently associated with ribosomes. Isolated fusion peptide rebinds to yeast ribosomes with high affinity. Based on these results, we propose that at least one of the ribosome-binding sites of EF-3 resides at the COOH-terminal end of the protein.
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PMID:Functional subdomains of yeast elongation factor 3. Localization of ribosome-binding domain. 904 59


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