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)

Antigenic peptides bound to class I molecules of the major histocompatibility complex (MHC) are recognized by T-cell receptors during development of an antiviral immune response. T cells respond to peptides derived from cytoplasmic viral proteins as well as viral membrane proteins, indicating that a pathway exists for the transport of proteins or peptides from the cytosol into the compartment(s) where the MHC class I molecules assemble. To investigate this pathway, we have developed an in vitro assay for the transport of peptides into microsomal vesicles. This assay provides evidence for the transport of chemically synthesized peptides (13-21 amino acids) containing N-linked glycosylation acceptor sequences, which serve as glycosylation substrates. Their transport results in depletion of the pool of available dolichol high-mannose oligosaccharides in the lumen of the microsomal vesicles. We have observed transport of peptides derived from antigenic human immunodeficiency virus gag and influenza B nucleoprotein sequences, but transport of a third randomly selected peptide was not detected, suggesting specificity of the transport process. We were not able to demonstrate ATP dependence of this peptide transport process by using apyrase and an ATPase inhibitor. This result was unexpected in light of the recent identification of MHC-linked genes with homology to ATP-binding cassette transporters, which have been proposed to mediate peptide transport.
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PMID:Evidence for peptide transport across microsomal membranes. 157 Mar 12

Site-directed mutagenesis was used to change four amino acid residues (Q82, P152, L179, H192) in the MalK subunit of S. typhimurium maltose transport system which are highly conserved among members of the ATP-binding cassette (ABC) family. Replacement of H192 caused complete failure to complement the transport defect of a malK strain whereas changes of the other residues resulted in reduced or wild-type activity. The purified mutant proteins exhibited ATPase activity comparable to wild-type MalK.
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PMID:Characterization of site-directed mutations in conserved domains of MalK, a bacterial member of the ATP-binding cassette (ABC) family [corrected]. 159 14

The overexpression of the P-glycoprotein, the MDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [3H]vinblastine accumulation. Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate. In addition, the vinblastine transport was inhibited by verapamil and daunorubicin. These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter. The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.
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PMID:Purification and reconstitution of functional human P-glycoprotein. 762 47

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding cassette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete loss of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.
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PMID:Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB. 765 Nov 40

Sulfonylureas are a class of drugs widely used to promote insulin secretion in the treatment of non-insulin-dependent diabetes mellitus. These drugs interact with the sulfonylurea receptor of pancreatic beta cells and inhibit the conductance of adenosine triphosphate (ATP)-dependent potassium (KATP) channels. Cloning of complementary DNAs for the high-affinity sulfonylurea receptor indicates that it is a member of the ATP-binding cassette or traffic ATPase superfamily with multiple membrane-spanning domains and two nucleotide binding folds. The results suggest that the sulfonylurea receptor may sense changes in ATP and ADP concentration, affect KATP channel activity, and thereby modulate insulin release.
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PMID:Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. 771 39

The multidrug resistance gene product, P-glycoprotein or the multidrug transporter, confers multidrug resistance to cancer cells by maintaining intracellular levels of cytotoxic agents below a killing threshold. P-glycoprotein is located within the plasma membrane and is thought to act as an energy-dependent drug efflux pump. The multidrug transporter represents a member of the ATP-binding cassette superfamily of transporters (or traffic ATPases) and is composed of two highly homologous halves, each of which harbors a hydrophobic transmembrane domain and a hydrophilic ATP-binding fold. This review focuses on various biochemical and molecular genetic approaches used to analyze the structure, function, and mechanism of action of the multidrug transporter, whose most intriguing feature is its ability to interact with a large number of structurally and functionally different amphiphilic compounds. These studies have underscored the complexity of this membrane protein which has recently been suggested to assume alternative topological and quaternary structures, and to serve multiple functions both as a transporter and as a channel. With respect to the multidrug transporter activity of P-glycoprotein, progress has been made towards the elucidation of essential amino acid residues and/or polypeptide regions. Furthermore, the drug-stimulatable ATPase activity of P-glycoprotein has been established. The mechanism of drug transport by P-glycoprotein, however, is still unknown and its physiological role remains a matter of speculation.
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PMID:Molecular analysis of the multidrug transporter. 776 31

We have overproduced, partially purified, and characterized PrtD, the ATP-binding cassette (ABC) integral membrane component from the metalloproteases secretion system of the Gram-negative phytopathogenic bacterium Erwinia chrysanthemi. These metalloproteases are secreted independently of the general export pathway encoded by the sec genes. They are secreted via a C-terminal secretion signal and by a secretion apparatus composed of two inner membrane proteins, PrtD and PrtE, and one outer membrane protein PrtF. PrtD is specifically labeled by 8-azido-ATP both in whole membrane vesicles and upon purification. The purified protein displays a low level of P-type ATPase activity. This activity is almost completely and specifically inhibited by the cognate C-terminal secretion signal of the PrtG and PrtB metalloproteases (half inhibition at 0.1 microM) but not by a C-terminal secretion signal of a protein not secreted by the Prt translocator. A mutant PrtD protein bearing a point mutation in the ATP binding site (conserved lysine 370 of the Walker A box changed to arginine) has also been purified. It displays a lower level of ATPase activity which correlates with the lower level of secretion of the metalloproteases by a strain expressing this mutated protein.
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PMID:PrtD, the integral membrane ATP-binding cassette component of the Erwinia chrysanthemi metalloprotease secretion system, exhibits a secretion signal-regulated ATPase activity. 796 27

The periplasmic maltose-binding protein-dependent, maltose transport system of Escherichia coli is a well studied member of the ATP-binding cassette family of transport ATPases. In addition to the water-soluble maltose-binding protein, the system comprises three membrane proteins, MalF, MalG, and MalK, which form a heterotetrameric complex (FGK2) in the cytoplasmic membrane. The purified complex exhibits transport-associated ATPase activity. To characterize the requirements for nucleotide binding and hydrolysis by the FGK2 complex, we used plasmids to express different combinations of the individual subunits as well as mutant forms of the MalK subunit. Prior to measuring nucleotide binding, we examined membrane preparations for the presence of each subunit from strains that contained all possible permutations of the three structural genes, malF, malG, and malK. We found that when all three genes were present or when malF and malK were present together, the corresponding antigens were detected easily on Western immunoblots and were soluble in the non-ionic detergent, Triton X-100. In contrast, all other permutations resulted in decreased amounts of antigen or antigen that was Triton X-100-insoluble. We relied on photocross-linking with 8-azido-[32P]ATP and ATP hydrolysis as indicators of the ability of the transport complex to interact with purine nucleotides. 8-Azido-[32P]ATP was photocross-linked to the MalK subunit. Photolabeling of MalK was inhibited by ATP, ADP, and GTP and not by other nucleotides. Photolabeling of MalK required the presence of MalF but not MalG. Mutations in malK that affect amino acid residues thought to be directly involved in nucleotide binding did indeed abolish labeling and resulted in loss of transport activity without affecting protein stability. In general, ATP hydrolysis correlated with the photocross-linking. A notable exception is the MalK941 mutant protein which retained the ability to be labeled by 8-azido-[32P]ATP but was unable to catalyze detectable levels of ATP hydrolysis. Some, but not all, of the malK mutations were dominant to wild type. To study the mechanism of dominance we devised a means of measuring the ability of different wild-type and mutant MalK proteins to interact with the MalF and MalG subunits. This assay relies on the fact that, when a bifunctional MalK-LacZ hybrid protein is associated with the MalF and MalG subunits, it is membrane-bound. Excess MalK competed with the MalK-LacZ hybrid protein for sites in the membrane and resulted in the hybrid fractionating as a soluble protein.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of the structural requirements for assembly and nucleotide binding of an ATP-binding cassette transporter. The maltose transport system of Escherichia coli. 822 95

The haemolysin exporter HlyB and its homologues are central to the unconventional signal-peptide-independent secretion of toxins, proteases and nodulation proteins by bacteria. HlyB is a member of the ATP-binding cassette (ABC) or traffic ATPase superfamily, and resembles closely in structure and function mammalian exporters such as the multidrug-resistance P-glycoprotein, combining both integral membrane and cytosolic domains. Overproduction of the HlyB cytoplasmic domain as a C-terminal peptide fused to glutathione S-transferase allowed the direct affinity purification and concentration of 30-50 mg ml-1 of soluble protein (GST-Bctp) in an apparently dimeric form possessing both transferase and ATPase activity. GST-Bctp bound to ADP-agarose and was eluted specifically by ATP and ADP, affinity behaviour which was confirmed in both the full-length HlyB and the unfused HlyB cytoplasmic domain synthesized in vitro. The stoichiometry of binding to MgATP and MgADP was close to equimolar and both ligands induced substantial conformational change in the protein. Mg(2+)-dependent ATPase activity of GST-Bctp (Vmax 1 mumol min-1 mg-1, Km 0.2 mM) was comparable with the activity of the bacterial importer MalK and human P-glycoprotein reconstituted into proteoliposomes, and over an order of magnitude higher than in vitro measurements of disaggregated MalK purified from inclusion bodies. Activity was unaffected by inhibitors of F- and V-type ATPases, non-hydrolysable ATP analogues, or translocation substrate, but was severely inhibited by inhibitors of E1E2 (P-type) ATPases, and the acidic phospholipid phosphatidyl glycerol.
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PMID:ATPase activity and ATP/ADP-induced conformational change in the soluble domain of the bacterial protein translocator HlyB. 836 61

Maltose transport across the cytoplasmic membrane of Escherichia coli is catalyzed by a periplasmic binding protein-dependent transport system and energized by ATP. The maltose system, a member of the ATP-binding cassette or ABC transport family, contains two copies of an ATP-binding protein in a complex with two integral membrane proteins. ATP hydrolysis by the transport complex can be assayed following reconstitution into proteoliposomes in the presence of maltose binding protein and maltose. Mutations in the transport complex that permit binding protein-independent transport render ATP hydrolysis constitutive so that hydrolysis can also be assayed with the transport complex in detergent solution. We have used both of these systems to study the role of two ATP binding sites in ATP hydrolysis. We found that both the wild-type and the binding protein-independent systems hydrolyzed ATP with positive cooperativity, suggesting that the two ATP binding sites interact. Vanadate inhibited the ATPase activity of the transport complex with 50% inhibition occurring at 10 mum vanadate. In detergent solution, the degree of cooperativity in the binding protein-independent complex decreased with increasing pH. The loss of cooperativity was accompanied by a decrease in ATPase activity and a decrease in sensitivity to vanadate. Because reconstitution of the complex into a lipid bilayer prevented the loss of cooperativity, we expect that ATP hydrolysis is cooperative in vivo. The mutations leading to binding protein-independent transport do not significantly alter the affinity, cooperativity, vanadate sensitivity, or substrate specificity of the ATP binding sites during hydrolysis. These results justify the use of the binding protein-independent system to investigate the mechanism of transport and hydrolysis.
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PMID:The maltose transport system of Escherichia coli displays positive cooperativity in ATP hydrolysis. 861 56


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