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 complete sequence of the kdp gene region of Clostridium acetobutylicum has been determined. This part of the chromosome comprises two small open reading frames (orfZ and orfY), putatively encoding hydrophobic peptides, and the genes kdpA, kdpB, kdpC, and kdpX, followed by an operon encoding a pair of sensor-effector regulatory proteins (KdpD and KdpE). Except for orfZ, orfY, and kdpX, all genes showed significant homology to the kdp genes of Escherichia coli, encoding a high-affinity potassium transport ATPase and its regulators. The complete genome sequence of Synechocystis sp. strain PCC 6803 and a recently published part of the Mycobacterium tuberculosis genome indicate the existence of a kdp system in these organisms as well, but all three systems comprise neither a second orf upstream of kdpA nor an additional kdpX gene. Expression of the clostridial kdp genes, including the unique kdpX gene, was found to be inducible by low potassium concentrations. A transcription start point could be mapped upstream of orfZ. A promoter upstream of kdpD was active only under noninducing conditions. Lowering the potassium content of the medium led to formation of a common transcript (orfZYkdpABCXDE), with a putative internal RNase E recognition site, which could be responsible for the instability of the common transcript. Except for the two small peptides, all gene products could be detected in in vitro transcription-translation experiments.
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PMID:The kdp system of Clostridium acetobutylicum: cloning, sequencing, and transcriptional regulation in response to potassium concentration. 922 59

Proteins associated with the cell wall peptidoglycan (CW-Pr) of Mycobacterium tuberculosis H37Ra were isolated to evaluate their immunoreactivity and immunoprophylactic properties against experimental tuberculosis. Chemical treatment of the cell wall with trifluoromethanesulfonic acid: anisole (2:1) resulted in the release of three proteins of 71, 60 and 45 kDa as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparative study of immune responses elicited to individual proteins in mice immunized with CW-Pr emulsified in incomplete Freund's adjuvant showed the 71-kDa protein to be the most immunoreactive antigen. This 71-kDa protein was found to cross-react with the 70-kDa heat shock protein from M. leprae and possessed ATPase activity. Mice immunized with the 71-kDa protein exhibited significantly higher immune responses, on the basis of T and B cell reactivity, as compared to a M. bovis Bacillus Calmette Guerin (BCG)-vaccinated group. The culture supernatants collected from 71-kDa stimulated lymphocytes stimulated exhibited increased interferon-gamma and interleukin-2 production. The protective efficacy of the 71-kDa protein in comparison to BCG was determined by challenging the mice with a virulent strain M. tuberculosis H37Rv. The 71-kDa protein was found to be more protective in animals challenged at 8 and 16 weeks post immunization, shown by increased survival rates and decreased viable bacilli counts in the target organs as compared to BCG-vaccinated animals.
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PMID:Immunoprophylactic properties of 71-kDa cell wall-associated protein antigen of Mycobacterium tuberculosis H37Ra. 925 66

The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.
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PMID:Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors. 935 18

Vacuolar-type (V) ATPases are thought to be the main determinant of phagosomal acidification. In phagosomes containing mycobacteria, which ostensibly impair the delivery of V-ATPases to the phagosomal membrane, the pH would be expected to be near neutral. This prediction was tested by microfluorescence ratio imaging using macrophages from mice susceptible to mycobacterial infection. Although less acidic than their counterparts containing dead bacteria, phagosomes containing live Mycobacteria bovis were nearly 1 pH unit more acidic than the cytosol, suggesting the existence of alternate H+ transport mechanisms. We therefore investigated whether Na+/H+ exchange (NHE) contributes to phagosomal acidification. Immunoblotting, reverse transcriptase-polymerase chain reaction, and pharmacological studies indicated that NHE1 is the predominant isoform of the exchanger in macrophages. Fractionation revealed that NHE1 is incorporated into the phagosomal membrane, and measurements of pH indicated that it is functional in this location. Nevertheless, acidification of the lumen of phagosomes containing either latex beads or live M. bovis was insensitive to (3-methylsulfonyl-4-piperidinobenzoyl)-guanidine methanesulfonate, a potent inhibitor of NHE1. This may have been due to the absence of an appropriate lumen to cytosol Na+ gradient, because the phagosomal membrane was found to be devoid of Na+/K+ pumps. Unexpectedly, the acidification of M. bovis phagosomes was fully reversed by specific inhibitors of the vacuolar H+-ATPase, suggesting that ATPases are present only transiently or in reduced quantities in the phagosomal membrane. Alternatively, acid equivalents accumulated in endosomes by V-ATPases may be delivered to the mycobacterial phagosome by carrier vesicles devoid of ATPases.
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PMID:Regulation of phagosomal acidification. Differential targeting of Na+/H+ exchangers, Na+/K+-ATPases, and vacuolar-type H+-atpases. 936 53

We have charterized a Mycobacterium smegmatis gene encoding a homolog of the ATP-dependent protease Lon (La). Our identification of a Lon homolog, in conjunction with our previous work, identifies M. smegmatis as the first known example of a eubacterium containing both Lon and a complete 20S proteasome (containing both alpha- and beta-subunits). Despite the significant primary sequence divergence between M. smegmatis Lon (Ms-Lon) and E. coli Lon (Ec-Lon), expression of Ms-Lon was only moderately toxic to E. coli cells. The ability of E. coli cells to tolerate expression of Ms-Lon reveals that Ms-Lon does not recognize and degrade essential E. coli proteins. We conclude that discrimination against nonsubstrate proteins is broadly conserved between Ec-Lon and Ms-Lon. Additional conservation of substrate recognition was demonstrated by the ability of Ms-Lon to degrade efficiently RcsA, a natural substrate of Ec-Lon. Purified Ms-Lon displays chymotrypsin-like specificity in peptidase assays that are stimulated by unfolded protein and supported by nonhydrolyzed nucleotide analogs. Maximal peptidase activity requires ATP or dATP. Replacement of Ms-Lon's catalytic Ser with Ala (S675A), Thr (S675T), or Cys (S675C) reduced to background levels Ms-Lon's in vitro peptidase activity. However, by employing a sensitive in vivo assay, based on the degradation of RcsA, we demonstrated that the S675C variant retained specific protease activity. Finally, variants of Ms-Lon, with substututions at or near S675, reduce the enzyme's basal ATPase activity, suggesting a structural interaction between the peptidase and ATPase active sites of Ms-Lon.
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PMID:The lon protease from Mycobacterium smegmatis: molecular cloning, sequence analysis, functional expression, and enzymatic characterization. 942 59

Bacteria possess multiple mechanisms for the transport of metal ions. While many of these systems may have evolved in the first instance to resist the detrimental effects of toxic environmental heavy metals, they have since become adapted to a variety of important homeostatic functions. The 'P'-type ATPases play a key role in metal ion transport in bacteria. A Cu+-ATPase from the intracellular bacterium Listeria monocytogenes is implicated in pathogenesis, and similar pumps in Mycobacterium tuberculosis and M. leprae may play a comparable role. Intracellular bacteria require transition metal cations for the synthesis of superoxide dismutases and catalases, which constitute an important line of defence against macrophage-killing mechanisms. The macrophage protein Nramp1, which confers resistance to a variety of intracellular pathogens, has also been shown recently to be a divalent amphoteric cation transporter. Mycobacterial homologues have recently been identified by genomic analysis. These findings suggest a model in which competition for divalent cations plays a pivotal role in the interaction between host and parasite.
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PMID:Metal ion homeostasis and intracellular parasitism. 963 46

The mechanisms underlying the survival of intracellular parasites such as mycobacteria in host macrophages remain poorly understood. In mice, mutations at the Nramp1 gene (for natural resistance-associated macrophage protein), cause susceptibility to mycobacterial infections. Nramp1 encodes an integral membrane protein that is recruited to the phagosome membrane in infected macrophages. In this study, we used microfluorescence ratio imaging of macrophages from wild-type and Nramp1 mutant mice to analyze the effect of loss of Nramp1 function on the properties of phagosomes containing inert particles or live mycobacteria. The pH of phagosomes containing live Mycobacterium bovis was significantly more acidic in Nramp1- expressing macrophages than in mutant cells (pH 5.5 +/- 0.06 versus pH 6.6 +/- 0.05, respectively; P <0.005). The enhanced acidification could not be accounted for by differences in proton consumption during dismutation of superoxide, phagosomal buffering power, counterion conductance, or in the rate of proton "leak", as these were found to be comparable in wild-type and Nramp1-deficient macrophages. Rather, after ingestion of live mycobacteria, Nramp1-expressing cells exhibited increased concanamycin-sensitive H+ pumping across the phagosomal membrane. This was associated with an enhanced ability of phagosomes to fuse with vacuolar-type ATPase-containing late endosomes and/or lysosomes. This effect was restricted to live M. bovis and was not seen in phagosomes containing dead M. bovis or latex beads. These data support the notion that Nramp1 affects intracellular mycobacterial replication by modulating phagosomal pH, suggesting that Nramp1 plays a central role in this process.
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PMID:Host resistance to intracellular infection: mutation of natural resistance-associated macrophage protein 1 (Nramp1) impairs phagosomal acidification. 967 47

Lon protease homologues contain a poorly conserved N-terminal region of variable length. To better understand the role of the N-terminal region of Lon in the complicated reaction cycle of ATP-dependent protein degradation, we expressed and characterized mutants of the Lon protease from Mycobacterium smegmatis (Ms-Lon) lacking 90, 225, and 277 N-terminal residues (N-G91, N-E226, and N-I278, respectively). N-I278 displayed neither peptidase nor ATPase activity despite the fact that it was stable and soluble in vivo, had a near-wild-type CD spectrum, and the deleted residues included neither the catalytic nucleophile for peptide bond hydrolysis (S675) nor the ATP binding regions. N-G91 and N-E226 retained peptidase activities against small unstructured peptides that were stimulated, to near-wild-type levels, by the Ms-Lon substrate protein alpha-casein. By contrast, N-G91 and N-E226 retained basal ATPase activities, but these activities were only stimulated weakly by alpha-casein. Ms-Lon, N-E226, and N-G91 all exhibited low-level peptidase activity in assays containing nonhydrolyzed nucleotide analogues. However, these peptidase activities were stimulated strongly by alpha-casein in the case of Ms-Lon but weakly by alpha-casein in the cases of N-G91 and N-E226. Strikingly, despite the near-wild-type peptidase activities of N-G91 and N-E226, both were severely impaired in their degradation of the Ms-Lon protein substrates alpha-casein in vitro and RcsA in vivo. Overall, N-G91 and N-E226 displayed catalytic properties similar to Escherichia coli Lon (Ec-Lon) in the presence of the PinA inhibitor, suggesting that PinA inhibits Ec-Lon protease by inhibiting the function of Ec-Lon's N-terminal region. In vivo protease assays further revealed that, in contrast to the inactive Ms-Lon point mutant S675A, N-G91 and N-E226 did not reduce the cellular activity of RcsA. This same defect was observed previously for Ms-Lons with multiple mutations in their peptidase active sites. We conclude that proteolytically inactive mutants of Ms-Lon retain the ability to reduce the cellular activity of RcsA but that both the N-terminal region and the peptidase active site region of Ms-Lon are required for this activity of wild-type Ms-Lon. The inabilities of N-G91 and N-E226 to degrade larger protein substrates and to reduce the cellular activity of RcsA were not the result of drastic alterations in their quaternary structures. Gel filtration profiles of N-G91 and N-E226 revealed that each was primarily tetrameric, with an increased percentage of dimeric species and a decreased percentage of trimeric species relative to Ms-Lon. The observed shifts in the dimer/trimer ratios of the N-terminal truncation mutants suggest that the Ms-Lon tetramer contains two types of subunit-subunit interactions.
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PMID:Functional role of the N-terminal region of the Lon protease from Mycobacterium smegmatis. 969 72

We have examined the functional properties including autophosphorylation of the Mycobacterium leprae Hsp70 homologue. Recombinant M. leprae Hsp70 had pH optima for its adenosine triphosphatase and autophosphorylating activities which were near pH 8 and 6, respectively. Both these activities were inhibited by reduced and alkylated bovine pancreatic trypsin inhibitor, but not other tested substrates. Autophosphorylation was augmented by up to 25 mM Ca2+. Using site-directed mutagenesis to construct two Thr-->Ala mutants at positions 175 and 193, and phosphoamino acid analysis, it was shown that Thr175 was the dominant threonine residue autophosphorylated in M. leprae Hsp70. Phosphorylation led to an increased affinity for a model polypeptide substrate, reduced and alkylated bovine albumin. These properties are compared with those of the DnaK protein of Escherichia coli.
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PMID:Phosphorylation of Mycobacterium leprae heat-shock 70 protein at threonine 175 alters its substrate binding characteristics. 974 55

20S proteasomes were purified from Streptomyces coelicolor A3(2) and shown to be built from one alpha-type subunit (PrcA) and one beta-type subunit (PrcB). The enzyme displayed chymotrypsin-like activity on synthetic substrates and was sensitive to peptide aldehyde and peptide vinyl sulfone inhibitors and to the Streptomyces metabolite lactacystin. Characterization of the structural genes revealed an operon-like gene organization (prcBA) similar to Rhodococcus and Mycobacterium spp. and showed that the beta subunit is encoded with a 53-amino-acid propeptide which is removed during proteasome assembly. The upstream DNA region contains the conserved orf7 and an AAA ATPase gene (arc).
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PMID:The 20S proteasome of Streptomyces coelicolor. 976 79


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