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)

A high-yield yeast expression system for site-directed mutagenesis of the Neurospora crassa plasma membrane H(+)-ATPase has recently been reported (Mahanty, S. K., Rao, U. S., Nicholas, R. A., and Scarborough, G. A. (1994) J. Biol. Chem. 269, 17705-17712). Using this system, each of the eight cysteine residues in the ATPase was changed to a serine or an alanine residue, producing strains C148S and C148A, C376S and C376A, C409S and C409A, C472S and C472A, C532S and C532A, C545S and C545A, C840S and C840A, and C869S and C869A, respectively. With the exception of C376S and C532S, all of the mutant ATPases are able to support the growth of yeast cells to different extents, indicating that they are functional. The C376S and C532S enzymes appear to be non-functional. After solubilization of the functional mutant ATPase molecules from isolated membranes with lysolecithin, all behaved similar to the native enzyme when subjected to glycerol density gradient centrifugation, indicating that they fold in a natural manner. The kinetic properties of these mutant enzymes were also similar to the native ATPase with the exception of C409A, which has a substantially higher Km. These results clearly indicate that none of the eight cysteine residues in the H(+)-ATPase molecule are essential for ATPase activity, but that Cys376, Cys409, and Cys532 may be in or near important sites. They also demonstrate that the previously described disulfide bridge between Cys148 and Cys840 or Cys869 plays no obvious role in the structure or function of this membrane transport enzyme.
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PMID:Site-directed mutagenesis of the cysteine residues in the Neurospora crassa plasma membrane H(+)-ATPase. 855 May 88

Structural features of the putative helical hairpin region comprising transmembrane segments 1 (TM1) and 2 (TM2) of the yeast plasma membrane H(+)-ATPase were probed by site-directed mutagenesis. The importance of phenylalanine residues Phe-116, Phe-119, Phe-120, Phe-126, Phe-144, Phe-159, and Phe-163 was explored by alanine replacement mutagenesis. It was found that substitutions at all positions, except Phe-120 and Phe-144, produced viable enzymes, although a range of cellular growth phenotypes were observed like hygromycin B resistance and low pH sensitivity, which are linked to in vivo action of the H(+)-ATPase. Lethal positions Phe-120 and Phe-144, could be replaced with tryptophan to produce viable enzyme, although the F144W mutant was highly perturbed. ATP hydrolysis measurements showed that Km was not significantly altered for most mutant enzymes, whereas Vmax was moderately reduced with two mutants, F144W and F163A, showing less than 50% of the normal activity. Double Phe-->Ala mutations in TM1 and TM2 were constructed to examine whether such substitutions would result in a higher degree of enzyme destabilization. Mutant F116A/F119A was viable and gave a normal phenotype, while F159A/F163A was not viable. Other double mutants, F116A/F159A and F119AF/159A, which are predicted to lie juxtaposed on TM1 and TM2, produced non-functional enzymes. However, a viable F119V/F159A mutant was isolated and showed hygromycin B resistance. These results suggest that double mutations eliminating 2 phenylalanine residues strongly destabilize the enzyme. A putative proline kink at Gly-122/Pro-123 in TM1 is not essential for enzyme action since these residues could be variously substituted (G122A or G122N; P123A, P123G, or P123F) producing viable enzymes with moderate effects on in vitro ATP hydrolysis or proton transport. However, several substitutions produced prominent growth phenotypes, suggesting that local perturbations were occurring. The location of Pro-123 is important because Gly-122 and Pro-123 could not be exchanged. In addition, a double Pro-Pro created by a G122P mutation was lethal, suggesting that maintenance of an alpha-helical structure is important. Other mutations in the hairpin, including modification of a buried charged residue, E129A, were not critical for enzyme action. These data are consistent with the view that the helical hairpin comprising TM1 and TM2 has important structural determinants that contribute to its overall stability and flexibility.
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PMID:Genetic probing of the first and second transmembrane helices of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. 855 Jun 23

Calcium release from intracellular stores occurs in a graded manner in response to increasing concentrations of either inositol 1,4,5-trisphosphate or caffeine. To investigate the mechanism responsible for this quantal release phenomenon, [Ca2+] changes inside intracellular stores in isolated single smooth muscle cells were monitored with mag-fura 2. Following permeabilization with saponin or alpha-toxin the dye, loaded via its acetoxymethyl ester, was predominantly trapped in the sarcoplasmic reticulum (SR). Low caffeine concentrations in the absence of ATP induced only partial Ca2+ release; however, after inhibiting the calcium pump with thapsigargin the same stimulus released twice as much Ca2+. When the SR Ca(2+)-ATPase was rendered non-functional by depleting its "ATP pool," submaximal caffeine doses almost fully emptied the stores of Ca2+. We conclude that quantal release of Ca2+ in response to caffeine in these smooth muscle cells is largely due to the activity of the SR Ca(2+)-ATPase, which appears to return a portion of the released Ca2+ back to the SR, even in the absence of ATP. Apparently the SR Ca(2+)-ATPase is fueled by ATP, which is either compartmentalized or bound to the SR.
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PMID:The quantal nature of calcium release to caffeine in single smooth muscle cells results from activation of the sarcoplasmic reticulum Ca(2+)-ATPase. 856 21

The role of N-linked glycosylation in the functional properties of gastric H+,K+-ATPase has been examined with tunicamycin and I-deoxymannojirimycin, inhibitors in glycoprotein biosynthesis and glycoprotein processing respectively. Tunicamycin completely abolished both K+-stimulated and 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATPase activity and SCH 28080-sensitive phosphorylation capacity. The expression level of both H+,K+-ATPase subunits remained unaffected. 1-Deoxymannojirimycin clearly affected the structure of the N-linked oligosaccharide moieties without affecting specific phosphorylation capacity. Purification of the functional recombinant enzyme from non-functional H+,K+-ATPase subunits coincided with purification of glycosylated beta-subunits and not of non-glycosylated beta-subunits. Transport of the H+,K+-ATPase beta-subunit to the plasma membrane but not its ability to assemble with the alpha-subunit dependent on N-glycosylation events. We conclude that the acquisition, but not the exact structure, of N-linked oligosaccharide moieties, is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells.
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PMID:Glycosylation is essential for biosynthesis of functional gastric H+,K+-ATPase in insect cells. 902 Aug 75

The parA system of plasmid R1 consists of two genes, parM and parR, and a cis-acting centromere-like site parC. The ParM protein exhibits similarity with a superfamily of ATPases that includes actin, hsp70 and hexokinase. ParM was purified to near-homogeneity and assayed for in vitro ATPase activity. The wild-type ParM protein was found to posses ATPase activity. Mutant ParM derivatives that exhibited decreased in vitro ATPase activity were non-functional in vivo, indicating that the ATP turnover by ParM is essential for correct plasmid partitioning. The mutant ParM proteins exhibited trans-dominance, suggesting that ParM participates as a structural component of the partitioning apparatus. The ATPase activity of ParM was activated slightly by the presence of ParR and activated to a much greater extent when ParR was bound to the centromere-like parC region. An analysis using the yeast two-hybrid system indicated that ParM and ParR interact, and demonstrated that ParR interacts with itself. Thus our results suggest a direct interaction of ParM and ParR at the natural partition site parC, and that the ATPase activity of ParM is specifically stimulated by this interaction.
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PMID:Partitioning of plasmid R1. The ParM protein exhibits ATPase activity and interacts with the centromere-like ParR-parC complex. 921 56

In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein-binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate Delta8proOmpA, a proOmpA derivative with a non-functional signal sequence. Owing to the signal sequence mutation, Delta8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP-dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild-type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex.
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PMID:PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA-SecY interaction during the initiation of translocation. 964 33

In Escherichia coli, precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo, are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (delta8proOmpA) bearing a non-functional signal sequence. Delta8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prIA4 strain. SecB reduces the translocation of delta8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB-SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.
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PMID:Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA. 976 86

Here we present the first description of the genomic organization, transcriptional regulatory sequences, and adult and embryonic gene expression for the mouse p97(CDC48) AAA ATPase. Clones representing two distinct p97 genes were isolated in a genomic library screen, one of them likely representing a non-functional processed pseudogene. The coding region of the gene encoding the functional mRNA is interrupted by 16 introns and encompasses 20.4 kilobase pairs. Definition of the transcriptional initiation site and sequence analysis showed that the gene contains a TATA-less, GC-rich promoter region with an initiator element spanning the transcription start site. Cis-acting elements necessary for basal transcription activity reside within 410 base pairs of the flanking region as determined by transient transfection assays. In immunohistological analyses, p97 was widely expressed in embryos and adults, but protein levels were tightly controlled in a cell type- and cell differentiation-dependent manner. A remarkable heterogeneity in p97 immunostaining was found on a cellular level within a given tissue, and protein amounts in the cytoplasm and nucleus varied widely, suggesting a highly regulated and intermittent function for p97. This study provides the basis for a detailed analysis of the complex regulation of p97 and the reagents required for assessing its functional significance using targeted gene manipulation in the mouse.
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PMID:The mouse p97 (CDC48) gene. Genomic structure, definition of transcriptional regulatory sequences, gene expression, and characterization of a pseudogene. 1018 99

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are activated by the Ino2p/Ino4p transcription factor that binds to ICRE promoter motifs and mediates maximal gene expression in the absence of inositol. We identified the ino80 mutation causing inositol auxotrophy as a result of a defect in ICRE-dependent gene activation. The product of the corresponding wild-type gene INO80 (= YGL150C) shows significant similarity to the Snf2p family of DNA-dependent ATPases. Nevertheless, SNF2 in increased gene dosage did not suppress ino80 mutant phenotypes. Mutation of the Ino80p lysine residue corresponding to the NTP binding site of Snf2p led to a non-functional protein. In ino80 null mutants, gene activation mediated by an ICRE decreased to 16% of the wild-type level. Maximal expression of PHO5, GAL1, CYC1 and ICL1 was also significantly reduced. Thus, Ino80p affects several transcription factors involved in unrelated pathways. As demonstrated by gel filtration, Ino80p is part of a high-molecular-weight complex of more than 1 MDa. Similar to what was found for Snf2p, the Ino80p-containing complex may influence the transcriptional level of several unrelated structural genes by functioning as an ATPase that possibly acts on chromatin.
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PMID:The product of the SNF2/SWI2 paralogue INO80 of Saccharomyces cerevisiae required for efficient expression of various yeast structural genes is part of a high-molecular-weight protein complex. 1036 Dec 78

The Agrobacterium tumefaciens VirB4 ATPase functions with other VirB proteins to export T-DNA to susceptible plant cells and other DNA substrates to a variety of prokaryotic and eukaryotic cells. Previous studies have demonstrated that VirB4 mutants with defects in the Walker A nucleotide-binding motif are non-functional and exert a dominant negative phenotype when synthesized in wild-type cells. This study characterized the oligomeric structure of VirB4 and examined the effects of Walker A sequence mutations on complex formation and transporter activity. VirB4 directed dimer formation when fused to the amino-terminal portion of cI repressor protein, as shown by immunity of Escherichia coli cells to lambda phage infection. VirB4 also dimerized in Agrobacterium tumefaciens, as demonstrated by the recovery of a detergent-resistant complex of native protein and a functional, histidine-tagged derivative by precipitation with anti-His6 antibodies and by Co2+ affinity chromatography. Walker A sequence mutants directed repressor dimerization in E. coli and interacted with His-VirB4 in A. tumefaciens, indicating that ATP binding is not required for self-association. A dimerization domain was localized to a proposed N-terminal membrane-spanning region of VirB4, as shown by the dominance of an allele coding for the N-terminal 312 residues and phage immunity of host cells expressing cI repressor fusions to alleles for the first 237 or 312 residues. A recent study reported that the synthesis of a subset of VirB proteins, including VirB4, in agrobacterial recipients has a pronounced stimulatory effect on the virB-dependent conjugal transfer of plasmid RSF1010 by agrobacterial donors. VirB4'312 suppressed the stimulatory effect of VirB proteins for DNA uptake when synthesized in recipient cells. In striking contrast, Walker A sequence mutants contributed to the stimulatory effect of VirB proteins to the same extent as native VirB4. These findings indicate that the oligomeric structure of VirB4, but not its capacity to bind ATP, is important for the assembly of VirB proteins as a DNA uptake system. The results of these studies support a model in which VirB4 dimers or homomultimers contribute structural information for the assembly of a transenvelope channel competent for bidirectional DNA transfer, whereas an ATP-dependent activity is required for configuring this channel as a dedicated export machine.
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PMID:Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter. 1038 64


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