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:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In pursuit of the characterization of the recently discovered flippase mode of operation of the anion transporter (band 3, AE1) of the human erythrocyte membrane, the transbilayer translocation (flip) of a fluorescently labeled, membrane-intercalated long-chain alkyl phosphate, 10-(alpha-napthyl)-1-decyl-phosphate (NDP) was investigated. In contrast to the alkyl sulfonates and esters of phosphatidic acid studied as yet, NDP moves exclusively via band 3. NDP is, however, dephosphorylated at the inner membrane surface by a cytoplasmic phosphatase likely to interact specifically with endofacial membrane structures of the erythrocyte. This phosphatase shares characteristic inhibitor sensitivities with protein tyrosine phosphatases present in the erythrocyte interior. Vanadate as an inhibitor of NDP dephosphorylation provided a means to study the kinetic properties and patterns of inhibition (by inhibitors of anion exchange) and stimulation (by proteolysis of band 3 and aliphatic alcohols) of the flip of NDP. NDP is also an inhibitor of the exchange of hydrophilic anions via band 3, while hydrophilic anions interfere with the flip of NDP. The results are compared with the characteristics of the flip, via Band 3, of other amphiphilic anions and of the exchange of hydrophilic anions. Attempts are presented to understand the low flip rate of long-chain amphiphilic anions on the basis of their molecular properties and the thermodynamics of the "transition state" of the flip process.
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PMID:Band 3-mediated flip-flop and phosphatase-catalyzed cleavage of a long-chain alkyl phosphate anion in the human erythrocyte membrane. 974 99

The role of the highly conserved residues in the gamma-phosphate binding site of myosin upon myosin motor function was studied. Each of five residues (Ser181, Lys185, Asn235, Ser236, and Arg238) in smooth muscle myosin was mutated. K185Q has neither a steady state ATPase nor an initial Pi burst. Although ATP and actin bind to K185Q, it is not dissociated from actin by ATP. These results indicate that the hydrolysis of bound ATP by K185Q is inhibited. S236T has nearly normal basal Mg2+-ATPase activity, initial Pi burst, ATP-induced enhancement of intrinsic tryptophan fluorescence, and ATP-induced dissociation from actin. However, the actin activation of the Mg2+-ATPase activity and actin translocation of S236T were blocked. In contrast S236A has nearly normal enzymatic properties and actin-translocating activity. These results indicate that 1) the hydroxyl group of Ser236 is not critical as an intermediary of proton transfer during the ATP hydrolysis step, and 2) the bulk of the extra methyl group of the threonine residue in S236T blocks the acceleration of product release from the active site by actin. Arg238, which interacts with Glu459 at the Switch II region, was mutated to Lys and Ile, respectively. R238K has essentially normal enzymatic activity and motility. In contrast, R238I does not hydrolyze ATP or support motility, although it still binds ATP. These results indicate that the charge interaction between Glu459 and Arg238 is critical for ATP hydrolysis by myosin. Other mutants, S181A, S181T, and N235I, showed nearly normal enzymatic and motile activity.
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PMID:Effects of mutations in the gamma-phosphate binding site of myosin on its motor function. 976 69

Site-directed mutations were made to the phosphate-binding loop threonine in the beta-subunit of the chloroplast F1-ATPase in Chlamydomonas (betaT168). Rates of photophosphorylation and ATPase-driven proton translocation measured in coupled thylakoids purified from betaT168D, betaT168C, and betaT168L mutants had <10% of the wild type rates, as did rates of Mg2+-ATPase activity of purified chloroplast F1-ATPase (CF1). The EPR spectra of VO2+-ATP bound to Site 3 of CF1 from wild type and mutants showed that EPR species C, formed exclusively upon activation, was altered in CF1 from each mutant in both signal intensity and in 51V hyperfine parameters that depend on the equatorial VO2+ ligands. These data provide the first direct evidence that Site 3 is a catalytic site. No significant differences between wild type and mutants were observed in EPR species B, the predominant form of the latent enzyme. Thus, the phosphate-binding loop threonine is an equatorial metal ligand in the activated conformation but not in the latent conformation of Site 3. The metal-nucleotide conformation that gives rise to species B is consistent with the Mg2+-ADP complex that becomes entrapped in a catalytic site in a manner that regulates enzymatic activity. The lack of catalytic function of CF1 with entrapped Mg2+-ADP may be explained in part by the absence of the phosphate-binding loop threonine as a metal ligand.
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PMID:EPR spectroscopy of VO2+-ATP bound to catalytic site 3 of chloroplast F1-ATPase from Chlamydomonas reveals changes in metal ligation resulting from mutations to the phosphate-binding loop threonine (betaT168). 1006 66

Three conserved glycine residues in the reactive thiol region of Dictyostelium discoideummyosin II were replaced by alanine residues. The resulting mutants G680A, G684A, and G691A were expressed in the soluble myosin head fragment M761-2R [Anson, M., Geeves, M. A., Kurzawa, S. E., and Manstein, D. J. (1996) EMBO J. 15, 6069-6074] and characterized using transient kinetic methods. Mutant G691A showed no major alterations except for a marked increase in basal Mg2+-ATPase activity. Phosphate release seemed to be facilitated by this mutation, and the addition of actin to G691A stimulated ATP turnover not more than 3-fold. In comparison to M761-2R, mutant constructs G691A and G684A showed a 4-fold reduction in the rate of the ATP cleavage step. Most other changes in the kinetic properties of G684A were small ( approximately 2-fold). In contrast, substitution of G680 by an alanine residue led to large changes in nucleotide binding. Compared to M761-2R, rates of nucleotide binding were 20-30-fold slower and the affinity for mantADP was approximately 10-fold increased due to a 200-fold reduction in the dissociation rate constant of mantADP. The ATP-induced dissociation of actin from the acto.680A complex was normal, but the communication between ADP and actin binding was altered such that the two sites are thermodynamically uncoupled but kinetically actin still accelerates ADP release.
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PMID:Kinetic analysis of Dictyostelium discoideum myosin motor domains with glycine-to-alanine mutations in the reactive thiol region. 1032 Mar 39

This study reports the organization of the wbp gene cluster and characterization of a number of genes that are essential for B-band O antigen biosynthesis in the clinically prevalent Pseudomonas aeruginosa serotype 06. Twelve genes were identified that share homology with other LPS and polysaccharide biosynthetic genes. This cluster contains homologues of wzx (encoding the O antigen flippase/translocase) and wzz (which modulates O antigen chain length distribution) genes, typical of a wzy-dependent pathway. However, a complete wzy gene (encoding the O-polymerase) was not found within the cluster. Four biosynthetic genes, wbpO, wbpP, wbpV and wbpM, and four putative glycosyltransferase genes, wbpR, wbpT, wbpU and wbpL, were identified in the cluster. To characterize their roles in LPS biosynthesis, null mutants of wbpO, wbpP, wbpV, wbpL and wbpM were generated using a gene-replacement strategy. Mutations in each of these genes caused deficiency in B-band synthesis. The wbpL mutant was deficient in both A-band and B-band LPS. WbpL(O6) is a bi-functional enzyme which could initiate B-band synthesis through the addition of QuiNAc to undecaprenol phosphate, and A-band synthesis by transferring either a GalNAc or a GlcNAc residue. Another approach used to assign function to the wbp(O6) genes was by complementation analysis. Two genes from Salmonella typhi, wcdA and wcdB, responsible for the synthesis of a homopolymer of GalNAcA called Vi antigen were used in complementation experiments to verify the functions of wbpO and wbpP. wcdA and wcdB restored B-band synthesis in wbpO and wbpP mutants respectively, implying that wbpO and wbpP are involved in UDP-GalNAcA synthesis. Although wbpV has homology to wbpK of the serotype O5 B-band LPS synthesis cluster, complementation analysis using the respective null mutants showed that the genes are not interchangeable. A knockout mutation of wbpN (located downstream of wbpM) did not abrogate LPS synthesis in either 05 or 06; therefore, it has been renamed orf48.5. These results establish the organization of genes involved in P. aeruginosa B-band O antigen synthesis and provide the evidence to assign functions to a number of LPS biosynthetic genes.
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PMID:Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide. 1062 48

The activities of calcium-activated ATPase (Ca2+-ATPase) and calcium magnesium-activated ATPase (Ca2+-Mg2+-ATPase) in the shell gland, and concentrations of 17beta-estradiol (E2) and progesterone in serum were monitored, respectively, from hens orally dosed with tri-o-cresyl phosphate (TOCP) (750 mg/kg). Treated birds were monitored daily for laying and development of delayed neurotoxicity, and activities of Ca2+-ATPase and Ca2+-Mg2+-ATPase were measured at 7 and 10 days after dosing. TOCP-treated birds manifested motor deficit by 7-9 days postdosing, while hens administered vehicle exhibited no signs of delayed neurotoxicity. Ca2+-ATPase and Ca2+-Mg2+-ATPase activities of shell glands from TOCP-dosed hens were not significantly affected (P > 0.05). The serum E2 concentration was significantly reduced in TOCP-treated hens (P < 0.01); however, progesterone levels were unaffected.
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PMID:Effects of tri-o-cresyl phosphate on serum estrogen and progesterone concentration and ATPase activity in the shell gland of adult hens. 1081 99

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.
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PMID:Detection and localization of a Ca2+-ATPase activity in Toxoplasma gondii. 1134 3

Galactosaemia is an inborn error of metabolism characterized by irreversible damage to neural tissue. To evaluate whether galactose metabolic disorders, (e.g. classical galactosaemia, galactokinase deficiency galactosaemia), is implicated for alterations of brain Mg2+-ATPase activity, various concentrations (1-16 mM) of galactose, galactose-1-phosphate, galactitol, glucose-1-phosphate or glucose were preincubated with whole brain homogenates of suckling rats at 37 degrees for 1 hr. Mg2+-ATPase activities were determined according to Bowler & Tirri's (1974). Galactose-1-phosphate or glucose-1-phosphate excessively activated the brain Mg2+-ATPase in a concentration-dependent way. Additionally, galactitol, galactose or glucose stimulated the enzyme up to 35-45% (P < 0.001) at concentrations >4 mM. A mixture of galactose-1-phosphate (2 mM), glactitol (2 mM) and galactose (4 mM), concentrations commonly found in blood and brain of untreated patients with classical galactosaemia, resulted in a 500% enzyme activation (P < 0.001) as compared to control. Moreover, a mixture of galactitol (2 mM) and galactose (1 mM), concentrations measured in patients with galactokinase deficiency, caused an enzyme stimulation (35%, P < 0.001). These findings suggest: a) The great Mg2+-ATPase activation by galactose-1-phosphate or glucose-1-phosphate may be due to the epimer of galactose and the presence of phosphorus. b) The brain Mg2+-ATPase stimulation by galactose and its derivatives could be toxic by modulating the Mg2+ concentration, the ATP availability, the activity of other ATP- and Mg2+-dependent enzymes as well as the rates of protein synthesis and cell growth.
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PMID:The in vitro effects of galactose and its derivatives on rat brain Mg2+-ATPase activity. 1257 32

Coordination between the nucleotide-binding site and the converter domain of myosin is essential for its ATP-dependent motor activities. To unveil the communication pathway between these two sites, we investigated contact between side chains of Phe-482 in the relay helix and Gly-680 in the SH1-SH2 helix. F482A myosin, in which Phe-482 was changed to alanine with a smaller side chain, was not functional in vivo. In vitro, F482A myosin did not move actin filaments and the Mg2+-ATPase activity of F482A myosin was hardly activated by actin. Phosphate burst and tryptophan fluorescence analyses, as well as fluorescence resonance energy transfer measurements to estimate the movements of the lever arm domain, indicated that the transition from the open state to the closed state, which precedes ATP hydrolysis, is very slow. In contrast, F482A/G680F doubly mutated myosin was functional in vivo and in vitro. The fact that a larger side chain at the 680th position suppresses the defects of F482A myosin suggests that the defects are caused by insufficient contact between side chains of Ala-482 and Gly-680. Thus, the contact between these two side chains appears to play an important role in the coordinated conformational changes and subsequent ATP hydrolysis.
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PMID:Requirement of domain-domain interaction for conformational change and functional ATP hydrolysis in myosin. 1275 55

Lead interferes with cellular energy metabolism by inhibiting ATP (Adenosine triphosphate) synthesis and hydrolysis. This study was conducted to determine in vitro effects of lead on Na+, K+-ATPase activity in four regions of adult rat brain: the cerebellum, the hippocampus, the frontal cortex and the brain stem. Male rats (Wistar strain) weighing 125-150 g were sacrificed, whole brain excised and the four regions were isolated. Each tissue was homogenized separately in sucrose (0.25 M) and imidazole (10 mM) buffer (pH 7.5) and P2 fraction was prepared by following established methods. The activity of ATPase was determined by measuring inorganic phosphate (Pi) liberated from ATP hydrolysis. The delineation of Na+, K+-activated component of ATPase was obtained by the difference between total ATPase and Mg2+-ATPase using 1 mM ouabain. The P2 fraction was incubated with 0, 5, 10, 25, 50 and 100 microM of lead at 37 degrees C for 10 min. The enzyme activity was expressed as micromoles of Pi liberated/mg protein/hr. The results indicated a concentration-dependent and region-specific response to lead. In vitro lead at 50 and 100 microM significantly inhibited ATPase activity in all regions of the brain. It was also observed that in the control rats, the enzyme activity was high in cerebellum and hippocampus regions of the brain. In vitro dithiothreitol (DTT) protected the enzyme activity from IC50 lead in four regions of brain. In cerebellum and hippocampus, a 5 microM DTT provided 100% protection against IC50 lead. These results suggest that lead interferes with the ion transport mechanism and cellular energy metabolism of the brain and this effect is region specific.
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PMID:In vitro effect of lead on Na+, K+-ATPase activity in different regions of adult rat brain. 1281 96


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