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)

Nucleotide and DNA coeffector substrate binding site characterizations were performed on two HSV-1 DNA helicases fulfilling different roles in DNA replication. Single ATP-binding sites were identified for helicase-primase and UL9 protein (Km(ATP) 0.62 mM and 0.54 mM, respectively). Analysis of structural requirements for DNA-dependent NTP hydrolysis revealed comparatively stringent requirements for helicase-primase in accommodating base-modified NTP analogs whereas the UL9 protein was much more permissive in this respect; neither enzyme was dependent on the ribose 2' or 3' hydroxyls for NTP hydrolysis. Both helicase-primase and UL9 protein ATPase activities were inhibited by ADP or GDP; this effect was competitive rather than allosteric. The enhancement of ATPase activity on a single stranded (ss) DNA substrate as opposed to double stranded (ds) DNA was much more marked for helicase-primase than for the UL9 protein (Km(dsDNA)/Km(ssDNA) 60 and 9, respectively). The triphosphates of the antiviral agents acyclovir and penciclovir were not effective substrates for either helicase-primase or UL9 protein.
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PMID:Characterisation of the nucleotide and DNA coeffector binding sites of the herpes simplex virus type 1 (HSV-1) encoded helicase-primase complex and UL9 origin binding protein. 814 77

In these studies, we have characterized calcium movement due to guanosine triphosphate (GTP) hydrolysis from an ATP-sequestered intracellular calcium pool in cultured gastric smooth muscle. GTP (1-100 microM), when added to an ATP-regenerating medium, resulted in a concentration-dependent and irreversible efflux of calcium from an organellar calcium pool. GTP-induced calcium efflux was not affected by variation of the ATP/ADP ratio (8.5-155.0), indicating that GTP did not act by inhibiting calcium influx via calcium adenosinetriphosphatase. To assess whether the calcium increase was necessarily associated with GTP hydrolysis, experiments were performed with the nonhydrolyzable guanine nucleotide analogues guanosine 5'-[beta-thio]diphosphate (GDP beta S), 5'-guanylyl imidodiphosphate guanosine (GppNHp), and 5'-O-(3-thiotriphosphate) (GTP gamma S). Administration of GDP beta S and GppNHp resulted in no significant calcium efflux. GTP gamma S caused a small steady-state calcium increase (20% of that induced by the hydrolyzable nucleotide) but irreversibly inhibited all subsequent calcium increase due to GTP. The possibility that GTP may either modify the concentration of mobilizable calcium in inositol trisphosphate (IP3)-sensitive calcium stores or the responsivity of IP3-associated calcium channels was assessed by two experiments: 1) prior administration of GTP at concentrations < or = 100 microM had no effect on IP3-induced calcium release, and 2) heparin, which competitively inhibits IP3 binding to its receptor on the endoplasmic reticulum, did not affect GTP-associated calcium increase. These results demonstrate that, in gastric smooth muscle, GTP causes calcium efflux from an intracellular pool that is functionally independent from that pool sensitive to IP3.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Calcium efflux from an intracellular pool activated by GTP hydrolysis in cultured gastric smooth muscle. 816 79

In granulocytes, emptying of intracellular Ca2+ stores activates Ca2+ influx across the plasma membrane. To study the putative role of GTP-binding proteins in this process, we have introduced non-hydrolyzable guanosine phosphate analogues into the cytosol of non-permeabilized HL-60 granulocytes using an endocytosis-hypoosmotic shock procedure. At the cytosolic concentrations obtained (100-500 microM), neither guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) nor guanosine 5'-3-O-(thio)diphosphate (GDP beta S) affected basal [Ca2+]i. Ca2+ release in response to the receptor agonist fMet-Leu-Phe, the Ca(2+)-ATPase inhibitor thapsigargin, or the Ca2+ ionophore ionomycin was also unaffected by GTP gamma S or GDP beta S. In contrast, the activation of the Ca2+ influx pathway by fMet-Leu-Phe or by thapsigargin was blocked by GTP gamma S but not by GDP beta S. The GTP gamma S effect was mimicked by NaF. The GTP gamma S and NaF effects were independent of protein kinase C activation and actin polymerization. Our results demonstrate that a GTP-sensitive element is involved in the signaling between intracellular Ca2+ stores and plasma membrane Ca2+ channels. The identical effects of GTP gamma S and NaF suggest that the GTP-sensitive element is a heterotrimeric G-protein.
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PMID:The regulation of store-dependent Ca2+ influx in HL-60 granulocytes involves GTP-sensitive elements. 825 21

The F-ATPase of chloroplasts couples proton flow to ATP synthesis, but is leaky to protons in the absence of nucleotides. This "proton slip" can be blocked by small concentrations of ADP or by inhibitors of the channel portion, CF0. We studied charge flow through the ATPase by flash spectrophotometry and analyzed the inhibition of proton slip by nucleotides, phosphate/arsenate, and insufficient proton motive force. The following inhibition constants (at given background concentrations) were observed: ADP, 0.2 microM (0.5 mM P(i)); ADP, 13.4 microM (no P(i)); P(i), 43 microM (1 microM ADP); GDP, 2.5 microM (0.5 mM P(i)); ATP, 2 microM. ADP and P(i) mutually lowered their respective inhibition constants. Phosphate could be replaced by arsenate. Proton slip occurred only if the proton motive force exceeded a certain threshold, similar to that for ATP synthesis. The inhibition of proton slip by ADP and GDP qualified the respective nucleotide binding sites as belonging to the subset of two (or three) potentially catalytic sites out of the total of six. We interpreted the ADP-induced transition between different conduction states of the ATPase from "slipping" to "closed" to "coupled" as a consequence of the alternating site mechanism of catalysis. Whereas the proton translocator idles in the absence of nucleotides, the high-affinity binding of the first ADP/P(i) couple to one site clutches proton flow to some (conformational) change that can only be executed after the binding of another ADP/P(i) couple to a second site. From there on these sites alternate in the catalytic cycle. An entropic machine is presented which likewise models proton slip, unisite, and multisite ATP synthesis and hydrolysis.
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PMID:Proton slip of the chloroplast ATPase: its nucleotide dependence, energetic threshold, and relation to an alternating site mechanism of catalysis. 839 25

Glutamine 170 to tyrosine mutation in the beta-subunit from Schizosaccharomyces pombe mitochondrial F1 was found to increase both affinity for ADP, apparent negative cooperativity of ATPase activity, and sensitivity to azide inhibition (Falson, P., Di Pietro, A., Jault, J.-M., Gautheron, D.C., and Boutry, M. (1989) Biochim. Biophys. Acta 975, 119-126). The mutation is shown here to increase the affinity for GDP, IDP, and guanosine 5'-(beta,gamma-imidotriphosphate), which are competitive inhibitors of GTPase and ITPase activities. Various fluorescence approaches also reveal an increased affinity of the catalytic site in mutant as compared with wild-type enzyme for GDP, IDP, and 2'(3')-N-methylanthraniloyl GDP. The mutation alters the maximal rates and pH dependence of GTPase and ITPase activities, whereas wild-type F1 exhibits single optima at pH 7.5-8.0. The pH activity profiles of the mutant enzyme for these substrates are biphasic, with optima at pH 8.5-9.0 and below 6.5. The mutation increases the sensitivity of GTPase and ITPase activities to azide inhibition, which increases with decreasing pH. At pH 6.0-7.0, an apparent negative cooperativity is observed when mutant F1 hydrolyzes GTP or ITP, whereas the wild-type enzyme shows Michaelian kinetics. Addition of bicarbonate at pH 7.0 substantially stimulates GTP or ITP hydrolysis and abolishes the apparent negative cooperativity by the mutant enzyme; on the contrary, the anion produces a slight inhibition of these activities catalyzed by wild-type F1. The overall results suggest that apparent negative cooperativity can be observed with GTP or ITP hydrolysis provided that the release of the respective diphosphate is a rate-limiting step.
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PMID:Glutamine 170 to tyrosine substitution in yeast mitochondrial F1 beta-subunit increases catalytic site interaction with GDP and IDP and produces negative cooperativity of GTP and ITP hydrolysis. 840 1

Chemical modification of mitochondrial F1-ATPase from Schizosaccharomyces pombe by the tryptophan-specific reagent N-bromosuccinimide (NBS) at pH 5.0 in the presence of 20% glycerol produced a characteristic lowering in both enzyme absorbance at 280 nm and intrinsic fluorescence at 332 nm that varied with NBS/F1 molar ratio up to a value of 130. Fluorometric titration of tryptophans and correlation to residual ATPase activity showed that modification of three reactive residues among the seven present on alpha- and epsilon-subunits did not markedly modify the enzyme activity but efficiently released endogenous ATP and abolished the fluorescence quenching related to GDP or ATP binding to the catalytic site. Additional modification of one, less reactive, tryptophan altered both negative cooperativity of ATP hydrolysis and sensitivity to azide inhibition and produced a nearly complete inactivation at high NBS/F1 molar ratio. NBS-induced inactivation of F1 was favored by catalytic-site saturation with GDP or low ATP concentration and on the contrary was prevented by noncatalytic-site saturation with ADP or high ATP concentration. When reactive tryptophans were selectively modified by NBS in the presence of ADP, and subunits were isolated after guanidine hydrochloride dissociation by one-step purification on reversed-phase HPLC, the absorbance of alpha-subunit at 280 nm was decreased, whereas that of epsilon-subunit was unchanged. Cyanogen bromide cleavage of alpha-subunit and fragments separation by reversed-phase HPLC showed that one peptide of 3 kDa apparent molecular mass had decreased absorbance. N-Terminal sequencing allowed its identification to fragment 255-282 that contains tryptophan257.
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PMID:Chemical modification of alpha-subunit tryptophan residues in Schizosaccharomyces pombe mitochondrial F1 adenosine 5'-triphosphatase: differential reactivity and role in activity. 842 30

We have described a thyroid hormone receptor in synaptosomes of the chick embryo brain. To understand how the hormones exert their actions at this level, we performed a series of studies to demonstrate that this receptor could be linked to G proteins. Guanosine 5'-[gamma-thio]triphosphate (GTP gamma S)(100 muM) lowered the binding capacity of the receptor high affinity site from 8.9 +/- 1.3 to 3.4 +/- 1.3 ng T3/mg protein, a finding consistent with the coupling of receptor to G proteins. Furthermore, ADP ribosylation with pertussis toxin showed that thyroid hormones induced a dose-dependent increase in the inactive alpha 0-subunit of the G0 protein. This effect was detected at 10 pM, with a maximal increase (mean +/- SEM, 50 +/- 3.6%) at 100 nM, and T4 was as effective as T3. Both hormones also decreased the intrinsic guanine triphosphatase activity of G proteins by lowering the binding of GTP to the alpha-subunit and their rate of hydrolysis. This inhibition was greater with T4 (25 +/- 5%) than with T3 (14 +/- 2%), suggesting that the former could be the more active hormone at the synaptosomal level. The effect on guanine triphosphatase activity confirms that the synaptosomal thyroid hormone receptor is coupled to a G(zero) protein. These results demonstrate that thyroid hormones increase or favor the ADP ribosylation of G alpha(zero) by pertussis toxin. Thus, they enhance the alpha(zero)-GDP form of the G(zero) protein, namely its inactive conformation. By decreasing the activity of this protein, these hormones may modulate the formation of second messengers in synaptosomes and intervene in the regulation of neuronal proliferation and differentiation induced by several factors. Therefore, thyroid hormones may exert their action on brain maturation at least in part by modulating G alpha(zero) through their synaptosomal receptor.
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PMID:Effect of thyroid hormones on G proteins in synaptosomes of chick embryo. 864 Dec 9

The Rho family small G proteins are implicated in various cell functions, such as cell morphological change, cell motility, and cytokinesis. However, their modes of action in regulating these cell functions remain to be clarified. In the present study, we have isolated a cDNA encoding a protein which interacts with the GTP-bound form, but not with the GDP-bound form, of the Rho family members, including RhoA, Racl, and Cdc42, by the yeast two-hybrid method. This protein is kinectin, known to be a vesicle membrane anchoring protein of kinesin, which is an ATPase motor transporting vesicles along microtubules.
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PMID:Interaction of the Rho family small G proteins with kinectin, an anchoring protein of kinesin motor. 876 96

Hepatocyte plasma membranes contain a glycosylated 230-kDa Ca(2+) -dependent, Mg(2+)-stimulated ATPase (pgp230), which consists of two subunits, one of 120 kDa and the other of 110 kDa. pgp230 can be enriched by the use of affinity chromatography on Concanavalin A-Sepharose, wheat germ lectin-Sepharose, and 5'-AMP-Sepharose. It has a high-affinity Ca2+ binding site. In the presence of Ca2+, it forms a phosphorylated intermediate by autocatalytic transfer of the terminal phosphate residue from ATP. Maximal Ca(2+)-dependent autophosphorylation is observed at pH 5-6. Photoaffinity labeling using 8-azido-[alpha-32P]ATP or [y-32P]ATP confirms the presence of ATP binding sites. Incubation with [alpha-32P]ATP leads to a rapid but transient labeling of pgp230. Various nucleotides, nucleotide receptor agonists, or antagonists inhibit Ca(2+)-dependent phosphorylation by [y-32P]ATP. The concentrations of half-maximal inhibition range from 10(-7) M to 10(-3) M. The rank order of inhibitory potency is: ATP > alpha,beta-methylene-ATP > CTP = TTP > y-4-amino-phenyl-ATP = 2-methyl-thio-ATP > UTP = GTP > GDP = ADP = beta,y-methylene-ATP = beta, y-methylene-TTP = beta,y-methylene-GTP = adenosine-5'-O-2-thiodiphosphate = CMP = AMP > adenosine > cytidine > guanosine = suramin > Reactive blue 2 > iso-butyl-methyl-xanthine > thymidine > uridine. These data suggest a nucleotide binding capacity of this new hepatocyte membrane glycoprotein. Further investigations should be carried out to reveal its biological function.
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PMID:Partial characterization of a new nucleotide binding glycoprotein of hepatocyte plasma membrane. 878 41

Smg GDS is a regulator having two activities on a group of small G proteins including the Rho and Rap1 family members and Ki-Ras; one is to stimulate their GDP/GTP exchange reactions, and the other is to inhibit their interactions with membranes. Structurally, it has 11 Arm repeats, a protein interaction motif, found in the Drosophila Armadillo protein, a homolog of mammalian beta-catenin. We have isolated here an Smg GDS-interacting protein from a human brain cDNA library by use of the yeast two-hybrid method and named it SMAP (Smg GDS-associated protein). SMAP was a protein with a Mr of 91,189 and 792 amino acids. SMAP had 9 Arm repeats. Recombinant SMAP interacted with recombinant Smg GDS but did not affect the two activities of Smg GDS on RhoA. SMAP was tyrosine phosphorylated by v-Src, and this phosphorylation reduced the affinity of SMAP for Smg GDS. Tissue and subcellular distribution analyses indicated that SMAP was ubiquitously expressed and highly concentrated at the endoplasmic reticulum area. Searches for sequence homology to SMAP revealed that SMAP was significantly homologous to sea urchin SpKAP115, suggesting that SMAP is a mammalian counterpart of SpKAP115 or its related protein. SpKAP115 is an accessory subunit of sea urchin kinesin II, an ATPase motor that transports vesicles along microtubules. These results suggest that SMAP serves as an adaptor for both Smg GDS and kinesin II or its related protein and links them with both the Smg GDS-regulated small G protein and Src tyrosine kinase signalings.
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PMID:SMAP, an Smg GDS-associating protein having arm repeats and phosphorylated by Src tyrosine kinase. 890 Jan 89


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