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)

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

We have isolated an ADP-ribosylation factor (ARF) gene from the human malarial parasite, Plasmodium falciparum. The gene (P. falciparum arf1) has four introns and the exons encode a protein of 181 amino acids with high similarity to the mammalian class I ARF proteins 1-3 (> or = 74% amino acid identity). Southern hybridization suggests there is at least one additional arf in the P. falciparum genome. Northern analysis identified a single P. falciparum arf1 mRNA of 1.8 kb in the asexual blood stage form of the parasite. The P. falciparum arf1 mRNA levels are developmentally regulated, reaching a maximum during nuclear division towards the end of the intraerythrocytic cycle. P. falciparum arf1 cDNA was isolated by reverse-transcriptase polymerase chain reaction and used to express a recombinant protein in Escherichia coli. Recombinant P. falciparum ARF1 protein was purified with stoichiometric amounts of bound GDP, although intrinsic guanose triphosphatase activity of the protein could not be detected. The protein stimulated cholera-toxin-catalyzed ADP-ribosyltransferase activity in a reaction that was dependent upon the addition of both dimyristoylglycerophosphocholine and cholate. The protein bound GTP with first-order kinetics with an apparent rate constant, k', of 0.0145 (+/- 0.0019) min-1. These results suggest that P. falciparum ARF1 is a member of the class 1 ARF family and provide additional evidence for the existence of a classical secretory pathway in P. falciparum.
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PMID:Isolation, expression and characterization of the gene for an ADP-ribosylation factor from the human malaria parasite, Plasmodium falciparum. 895 60

Autotaxin (ATX) is an extracellular enzyme and an autocrine motility factor that stimulates pertussis toxin-sensitive chemotaxis in human melanoma cells at picomolar to nanomolar concentrations. This 125-kDa glycoprotein contains a peptide sequence identified as the catalytic site in type I alkaline phosphodiesterases (PDEs), and it possesses 5'-nucleotide PDE (EC 3.1.4.1) activity (Stracke, M. L., Krutzsch, H. C., Unsworth, E. J., Arestad, A., Cioce, V., Schiffmann, E., and Liotta, L. (1992) J. Biol. Chem. 267, 2524-2529; Murata, J., Lee, H. Y., Clair, T., Krutsch, H. C., Arestad, A. A., Sobel, M. E., Liotta, L. A., and Stracke, M. L. (1994) J. Biol. Chem. 269, 30479-30484). ATX binds ATP and is phosphorylated only on threonine. Thr210 at the PDE active site of ATX is required for phosphorylation, 5'-nucleotide PDE, and motility-stimulating activities (Lee, H. Y., Clair, T., Mulvaney, P. T., Woodhouse, E. C., Aznavoorian, S., Liotta, L. A., and Stracke, M. L. (1996) J. Biol. Chem. 271, 24408-24412). In this article we report that the phosphorylation of ATX is a transient event, being stable at 0 degrees C but unstable at 37 degrees C, and that ATX has adenosine-5'-triphosphatase (ATPase; EC 3.6.1.3) and ATP pyrophosphatase (EC 3.6.1.8) activities. Thus ATX catalyzes the hydrolysis of the phosphodiester bond on either side of the beta-phosphate of ATP. ATX also catalyzes the hydrolysis of GTP to GDP and GMP, of either AMP or PPi to Pi, and the hydrolysis of NAD to AMP, and each of these substrates can serve as a phosphate donor in the phosphorylation of ATX. ATX possesses no detectable protein kinase activity toward histone, myelin basic protein, or casein. These results lead to the proposal that ATX is capable of at least two alternative reaction mechanisms, threonine (T-type) ATPase and 5'-nucleotide PDE/ATP pyrophosphatase, with a common site (Thr210) for the formation of covalently bound reaction intermediates threonine phosphate and threonine adenylate, respectively.
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PMID:Autotaxin is an exoenzyme possessing 5'-nucleotide phosphodiesterase/ATP pyrophosphatase and ATPase activities. 899 94

Polycystic kidney disease progresses more rapidly in men than in women. To investigate the basis for this sexual dimorphism, we exposed Madin-Darby canine kidney (MDCK) cells grown on collagen-coated cell culture inserts to control media, or to estradiol or testosterone (1 nM-1 microM). Compared to control and estradiol-treated cells, testosterone stimulated fluid secretion in a dose-dependent manner, enhancing fluid secretion 4.8-fold at 1 nM and 19.7-fold at 1 microM (0.59 +/- 0.18 vs. 0.03 +/- 0.01 microliter/cm2/hr, P < 0.001). Chloride transport paralleled fluid secretion. Testosterone increased cellular cyclic AMP levels 3.2-fold at 1 nM and 12.3-fold at 1 microM (81.3 +/- 30.7 vs. 6.6 +/- 3.3 pmol/mg protein, P < 0.001). GDP beta S (500 microM), an inhibitor of Gs, and 2',3'-dideoxyadenosine (10 microM), an inhibitor of the catalytic subunit of adenylate cyclase, suppressed testosterone-induced fluid and solute secretion. Neither testosterone nor estradiol had any effect on microsomal Na,K-ATPase activity, cellular proliferation or cellular total protein content. Our studies show that testosterone stimulates fluid secretion and solute transport by MDCK cells by increasing cAMP generation. In vivo, testosterone may contribute to cyst expansion by enhancing fluid secretion. This observation may help explain the worse prognosis of polycystic kidney disease observed in men.
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PMID:Effects of sex hormones on fluid and solute transport in Madin-Darby canine kidney cells. 915 Apr 70

To define the requirements for the homotypic fusion of mammalian endoplasmic reticulum (ER) membranes, we have developed a quantitative in vitro enzyme-linked immunosorbent assay. This assay measures the formation of IgG (H2L2) following the fusion of ER microsomes containing either the heavy or light chain subunits. Guanine nucleotide dissociation inhibitor (GDI), a protein that extracts Rab GTPases in the GDP-bound form from membranes, potently inhibits fusion. Inhibition was not observed using GDI mutants defective in Rab binding. Kinetic analysis of the inhibitory effects of GDI revealed that Rab activation is required immediately preceding or coincident with fusion and that this step is preceded by a priming event requiring a member of the AAA ATPase family. Our results suggest that homotypic fusion of ER membranes requires Rab and that Rab activation is a transient event necessary for the formation of a fusion pore leading to the mixing of luminal contents of ER microsomes.
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PMID:A Rab GTPase is required for homotypic assembly of the endoplasmic reticulum. 915 91

1. This study was designed to investigate the mechanism of the relaxation induced by vasoactive intestinal peptide (VIP) in medial strips of the porcine coronary artery, by determining the effect on the cytosolic Ca2+ concentration ([Ca2+]i), the [Ca2+]i-force relation and the involvement of G-protein. 2. Front-surface fluorometry of fura-2 revealed that U46619, a thromboxane A2 analogue, and the high K(+)-depolarization induced increases in both the [Ca2+]i and force of the medial strips. At a steady state of contraction, the extent of an increase in [Ca2+]i induced by 100 nM U46619 was similar to that induced by 30 mM K(+)-depolarization. VIP concentration-dependently (1 nM-1 microM) induced transient decreases in both the [Ca2+]i and force of the medial strips precontracted with 100 nM U46619. The decreases in the [Ca2+]i and force induced by VIP during the contraction with U46619 were much greater than those with 30 mM K(+)-depolarization. 3. The VIP-induced decreases in the [Ca2+]i and force were attenuated by K+ channel blockers such as tetrabutylammonium (TBA: non-selective K+ channel blocker), charybdotoxin (large conductance Ca(2+)-activated K+ channel blocker), and 4-aminopyridine (4-AP: voltage-dependent K+ channel blocker). However, neither glibenclamide (ATP-sensitive K+ channel blocker) nor apamin (small conductance Ca(2+)-activated K+ channel blocker) had any significant inhibitory effect. 4. In the 30 mM K(+)-depolarized strips, pretreatment with thapsigargin, a specific Ca(2+)-ATPase inhibitor of the Ca2+ store sites, completely abolished the VIP-induced decrease in [Ca2+]i, but partially attenuated the VIP-induced decrease in force. 5. VIP shifted the [Ca2+]i-force relation of the U46619-induced contractions to the right in a concentration-dependent manner. In the alpha-toxin-permeabilized strips, VIP decreased the force development at a constant [Ca2+]i level (pCa = 6.5) in a GTP-dependent manner, which was antagonized by guanosine-5'-O-(beta-thiodiphosphate) (GDP beta S). 6. We thus conclude that VIP relaxes the coronary artery via three mechanisms: (1) a decrease in [Ca2+]i by inhibiting the Ca2+ influx presumably through the membrane hyperpolarization mediated by the activation of the large conductance Ca(2+)-activated (charybdotoxin-sensitive) K+ channels and voltage-dependent (4-AP-sensitive) K+ channels; (2) a decrease in [Ca2+]i by sequestrating cytosolic Ca2+ into thapsigargin-sensitive Ca2+ store sites; and (3) a decrease in the Ca(2+)-sensitivity of the contractile apparatus through the activation of G-protein.
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PMID:The mechanisms of the relaxation induced by vasoactive intestinal peptide in the porcine coronary artery. 922 56

CFTR-NBF-2 expressed and purified in fusion with the maltose-binding protein was shown to catalyse the reaction ATP-->ADP+Pi by three different assays, monitoring ATP turnover, formation of ADP and release of Pi (Km 86 microM, rate constant 0.37 min(-1)). The reaction product ADP inhibits this ATPase activity. In a similar manner the hydrolysis of GTP to GDP and Pi was demonstrated (Km 40 microM, rate constant 0.29 min(-1)). In the presence of AMP the ATPase reaction was superseded by the formation of two ADP from ATP and AMP. As typical for adenylate kinases a distinct AMP-binding site could be verified for CFTR-NBF-2 by the inability of TNP-ATP and AMP to compete for binding. All three enzymatic activities were inhibited by the symmetric double-substrate-mimicking inhibitor Ap5A. As NBF-2 plays a central role in CFTR channel opening and closing the results reported here are fundamental in understanding mechanisms of CFTR channel activity regulation.
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PMID:A recombinant polypeptide model of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator functions as an active ATPase, GTPase and adenylate kinase. 923 25

Hsp70 is a multifunctional molecular chaperone whose interactions with protein substrates are regulated by ATP hydrolysis and ADP-ATP exchange. We show here that, in addition to ATPase activity, purified Hsp70 free from nucleoside-diphosphate (NDP) kinase exhibits intrinsic ADP-ATP exchange activity. The rate constants for ATP hydrolysis and ATP synthesis were in a similar range at the optimum pH of 7.5-8.5 in the presence of 5 mM ATP and 0.5 mM ADP. Hsp70 exhibited a considerably strict preference for ATP as a phosphate donor, and a biased substrate specificity, unlike NDP kinase; ADP, UDP, CDP > dTDP, dCDP > GDP, dGDP. During the reaction, Hsp70 formed an acid-labile autophosphorylated intermediate, and nucleoside diphosphate-dependent dephosphorylation of the latter then occurred. These properties of Hsp70 are not identical but similar to those of NDP kinase, but are not similar to those of adenylate kinase and ATP synthase.
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PMID:Intrinsic ADP-ATP exchange activity is a novel function of the molecular chaperone, Hsp70. 948 62

The 2.4-A resolution crystal structure of a dominantly active form of the small guanosine triphosphatase (GTPase) RhoA, RhoAV14, complexed with the nonhydrolyzable GTP analogue, guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), reveals a fold similar to RhoA-GDP, which has been recently reported (Wei, Y., Zhang, Y., Derewenda, U., Liu, X., Minor, W., Nakamoto, R. K., Somlyo, A. V., Somlyo, A. P., and Derewenda, Z. S. (1997) Nat. Struct. Biol. 4, 699-703), but shows large conformational differences localized in switch I and switch II. These changes produce hydrophobic patches on the molecular surface of switch I, which has been suggested to be involved in its effector binding. Compared with H-Ras and other GTPases bound to GTP or GTP analogues, the significant conformational differences are located in regions involving switches I and II and part of the antiparallel beta-sheet between switches I and II. Key residues that produce these conformational differences were identified. In addition to these differences, RhoA contains four insertion or deletion sites with an extra helical subdomain that seems to be characteristic of members of the Rho family, including Rac1, but with several variations in details. These sites also display large displacements from those of H-Ras. The ADP-ribosylation residue, Asn41, by C3-like exoenzymes stacks on the indole ring of Trp58 with a hydrogen bond to the main chain of Glu40. The recognition of the guanosine moiety of GTPgammaS by the GTPase contains water-mediated hydrogen bonds, which seem to be common in the Rho family. These structural differences provide an insight into specific interaction sites with the effectors, as well as with modulators such as guanine nucleotide exchange factor (GEF) and guanine nucleotide dissociation inhibitor (GDI).
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PMID:Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue. 954 99


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