Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0155339 (Brown)
 12,436 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An enzyme that uses GTP as substrate for the formation in stoichiometric quantities of formate, inorganic pyrophosphate, and 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine-5'-phosphate has been purified 2200-fold from extracts of Escherichia coli B. This enzyme is named GTP cyclohydrolase II to distinguish it from a previously studied E. coli enzyme, named GTP cyclohydrolase (and called GTP cyclohydrolase I in this paper), that catalyzes the first of a series of enzymatic reactions leading to the biosynthesis of the pteridine portion of folic acid (Burg, A. W., and Brown, G. M. (1968) J. Biol. Chem. 243, 2349-2358). Some of the properties of GTP cyclohydrolase II are: (a) divalent cations are required for activity (Mg2+ is most effective); (b) its molecular weight, estimated by filtration on Sephadex G-200, is 44,000; (c) the K-m for GTP is 41 mum; (d) its pH optimum is 8.5; and (e) its activity is inhibited by inorganic pyrophosphate, one of the products of the reaction. Compounds not used as substrate are: GDP, GMP, guanosine, dGTP, ATP, ITP, and XTP. Properties a, b, c, and e (above), as well as the nature of the products, distinguish this enzyme from GTP cyclohydrolase I. Since GTP cyclohydrolase II apparently is not concerned with the biosynthesis of folic acid, the possible physiological role of this enzyme in the biosynthesis of riboflavin is considered in the light of the present investigations and the previously published work on riboflavin biosynthesis by other investigators.
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PMID:Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli. 23 52

Recently, we have reported that the isolated guanine nucleotide-binding regulatory protein, Gh, couples to the alpha 1-adrenergic receptor (Im, M.-J., and Graham, R. M. (1990) J. Biol. Chem. 265, 18944-18951 and Im, M.-J., Riek, R.P., and Graham, R. M. (1990) J. Biol. Chem. 265, 18952-18960) and has a molecular mass of approximately 74 kDa, and the approximately 50-kDa protein which is copurified probably regulates guanine nucleotide binding of the 74-kDa GTP-binding protein. In this paper, we describe the role of purified Gh in the regulation of phospholipase C in the reconstitution system. The stimulation of phospholipase C activity by Gh effectively occurred at a low calcium concentration (less than or equal to 2 microM), but the phospholipase C (PLC) itself required at least 50-100 times more calcium to become fully activated. The characteristic nature of phospholipase C stimulation by Gh is its response to the calcium concentration. Thus, the enzyme activity changes in narrow submicromolar ranges and reaches maximal stimulation, but it does not extend to the levels above those stimulated by calcium alone. The calcium concentrations for the maximal stimulation of phospholipase C activity were 10-20 microM with phospholipid vesicles and 100-200 microM with detergent solution. These calcium concentrations were further decreased when Gh and phospholipase C were co-reconstituted into the phospholipid vesicles or in the detergent solution. The maximal stimulations of the PLC activity were reached at less than 5 microM calcium in both the vesicles and the detergent solution. The changes of calcium concentration for the activation of PLC are quite different from those obtained by reconstituting PLC-beta 1 with Gq-like G-proteins (Smarcka, A. V., Hepler, J. R., Brown, K. O., and Sternweis, P. C. (1991) Science 251, 804-807 and Taylor, S. J., Chae, H. Z., Rhee, S. G., and Exton, J. H. (1991) Nature 350, 516-518). The phospholipase C activity was stimulated in a Gh concentration-dependent manner in the presence of GTP gamma S. The phospholipase C activity was activated by Gh alpha in the presence of aluminum fluoride, but not by Gh beta. Furthermore, a Gh.PLC complex can be induced by incubation with aluminum fluoride in a detergent solution and partially purified without the dissociation of related proteins. Thus, our reconstitution studies show that the pattern of stimulation of PLC by AIF-4-activated Gh in the ternary complex is similar to the stimulation of PLC activated by Gh in both detergent solution and phospholipid vesicles.
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PMID:Characterization of a phospholipase C activity regulated by the purified Gh in reconstitution systems. 157 27

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CAMP-NeuAc synthetase) from rat liver catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid from CTP and NeuAc. We have purified this enzyme to apparent homogeneity (241-fold) using gel filtration on Sephacryl S-200 and two types of affinity chromatographies (Reactive Brown-10 Agarose and Blue Sepharose CL-6B columns). The pure enzyme, whose amino acid composition and NH2-terminal amino acid sequence are also established, migrates as a single protein band on non-denaturing polyacrylamide gel electrophoresis. The molecular mass of the native enzyme, estimated by gel filtration, was 116 +/- 2 kDa whereas its Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 58 +/- 1 kDa. CMP-NeuAc synthetase requires Mg2+ for catalysis although this ion can be replaced by Mn2+, Ca2+, or Co2+. The optimal pH was 8.0 in the presence of 10 mM Mg2+ and 5 mM dithiothreitol. The apparent Km for CTP and NeuAc are 1.5 and 1.3 mM, respectively. The enzyme also converts N-glycolylneuraminic acid to its corresponding CMP-sialic acid (Km, 2.6 mM), whereas CMP-NeuAc, high CTP concentrations, and other nucleotides (CDP, CMP, ATP, UTP, GTP, and TTP) inhibited the enzyme to different extents.
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PMID:Purification and characterization of the nuclear cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from rat liver. 157 59

The alpha subunits of heterotrimeric GTP-binding (G) proteins act upon ion channels through both cytoplasmic and membrane-delimited pathways (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213). The membrane pathway may involve either a direct interaction between G protein and ion channel or an indirect interaction involving a membrane-delimited second messenger. To distinguish between the two possibilities, we tested whether a purified G protein could interact with a purified channel protein in a defined system to produce changes in channel currents. We selected the alpha subunit of Gs and the dihydropyridine (DHP)-sensitive Ca2+ channel of skeletal muscle T-tubules, the DHP binding protein (DHPBP), because: 1) a membrane-delimited interaction between the two has been shown (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213; Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895); and 2) at the present time, these Ca2+ channels are the only putative G protein channel effectors which, following purification, still retain channel function. We used a defined system in which purified components were studied by direct reconstitution in planar lipid bilayers. Just as we had found in crude skeletal muscle T-tubule membranes (Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895), alpha*s but not alpha*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gs alpha and Ca2+ channels. To establish whether the two proteins were physically associated in the native T-tubule membrane, we examined the ability of either endogenous G proteins or exogenous alpha*s to purify with detergent-solubilized DHPBP through a wheat germ agglutinin affinity column and a sucrose gradient. Small amounts of a labeled G protein were found to co-purify with DHPBP. In addition, partially purified DHPBP increased the sedimentation rate of purified alpha*s but not alpha*i-3. G proteins were immunoprecipitated with an antibody to the alpha 1 subunit of the DHPBP, and, in addition, both alpha s and the beta subunit of Gs were detected in Western blots of the partially purified DHPBP. The results suggest that Gs and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for Gs.
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PMID:Evidence for direct interaction of Gs alpha with the Ca2+ channel of skeletal muscle. 165 89

Aluminum fluoride (AlF4-) activates the heterotrimeric G protein Gs (stimulatory G protein of adenylylcyclase) (Sternweis, P. C., and Gilman, A. G. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4888-4891) and GT (transducin), and for GT, Bigay et al. (Bigay, J., Deterre, P., Pfister, C., and Chabre, M. (1985) FEBS Lett. 191, 181-185) have made the intriguing proposal that AlF4- acts by mimicking the gamma-phosphate of GTP. The endogenous G protein (probably G alpha i-2 or G alpha i-3 (Yatani, A., Mattera, R., Codina, J., Graf, R., Okabe, K., Padrell, E., Iyengar, R., Brown, A. M., and Birnbaumer, L. (1988) Nature 336, 680-682) that stimulates the muscarinic atrial K+ (K+[ACh]) channel is also thought to be activated by AlF4- (Kurachi, Y., Nakajima, T., and Ito, H. (1987) Circulation 76, 105P). To investigate the AlF4- mechanism, we applied potassium fluoride (KF) to the cytoplasmic face of inside-out membrane patches excised from guinea pig atria. We found that KF activated single K+[ACh] channel currents in both a concentration- and a Mg(2+)-dependent manner. Activation persisted following removal of KF, but unlike activation by guanosine 5'-(3-thiotriphosphate) (GTP gamma S), was fully reversed by removal of Mg2+. Evidence for Al3+ involvement was that the Al3+ chelator deferoxamine (500 microM) inhibited KF activation and that at low concentrations of KF (less than 1 mM), micromolar AlCl3 concentrations potentiated KF stimulation. The rate of activation produced by KF was far slower than the rate produced by GTP or GTP gamma S, and unlike these guanine nucleotides, the rate was unchanged in the presence of agonist. To test the gamma-phosphate-mimicking hypothesis, we evaluated the requirement for GDP; and to accomplish this, it was necessary to establish a condition that ensured exchange of guanine nucleotides. This condition was satisfied by using the muscarinic agonist carbachol because both the rate and the extent of activation of the K+[ACh] channels produced by GTP were much faster in carbachol, and both were greatly slowed when GDP was added along with GTP. By contrast, the effects of KF were unchanged by carbachol in the presence or absence of GDP. Further evidence that GDP is not essential for activation by AlF4- was provided by the observation that during carbachol activation and following extensive washing with GMP, guanosine 5'-O-(2-thiodiphosphate) at blocking concentrations had no effect on activation produced by KF.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanism of fluoride activation of G protein-gated muscarinic atrial K+ channels. 174 80

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.
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PMID:Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms. 190 16

Rap1A is a Ras-related GTP binding protein which has an amino acid sequence identical to that of Ras in the putative "effector" domain (amino acids 32-40). The binding of Rap1A to Ras-GTPase activating protein (GAP) through this domain is a potential mechanism for explaining the observation that Rap1A can antagonize the ability of oncogenic Ras to transform cells. It was recently shown (Yatani, A., Okabe, K., Polakis, P., Halenbeck, R., McCormick, F., and Brown, A. M. (1990) Cell 61, 769-776) that the activation of M2-muscarinic receptor-coupled K+ channels in heart is inhibited by the addition of exogenous Ras and Ras-GAP. We have made use of this system in the present paper to show that Rap1A is able to effectively block this inhibitory action of Ras-GAP. We observed that both Rap1A-GDP and Rap1A-guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) were able to block the inhibitory effect of Ras-GAP upon channel activation. This effect occurred at picomolar concentrations of Rap1A, and the GTP gamma S-bound form of the protein was consistently found to be more potent than the GDP form. A Rap1A Thr35----Ala mutation which bound GTP gamma S did not prevent K+ channel inhibition by Ras-GAP, suggesting that the antagonism by wild type Rap1A involves an interaction with GAP in the effector domain. The effectiveness of Rap1A to inhibit Ras-GAP is dependent upon the amount of Ras-GAP present in the assay and can also be overcome by the addition of GTP-bound N-Ras (GC-43), suggesting a competitive mechanism is operative. Finally, a truncated form of Ras-GAP (GAP32) which is no longer dependent upon Ras for inhibition of the M2-activated K+ channel is also no longer sensitive to blockade by added Rap1A. These data support the concept of GAP as an effector of Ras action and indicate that Rap1A can serve as an inhibitor of Ras action in a system distinct from cell transformation by a competitive mechanism involving the GAP binding domain of Rap1A.
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PMID:Rap1A antagonizes the ability of Ras and Ras-Gap to inhibit muscarinic K+ channels. 193 45

Gp is a major GTP-binding protein of human placenta and platelets [Evans, T., Brown, M. L., Fraser, E. D., & Northup, J. K. (1986) J. Biol. Chem. 261, 7052-7059]. High-affinity guanine nucleotide binding is associated with a polypeptide migrating identically with H-ras on SDS-PAGE. We have characterized the interactions of preparations of purified human placental Gp with guanine nucleotides in detergent solution. Equilibrium binding studies with [35S]GTP gamma S, [3H]Gpp(NH)p, and [3H]GTP identified a single class of sites with a dissociation constant of 10 +/- 1, 153 +/- 61, and 125 +/- 77 nM for the ligands, respectively. These three ligands were mutually competitive with Ki values consistent with the Kd values from direct binding experiments. Competition for the binding of [3H]Gpp(NH)p was used to determine the specificity of the site. Ki values determined from this assay were 14 nM for GTP gamma S, 143 nM for Gpp(NH)p, 3.3 microM for GDP beta S, 69 nM for GTP, and 64 nM for GDP. ATP, ADP, cAMP, cGMP, and NAD+ had no detectable affinity for this site. While the equilibrium binding data fit well to a single class of sites, association kinetics of these ligands were better fit to two rate constants. Dissociation kinetics, however, were not clearly resolved into two rates.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Unique guanine nucleotide binding properties of the human placental GTP-binding protein Gp. 212 Dec 70

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.
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PMID:The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system. 215 36

Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.
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PMID:Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP. 222 78


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