Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

GTP cyclohydrolase I, an enzyme that catalyzes the first reaction in the pathway for the biosynthesis of the pteridine portion of folic acid, was purified from Escherichia coli by 3,900-fold to apparent homogeneity. Its molecular weight is estimated at 210,000. At relatively high concentrations of salt (e.g. 0.3 M KCl) the enzyme can be dissociated into seemingly identical subunits of 51,000 molecular weight. Removal of the salt allows reassociation. GTP, ATP, and inorganic orthophosphate at concentration of 5 muM, 100muM, and 0.2 mM, respectively, promote the reassociation of the subunits even in the presence of 0.3 M salt. The subunits have little or no catalytic activity. When the enzyme was subjected to electrophoresis on polyacrylamide gel under denaturing conditions (in the presence of sodium dodecyl sulfate) only one protein band was evident; its molecular weight was estimated at 25,500. Proline was determined as the only NH2-terminal amino acid residue of the enzyme. These observations suggest that the enzyme consists of four identical subunits and that each subunit contains two identical polypeptide chains. Enough GTP was bound to the enzyme to suggest that each polypeptide contains one GTP binding site. The Km value for GTP IS 0.02 MuM. ATP, dGTP, and guanosine 5'-tetraphosphate are competitive inhibitors with Ki values of 0.25 muM, 0.24 muM, and 0.13 muM, respectively. Orthophosphate is an uncompetitive inhibitor. The enzyme is relatively heat-stable; its half-life at 82 degrees is 7 min. Salt (NaCl, KCl, NH4Cl) at a concentration of 0.1 M activates the enzyme by 4- to 5-fold. The only products of the action of the enzyme are formate and the triphosphoester of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropteridine (H2-neopterin-PPP). The evidence strongly suggests that this single enzyme catalyzes 4 independent chemical reactions in the conversion of GTP to H2-neopterin-PPP.
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PMID:Characteristics of guanosine triphosphate cyclohydrolase I purified from Escherichia coli. 82 48

The effects of crotin I and crotin II on the partial reactions of polypeptide chain elongation were investigated and compared with the known effects of ricin. Crotin II was a more powerful inhibitor than crotin I, but no qualitative differences between the two crotins were found. Rat liver ribosomes, preincubated with crotins and washed through sucrose gradients, remained inactive in protein synthesis. Among the individual steps of elongation, the peptidyltransferase reaction was unaffected by crotins, but some of the reactions that involve the interaction of elongation factors 1 and 2 with ribosomes were modified. A strong inhibition of the binding of elongation factor 2 to ribosomes and a stimulation of the elongation factor2-dependent GTP hydrolysis were observed; this indicates the formation of a very unstable elongation factor 2--GDP--ribosome complex, which, however, allows a single round of translocation to take place in the presence ofelongation factor 2 and added GTP. The elongation factor 1-dependent GTP hydrolysis was inhibited by crotins, whereas the enzymic binding of aminoacyl-tRNA, to both rat liver and Artemia salina ribosomes, was scarcely affected. In a protein-synthesizing system the inhibition by crotins and by ricin leads to a block of the nascent peptides on the ribosomal aminoacyl-tRNA site, an effect consistent with inhibition at the level of translocation. The mechanism of action of crotins appears to be very similar to that of ricin.
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PMID:Inhibition of protein synthesis in vitro by crotins and ricin. Effect on the steps of peptide chain elongation. 94 4

The rate of initiation of protein synthesis in rabbit reticulocyte lysates is regulated by a translational inhibitor protein which is activated in the absence of added haemin. The effects of this inhibitor on amino acid incorporation are overcome by the protein synthesis initiation factor IF-MP which binds Met-tRNAf in a ternary complex with GTP and which can transfer this complex to small ribosomal subunits. Addition of this factor to haemin-deficient lysates prevents loss of polysomes and regenerates polysomes from 80-S single ribosomes, thus confirming an effect at the level of polypeptide initiation. The ability of the initiation factor to overcome the effects of various concentrations of the translational inhibitor suggests that the inhibitor inactivates the factor catalytically rather than stoichiometrically. In a system in vitro consisting of salt-washed 40-S ribosomal subunits, initiator Met-tRNAf and GTP, the initiation factor IF-MP transfers Met-tRNAf to the subunits in the absence of any other factor or mRNA. Equilibrium buoyant density gradient analysis in CsCl shows that formaldehyde-fixed subunits carrying Met-tRNAf bound under these conditions have a buoyant density approximately 0.02 g/cm3 lower than the bulk of salt-washed subunits, suggesting that approximately 100000 daltons of additional protein are associated with these subunits. This is in marked contrast to the amounts of protein bound to subunits incubated with Met-tRNAf and GTP in the presence of a crude ribosomal salt-wash fraction. The translational inhibitor has no effect on formation of the ternary complex IF-MP-Met-tRNAf-GTP but does impair the factor-catalysed transfer of Met-tRNAf to washed subunits. The possible mechanisms of action of the inhibitor on polypeptide chain initiation are reviewed in the light of these results.
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PMID:Functional relationships between a reticulocyte polypeptide-chain-initiation factor (IF-MP) and the translational inhibitor involved in regulation of protein synthesis by haemin. 94 56

1. A factor, which makes a ternary complex with GTP and eukaryotic initiator tRNA (Met-tRNAi), has been purified 100-fold from developed cysts of Artemia salina. Some of the properties of the purified factor have been studied. 2. Mg2+ appears to inhibit ternary complex formation. 3. Little or no ternary complex is formed when 5 muM GTP is replaced by an identical concentration of UTP, CTP or ATP. The analog, guanosine 5'-(beta, gamma-imino)triphosphate [GMP-P(NHP)] seems to be a much better substitute for GTP than guanosine 5'-(beta, gamma-methylene)triphosphate [GMP-P(CH2)P]. Since GMP-P(NH)P is as effective as GTP in ternary complex formation, it would appear that GTP plays the role of an allosteric effector in this step of eukaryotic polypeptide chain initiation. 4. GDP inhibits both the rate and extent of ternary complex formation. The inhibition is largely reversed by adding a 5-fold molar excess of GTP over GDP. DGDP is slightly less inhibitory than GDP. UDP and CDP are much less inhibitory than GDP and very little inhibition is obtained with ADP. 5. The preformed ternary complex is rapidly and completely destroyed in the presence of N-ethylmaleimide. The results suggest that free--SH groups of the factor may be essential for maintaining the integrity of the ternary complex.
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PMID:Role of GTP in eukaryotic polypeptide-chain initiation. Purification and properties of a factor from Artemia salina embryos which interacts with initiator transfer RNA and guanine nucleotides. 97 64

Initiation factor IF-E2 was isolated from rabbit reticulocytes and purified 120-fold to near homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and phosphocellulose, and, when suitable, by sucrose density gradient centrifugation. The factor is a complex protein containing three nonidentical polypeptides of molecular weight 57,000, 52,000, and 36,000. It behaves as a complex throughout its purification and during polyacrylamide gel electrophoresis in nondenaturing buffer but its thress components are readily separated by electrophoresis in denaturing buffers. None of its components corresponds to any of the polypeptides of the other initiation factors or to any proteins of ribosomes washed in buffers containing a high salf concentration. A stoichiometric ratio of 1:1:1 was determined for the three polypeptides; based on the assumption of one copy each per complex, the calculated factor molecular weight is 145,000, a value in agreement with the measured value of 160,000. Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase also isolated from rabbit reticulocytes. Neither procedure causes a measurable change in the ability of the factor to form a ternary complex with GTP and the initiator methionyl-tRNA. 5'-Guanylyl-methylenediphosphonate may substitute for GTP, but only at relatively high concentrations. The binding of labeled initiation factor IF-E2 and methionyl-tRNA to the 40 S ribosomal subunit was studied by sucrose density gradient centrifugation. Appreciable binding of the factor is seen only when all three components of the ternary complex are included in the reaction mixture. The binding of either the factor or methionyl-tRNA was not stimulated by the addition of globin messenger RNA and initiation factor IF-E3. It was shown that all three polypeptide components of initiation factor IF-E2 are bound to these nascent initiation complexes.
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PMID:Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes. 100 8

Microsomes isolated from the fibroin region of the Bombyx mori silkgland synthesize in the cell-free system a glycin rich polypeptide or polypeptides presumably representing fibroin precursors. Besides microsomes the system requires ATP and ATP-generating system, GTP, soluble protein fraction and tRNA, glycine incorporation is inhibited by puromycin and cycloheximide. It is shown that the synthesis of a polypeptide with high Gly/Lys ratio requires soluble protein fraction isolated from the silk gland at the end of the instar V. When the soluble protein fraction from the larvoe at the early instar V is used the Gly/Lys ratio in the product is markedly lower. These results permit to suggest that fibroin synthesis may be regulated at the level of tRNA aminoacylation.
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PMID:[Properties of the cell-free protein synthesizing system from the fibroin region of the Bombyx mori silkgland]. 105 90

The heme-regulated translational inhibitor (HRI) has been purified 4800-fold. On electrophoresis in sodium dodecyl sulfate/polyacrylamide gel, the purified HRI showed one major polypeptide band. The purified HRI inhibits protein synthesis in lysates containing optimal levels of hemin with inhibition kinetics which parallel those observed in heme-deficiency. Data are presented which are consistent with an enzymatic function of HRI in the inhibition of protein synthesis. The HRI is an adenosine 3':5'-cyclic monophosphate independent protein kinase which phosphorylates the small subunit (38,000) but not the large subunits (52,000 and 50,000) of the initiation factor which forms a ternary complex with Met-tRNAf and GTP. This evidence supports the hypothesis that inhibition of protein synthesis by HRI involves the phosphorylation of the initiation factor. These findings are discussed in relation to various models for the regulation of protein kinase activity by heme. (see article).
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PMID:Regulation of protein synthesis in rabbit reticulocyte lysates: purification and initial characterization of the cyclic 3':5'-AMP independent protein kinase of the heme-regulated translational inhibitor. 106 87

The interaction of protein synthesis elongation factor 1 (EF-1) from wheat embryos and elongation factor Tu from Escherichia coli with cytidylyl(5'-3')guanosine 5'-triphosphate(pppGpC) has been studied. The dinucleotide 5'-triphosphate interacts strongly with EF-1 as evidenced by its capacity to inhibit the binding of [3H]GTP to the factor. The analogs pGpC and GpC do not interfere with GTP binding to EF-1 but guanosine 5'-triphosphate cyclic 2',3'-monophosphate and ppGpC are also potent inhibitors. The binding of the dinucleotide 5'-triphosphate to EF-1 was also demonstrated directly by the nitrocellulose retention method and by Sephadex G-50 fractionation using a radioactive analog iodinated with 125I in the 5 position of the cytosine of pppGpC. The dinucleotide triphosphate can replace GTP in the formation of a ternary complex EF-1-aminoacyl-tRNA-GTP and in its requirement for the binding of aminoacyl-tRNA to ribosomes catalyzed by EF-1. The absolute requirement for GTP in an in vitro polypeptide-synthesizing system can also be met by pppGpC and by guanosine 5'-triphosphate cyclic 5',3'-monophosphate. The bacterial factor EF-Tu differs drastically from eukaryotic EF-1 in its nucleotide specificity since EF-Tu only interacts slightly (if at all) with pppGpC. The low inhibition of [3H]GTP binding to EF-Tu by pppGpC could be due to a slight contamination in the latter compound.
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PMID:The activity of oligonucleotides containing guanosine 5'-triphosphate in protein synthesis. I. The interaction of protein synthesis elongation factor I with cytidylyl (5'-3')-guanosine 5'-triphosphate. 109 Jun 16

Fusidic acid inhibits polypeptide chain elongation by binding to the ribosome - elongation factor-G - GDP complex and thereby preventing its dissociation. The experiments reported here quantitate the interaction of the antibiotic [3H]-24,25-dihydrofusidic acid, an active analog of fusidic acid, with the ribosome - elongation factor-G - GDP comples. All components of the complex are essential for [3H]-24,25-dihydrofusidic acid binding. The stoichiometry of the interaction is ca. 1:1, and the Ka apparent, as determined by equilibrium dialysis, is 2.6 times 10-6 M-minus 1. It is further shown that GTP and GDP are equally effective in forming complexes to which the antibiotic may bind, whereas GMP and beta,gamma-methyleneguanosine triphosphate will not form complexes to which the antibiotic may bind. In order to examine the structural basis of the mode of antibiotic action shown by fusidic acid, we have considered two activities of 21 structural analogs of this antibiotic: ability to bind to the aforementioned ternary complex and ability to stabilize this complex. The comparative binding capability of the analogs were extablished through competition experiments with [3H]-24,25-dihydrofusidic acid. The data obtained from these experiments can be summarized as follows. (1) The C17-20 double bond of fusidic acid appears to be critical for both binding and complex stabilization activities. (2) A carboxyl group in the vicinity of the C20 carbon is also essential for both activities. (3) Modifications of other functional groups in the molecule can lead to significantly decreased stabilization of the ternary ribosome complex and/or ability to compete with [3H]-24,25-dihydrofusidic acid for binding to the complex, but do not demonstrate absolute structural requirements for either activity.
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PMID:Some characteristics of and structural requirements for the interaction of 24,25-dihydrofusidic acid with ribosome - elongation factor g Complexes. 109 41

Five enzyme complexes, which are concerned with electron transport and oxidative phosphorylation, have been isolated from beef heart mitochondria. Enzyme complexes I, II, III and IV are the electron transfer complexes discovered in 1961. Complex V is an energy-conserving complex. It catalyzes ATP-Pi exchange and ATP hydrolysis. The exchange reaction is sensitive to uncouplers, rutamycin, valinomycin plus K-+, dicyclorexylcarboditmide, arsenate, azide, and adenylyl imidodiphosphate. It is also specific for ATP; ITP, GTP and UTP are essentially ineffective. Studies with the photoaffinity labeling uncoupler, 2-azido-4-nitrophenol (NPA), have shown that the mitochondrial uncoupler-binding sites are located exclusively in complex V. Complexes I, III and IV, which carry the three coupling sites of the respiratory chain, had negligible capacity for the binding of NPA, whereas the uncoupler-binding capacity of complex V appeared to be increased two- to threefold as compared to mitochondria. Complexes I, II, III, IV and V are obtained from the same batch of mitochondria by a simple fractionation procedure, which employs cholate, deoxycholate, ammonium acetate and ammonium sulfate. Studies with NPA have shown that mitochondria contain per milligram protein about 0.6 nmole of uniformly reacting uncoupler binding site. All of the uncouplers tested appeared to interact competitively with this site. Photoaffinity labeling with tritiated NPA has shown that a major portion of NPA binds to a polypeptide of molecular weight between 26,000 and 30,000. Other studies on the mechanism of uncoupling have shown that picrate is a membrane-impermeable uncoupler. It cannot uncouple mitochondria. However, it is an effective uncoupler of ATP synthesis and ATP-induced transhydrogenation or reverse electron transfer when used in conjunction with sonicated submitochondrial particles, which have an inside-out orientation of the inner membrane with respect to the medium. In these particles, picrate binds to the same uncoupler-binding site as NPA and other uncouplers. However, unlike the membrane-permeable uncouplers, picrate is a poor protonophore. It has a very small effect on the proton permeability of phosphorylating submitochondrial vesicles, even at two to three times the concentration needed for complete uncoupling. The increase in the proton permeability of submitochondrial vesicles caused by such high concentrations of picrate (500 mum) can be achieved with approximately 5 mum 2,4-dinitrophenol. At this concentration, dinitrophenol results in only about 20% uncoupling.
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PMID:Mitochondrial ATP-Pi exchange complex and the site of uncoupling of oxidative phosphorylation. 109 89


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