Phosphoglycerate kinase from yeast synthesizes guanosine 5'-tetraphosphate

Yeast phosphoglycerate kinase (PGK; EC 2.7.2.3) synthesizes adenosine 5'-tetraphosphate (ppppA) from ATP and 1,3-bisphosphoglycerate. Using an HPLC assay, we have shown the synthesis of guanosine 5'-tetraphosphate (ppppG; 0.17 nmol.min-1.PGK unit-1) characterized by its u.v. spectrum, HPLC behavior, and enzymatic digestions. That the synthesis of ppppG is catalyzed by PGK itself and not by a contaminant was shown because it depended on 3-phosphoglycerate (as a source of 1,3-bisphosphoglycerate), coeluted with PGK activity upon gel filtration, and the thermal inactivation of the PGK and the ppppG synthetic activity were parallel.     

Inositol hexaphosphate guanosine diphosphate phosphotransferase from Phaseolus aureus

Inositol hexaphosphate guanosine diphosphate phosphotransferase which transfers phosphate from inositol hexaphosphate to guanosine diphosphate, synthesizing guanosine triphosphate, has been isolated from germinating mung bean. A purification of 86-fold with 33% recovery has been obtained and the protein was made homogeneous after polyacrylamide gel electrophoresis. The MW of this enzyme was ca 92000. The optimal pH was 7·0 and Mn²⁺ was stimulatory. Inositol hexaphosphate was the most active donor of the phosphoryl group (P) to GDP. Inositol penta- or tetra-phosphate (mixed) was partially active, but inositol pentaphosphate produced in this reaction did not act further as phosphate donor. The transfer of P from inositol hexaphosphate was mediated through a phosphoprotein. Polyphosphate (poly Pi), pyrophosphate (PPi) and orthophosphate (Pi) were inactive in this reaction. ADP, CDP and UDP could not substitute for GDP, neither could dGDP nor GMP accept P from inositolphosphate. GTP inhibited the reaction, but ATP did not interfere with the reaction. The products have been shown to be [GMP- ³²P] and inositol pentaphosphate by several criteria. The reaction is practically irreversible. K_m values for GDP and inositol hexaphosphate were 1·1 × 10⁻⁴ M and 1·6 × 10⁻⁶ M respectively.     

Synthesis of guanosine and its derivatives from AICA-riboside

A novel and convenient method for the synthesis of guanosine is described. The reaction of AICA-riboside with sodium methylxanthate gave 2-mercaptoinosine in almost quantitative yield. The latter was oxidized with hydrogen peroxide to afford inosine-2-sulfonic acids, which was readily animated to give guanosine in excellent yield. Similarly, the preparation of N²-methylguanosine and N²,N²-dimethylguanosine, minor constituents of transfer RNA, was also accomplished. Furthermore, this procedure was extended to the synthesis of 2′,3′-O-isopropylideneguanosine and the isopropylidene derivatives of various N²-substituted guanosines from 2′,3′-O-isopropylidene-AICA-riboside. Guanosine via 2′,3′-O-isopropylideneguanosine was successfully phosphorylated to give 5′-guanylic acid.     

Preparation of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) from Escherichia coli ribosomes

A means of preparative enzymatic synthesis of guanosine tetraphosphate (ppGpp), guanosine pentaphosphate (pppGpp), and related derivatives is deseribed. The Escherichia coli ribosomes can be recovered, stored, and used repeatedly as a source of synthetic activity. The procedure described affords a relatively simple means of synthesizing gram amounts of these nucleotides as well as some derivatives such as the β-γ methylenyl derivative of guanosine pentaphosphate (peppGpp).     

The encapsulation and release of guanosine from PEGylated liposomes

The encapsulation and release kinetics of guanosine from liposomes and polyethylene glycol (PEG)-modified liposomes are reported. Specifically, the influence of PEG chain length, PEGylation level, lipid type, drug-loading level, temperature, and solution conditions (i.e., salt and pH effects) on the rate and mechanism for release have been determined. Increasing PEGylation significantly reduced the guanosine release kinetics; this is more significant for greater molecular weight PEG and is correlated with the PEG layer thickness. Further, the mechanism for guanosine release changed from diffusion to interfacial control as the PEG level increased. The interfacial structure introduced by PEG also increased the activation energy required for guanosine transport across the lipid bilayer from 14 to 22 kJmol^?1. Findings from this study provide further insight into optimizing the formulation of Stealth liposomes.     

Conformation of acetylaminofluorene and aminofluorene modified guanosine and guanosine derivatives

Acetylaminofluorene and aminofluorene modified Guo, GMP, d(GpA) and d(ApG) have been studied by circular dichroism and ¹H nuclear magnetic resonance. Aminofluorene modified Guo is preferentially in the $\text{anti}$ conformation and acetylaminofluorene modified Guo in the $\text{syn}$ conformation. It is proposed that the $\text{anti}$ conformation of aminofluorene modified Guo is stabilized by an intra molecular hydrogen bond between the NH group of aminofluorene residue and the 5′-OH group of the sugar. The results on the modified dinucleoside monophosphates are analyzed according to this hypothesis.     

Electrocatalytic oxidation of guanine, guanosine, and guanosine monophosphate

The electrochemical behavior of guanine, guanosine, and guanosine monophosphate (GMP) at redox polymer film modified indium tin oxide electrodes is examined by voltammetry and redox titration. Utilizing the redox polymer-coated electrodes as indicator electrodes, a new method for measuring the oxidation potentials, based on monitoring their catalytic oxidation by different redox polymer coated electrodes at different pH, was proposed in this work. The oxidation potentials of 0.81 V and 1.02 V versus normal hydrogen electrode were determined for guanine and guanosine/GMP under physiological conditions, the lowest oxidation potentials ever reported, to our knowledge.     

Partial purification and properties of guanosine triphosphate cyclohydrolase from Drosophila melanogaster

The first enzyme (named GTP cyclohydrolase) in the pathway for the biosynthesis of pteridines has been partially purified from extracts of late pupae and young adults of Drosophila melanogaster. This enzyme catalyzes the hydrolytic removal from GTP of carbon 8 as formate and the synthesis of 2-amino-4-hydroxy-6-(d-erythro-1,2,3-trihydroxypropyl)-7,8-dihydropteridine triphosphate (dihydroneopterin triphosphate). Some of the properties of the enzyme are as follows: it functions optimally at pH 7.8 and at 42 C; activity is unaffected by KCl and NaCl, but divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺) are inhibitory; the K _m for GTP is 22 m; and the molecular weight is estimated at 345,000 from gel filtration experiments. Of a number of nucleotides tested, only GDP and dGTP were used to any extent as substrate in place of GTP, and these respective compounds were used only 1.8% and 1.5% as well as GTP.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929).     

Penicillin interaction with guanosine

Because of the widespread use of penicillins as antibacterial agents, the question of how penicillin affects the function and structure of nucleic acids becomes of biological importance. This communication reports a nuclear magnetic resonance study which shows that penicillin-G interacts with guanosine in dimethyl sulfoxide and can break the strong guanosine-cytidine pairing by forming a binary hydrogen-bonded complex of penicillin-guanosine. The binding sites in penicillin are the carboxylate and the carbonyl groups, while the NH and NH₂ groups of guanosine act as hydrogen donors.     

Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

Using highly purified calf brain tubulin bearing [8-14C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins (both components containing negligible amounts of nucleoside diphosphate kinase and nonspecific phosphatase activities), we have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly.     

Inactivation of phosphoenolpyruvate carboxykinase by the guanosine nucleotide analogue, 5'-p-fluorosulfonylbenzoyl guanosine

Rat liver cytosolic phosphoenolpyruvate carboxykinase is inactivated by incubation with 0.84 mM 5′-p-fluorosulfonylbenzoyl guanosine, but is not appreciably affected by the adenosine analogue, 5′-p-fluorosulfonylbenzoyl adenosine, in correspondance with the known nucleotide specificity of this enzyme. Marked protection against inactivation by 5′-p-fluorosulfonylbenzoyl guanosine is provided (either in the presence or absence of divalent metal cation) by GTP or GDP but not by ATP or phosphoenolpyruvate. The inactivation appears to be due to covalent reaction since radioactive reagent remains associated with the enzyme after extensive dialysis and gel filtration on Sephadex G-25. These results are consistent with affinity labeling of the nucleotide binding site of phosphoenolpyruvate carboxykinase by the guanosine nucleotide analogue 5′-p-fluorosulfonylbenzoyl guanosine.     

Purification and properties of guanosine triphosphate cyclohydrolase II from Escherichia coli.

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.     

Sequence-dependent effects on DNA stability resulting from guanosine replacements by inosine.

The effect of replacing a G.C base-pair with an I.C base-pair on DNA stability was investigated for a related set of 14-mers. DNA melting analysis of the 14-mers was used to determine delta Hzero, delta Szero and delta G(zero)37 of the double to single stranded transition. All 14mers were shown to have B-DNA character by circular dichroism analysis. 14mers substituted with a single inosine in place of guanosine at different positions showed that consequences on DNA stability are sequence-dependent. Large changes in delta Hzero and delta Szero result when inosine is substituted within the trinucleotide sequence d(TCG).d(CGA) while substitution within d(TCC).d(GGA) causes minor changes in enthalpy and entropy. Moreover, some 14-mers with two inosine substitutions five base-pairs apart showed non-additive free energy changes for the double to single stranded transition. These results clearly indicate that the structural consequences of replacing a single guanosine with an inosine are transmitted over a significant distance.     

Stathmin slows down guanosine diphosphate dissociation from tubulin in a phosphorylation-controlled fashion

Stathmin is an important protein that interacts with tubulin and regulates microtubule dynamics in a phosphorylation-controlled fashion. Here we show that the dissociation of guanosine 5'-diphosphate (GDP) from beta-tubulin is slowed 20-fold in the (tubulin)(2)-stathmin ternary complex (T(2)S). The kinetics of GDP or guanosine 5'-triphosphate (GTP) dissociation from tubulin have been monitored by the change in tryptophan fluorescence of tubulin upon exchanging 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-diphosphate (S6-GDP) for tubulin-bound guanine nucleotide. At molar ratios of stathmin to tubulin lower than 0.5, biphasic kinetics were observed, indicating that the dynamics of the complex is extremely slow, consistent with its high stability. The method was used to characterize the effects of phosphorylation of stathmin on its interaction with tubulin. The serine-to-glutamate substitution of all four phosphorylatable serines of stathmin (4E-stathmin) weakens the stability of the T(2)S complex by about 2 orders of magnitude. The phosphorylation of serines 16 and 63 in stathmin has a more severe effect and weakens the stability of T(2)S 10(4)-fold. The rate of GDP dissociation is lowered only 7-fold and 4-fold in the complexes of tubulin with 4E-stathmin and diphosphostathmin, respectively. Sedimentation velocity studies support the conclusions of nucleotide exchange data and show that the T(2)S complexes formed between tubulin and 4E-stathmin or diphosphostathmin are less compact than the highly stable T(2)S complex. The correlation between the effect of phosphorylation of stathmin on the stability of T(2)S complex measured in vitro and on the function of stathmin in vivo is discussed.