Isolation of inosine-5'-monophosphate from fish muscles

Conditions for transformation of tissue adenosine-5'-monophosphate (AMP) into inosine-5'-monophosphate (IMP) with the aid of endogenic AMP-aminohydrolase are developed resting on the studied properties of AMP-aminohydrolase (EC 3.5.4.6) from saltwater fish muscles (one of the enzymes participating in the nucleotide metabolism). Sorption of the nucleotide is performed on the activated charcoals A gamma-3 A gamma-5 which eluate IMP from acid solutions. It reduces the process of isolation, permits application of the acid wash solutions to remove salts; the alkaline ethyl alcohol-aid elution at the subsequent stages accelerates the process of nucleotide concentration by means of vacuum evaporation. The suggested approaches allow developing a simple method of IMP production from fish tissues which diminishes the cost of preparation.     

Antioxidative and radiation modulating properties of guanosine-5'-monophosphate

Employing enhanced chemiluminescence in luminol-p-iodophenol peroxidase system and coumarine-3-carboxylic acid, it was shown that guanosine-5'-monophosphate (GMP) appreciably reduces formation of H?O? and hydroxyl radicals induced by x-ray irradiation. Using immunoenzyme assay, we revealed that GMP lowered 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) formation in DNA in vitro after irradiation. The results of survival test have shown that mice being injected intraperitoneally with GMP after irradiation with a dose of 7 Gy had better survival rate than the control mice. GMP reduced leucopoenia and thrombocytopenia in irradiated mice. Obtained results give premises that GMP may be promising therapeutic agent for treatment of radiation injuries.     

Characteristics and crystal structure of bacterial inosine-5'-monophosphate dehydrogenase

IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the first step unique to GTP synthesis. To provide a basis for the evaluation of IMPDH inhibitors as antimicrobial agents, we have expressed and characterized IMPDH from the pathogenic bacterium Streptococcus pyogenes. Our results show that the biochemical and kinetic characteristics of S. pyogenes IMPDH are similar to other bacterial IMPDH enzymes. However, the lack of sensitivity to mycophenolic acid and the Km for NAD (1180 microM) exemplify some of the differences between the bacterial and mammalian IMPDH enzymes, making it an attractive target for antimicrobial agents. To evaluate the basis for these differences, we determined the crystal structure of the bacterial enzyme at 1.9 A with substrate bound in the catalytic site. The structure was determined using selenomethionine-substituted protein and multiwavelength anomalous (MAD) analysis of data obtained with synchrotron radiation from the undulator beamline (19ID) of the Structural Biology Center at Argonne's Advanced Photon Source. S. pyogenes IMPDH is a tetramer with its four subunits related by a crystallographic 4-fold axis. The protein is composed of two domains: a TIM barrel domain that embodies the catalytic framework and a cystathione beta-synthase (CBS) dimer domain of so far unknown function. Using information provided by sequence alignments and the crystal structure, we prepared several site-specific mutants to examine the role of various active site regions in catalysis. These variants implicate the active site flap as an essential catalytic element and indicate there are significant differences in the catalytic environment of bacterial and mammalian IMPDH enzymes. Comparison of the structure of bacterial IMPDH with the known partial structures from eukaryotic organisms will provide an explanation of their distinct properties and contribute to the design of specific bacterial IMPDH inhibitors.     

A kinetic alignment of orthologous inosine-5'-monophosphate dehydrogenases

IMP dehydrogenase (IMPDH) catalyzes two very different chemical transformations, a dehydrogenase reaction and a hydrolysis reaction. The enzyme toggles between the open conformation required for the dehydrogenase reaction and the closed conformation of the hydrolase reaction by moving a mobile flap into the NAD site. Despite these multiple functional constraints, the residues of the flap and NAD site are highly diverged, and the equilibrium between open and closed conformations ( K c ) varies widely. In order to understand how differences in the dynamic properties of the flap influence the catalytic cycle, we have delineated the kinetic mechanism of IMPDH from the pathogenic protozoan parasite Cryptosporidium parvum ( CpIMPDH), which was obtained from a bacterial source through horizontal gene transfer, and its host counterpart, human IMPDH type 2 (hIMPDH2). Interestingly, the intrinsic binding energy of NAD (+) differentially distributes across the dinucleotide binding sites of these two enzymes as well as in the previously characterized IMPDH from Tritrichomonas foetus ( TfIMPDH). Both the dehydrogenase and hydrolase reactions display significant differences in the host and parasite enzymes, in keeping with the phylogenetic and structural divergence of their active sites. Despite large differences in K c , the catalytic power of both the dehydrogenase and hydrolase conformations are similar in CpIMPDH and TfIMPDH. This observation suggests that the closure of the flap simply sets the stage for catalysis rather than plays a more active role in the chemical transformation. This work provides the essential mechanistic framework for drug discovery.     

Cloning and sequence analysis of the human and Chinese hamster inosine-5'-monophosphate dehydrogenase cDNAs

Inosine-5'-monophosphate dehydrogenase, a key enzyme in the regulation of guanine nucleotide biosynthesis, was purified to homogeneity; and a polyclonal antibody directed against the purified protein was used to isolate human and Chinese hamster IMP dehydrogenase cDNA clones. These clones were sequenced and found to contain an open reading frame of a protein containing 514 amino acids. A sequence of 35 amino acids obtained by analysis of the purified protein is identical to a segment of the protein sequence deduced from the IMP dehydrogenase cDNA. The molecular mass of the deduced protein is 56 kDa, which is the observed molecular mass of the purified protein and of the immunoprecipitated in vitro translation product. Comparison of the protein sequences deduced from the human and Chinese hamster cDNA clones indicates only eight amino acid differences, suggesting that IMP dehydrogenase is a highly conserved protein.     

Positional isotope exchange and kinetic experiments with Escherichia coli guanosine-5'-monophosphate synthetase

The kinetic mechanism of Escherichia coli guanosine-5'-monophosphate synthetase has been determined by utilizing initial velocity kinetic patterns and positional isotope exchange experiments. The initial velocity patterns of MgATP, XMP, and either NH3 or glutamine (as nitrogen source) were consistent with the ordered addition of MgATP followed by XMP and then NH3. The enzyme catalyzes the exchange of 18O from the beta-nonbridge positions of [beta,beta,beta gamma,gamma,gamma,gamma-18O6]ATP into the alpha beta-bridge position only in the presence of XMP and Mg2+. The exchange reaction did not require NH3. The isotope exchange reaction increased as the XMP concentration increased and then decreased at saturating levels of XMP. These results also support the ordered addition of MgATP followed by XMP. GMP synthetase catalyzes the hydrolysis of ATP to AMP and PPi along with an ATP/PPi exchange reaction in the absence of NH3. These data taken together support a mechanism in which the initial step in the enzymatic reaction involves formation of an adenyl-XMP intermediate. Psicofuranine, an irreversible inhibitor of the enzyme, acts by preventing the release or further reaction of adenyl-XMP with H2O or NH3 but does not suppress the isotope exchange or ATP/PPi exchange reactions. GMP synthetase has also been shown to require a free divalent cation for full activity. When Ca2+ replaces Mg2+ in the reaction, the positional isotope exchange reaction is enhanced but the reaction with NH3 to form GMP is greatly suppressed.     

Cyclic guanosine-3',5'-monophosphate and biopteridine biosynthesis in Nocardia sp

Nocardia sp. strain NRRL 5646 contains a nitric oxide synthase (NOS) enzyme system capable of generating nitric oxide (NO) from arginine and arginine-containing peptides. To explain possible roles of the NOS system in this bacterium, guanylate cyclase (GC) and tetrahydrobiopterin (H(4)B) biosynthetic enzymes were identified in cell extracts and in culture media. Cell extracts contained GC activity, as measured by the conversion of GTP to cyclic guanosine-3',5'-monophosphate (cGMP) at 9.56 pmol of cGMP h(-1) mg of protein(-1). Concentrations of extracellular cGMP in culture media were significantly increased, from average control levels of 45 pmol cGMP liter(-1) to a maximum of 315 pmol liter(-1), in response to additions of GTP, L-arginine, H(4)B, and sodium nitroprusside to growing Nocardia cultures. On the other hand, the NOS inhibitor N(G)-nitro-L-arginine and the GC inhibitor 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one both dramatically decreased extracellular cGMP levels. Activities for GTP-cyclohydrase-1, 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, enzymes essential for H(4)B biosynthesis, were present in Nocardia culture extracts at 77.5 pmol of neopterin and 45.8 pmol of biopterin h(-1) mg of protein(-1), respectively. In Nocardia spp., as in mammals, GTP is a key intermediate in H(4)B biosynthesis, and GTP is converted to cGMP by a GC enzyme system that is activated by NO.     

Effects of dopaminergic agonists and antagonists on cerebellar guanosine-3',5'-monophosphate (cGMP).

Apomorphine was found to cause an increase in cerebellar cGMP content. Bromocriptine, at a dose that caused stereotypies, neither elevated cGMP, nor blocked the apomorphine- induced rise in cGMP. The apomorphine-induced rise in cGMP was effectively blocked by haloperidol and some other neuroleptics, but not by sulpiride. These actions of the neuroleptics correlated with their ability to displace ³H-spiroperidol from striatal membranes, suggesting that dopamine receptor interactions were important in the cGMP changes noted. Based on the observation that haloperidol antagonized the increase induced by restraint, it is suggested that dopaminergic systems are involved in the reaction to stress.     

Uptake of cyclic guanosine-3',5'-monophosphate and its metabolism in 3T6 cells

Uptake of 3H-cGMP by cultured murine 3T6 cells was studied. The cells were shown to contain radioactivity 1 minute after its addition, with the level of radioactivity increasing during a 3 hour incubation period. By this time, the intracellular non-metabolized cGMP corresponded to 1-5% of the whole intracellular radioactivity. In the presence of theophylline the uptake of 3H-cGMP by cells was seen decreasing, however, the portion of non-metabolized cGMP reached 45-50% of the whole intracellular radioactivity. Thus, the presence of theophylline made it possible to maintain the high level of intracellular cGMP. It is concluded that the incubation of cell cultures in the medium with cGMP may be useful for achieving an elevating intracellular cGMP concentration and for studying the biological effect of cyclic nucleotide.     

Independence of the insulin effect from the guanosine-3',5'-monophosphate level in muscle tissue

The role of cyclic GMP in the insulin effect was investigated using isolated frog sartorii. A study was made of the effect of exogenous cyclic GMP, dibutyryl cyclic GMP, 8-bromo-cyclic GMP on xylose transport, glycogen synthesis and muscle respiration. Only dibutyryl cyclic GMP (1.10(-6) - 10(-4) M) alone was observed to have a stimulating effect on glycogen synthesis and respiration. The xylose transport was but slightly accelerated only following a 20 hours incubation of muscles in the cyclic GMP solution. Cyclic GMP was shown to penetrate the muscle fibres. The cyclic GMP content in muscles was equal to 22.7 +/- 2.0 pM per gram of wet weight. Insulin exerted no effect on cyclic GMP concentration in muscles. The data obtained do not allow to conclude that cyclic GMP may serve as a mediator in realization of the insulin effect on membrane and intracellular processes.     

Partial purification and characterization of adenosine- and guanosine-3',5'-monophosphate phosphodiesterases from human lung tissue.

Crude extracts of human lung tissue were examined for cyclic adenosine- and guanosine-3',5'-monophosphate (cAMP and cGMP) phosphodiesterase activities. Nonlinear reciprocal plots were observed for each substrate. DEAE-Sephadex chromatography of the extracts revealed four main fractions of activity, which were further purified by Sephadex gel filtration. The phosphodiesterase activity of the resulting individual fractions was partially characterized with respect to substrate specificity, kinetic parameters, apparent molecular weight (gel filtration), thermal stability at 30 and 37 degrees C, effect of the cyclic nucleotide not utilized as substrate, and the possible influence of Ca2+-dependent protein activator. The results indicate that the tissue contains phosphodiesterases with strict specificity and a high apparent affinity for each of the two cyclic nucleotides (the Km values determined were approximately 0.3-0.4 muM). The high affinity cAMP phosphodiesterase activity was enriched in two of the purified fractions; both activities probably represent fragments of the native high affinity cAMP specific enzyme. A third purified phosphodiesterase showed mixed substrate specificity. The Km value recorded for hydrolysis of either substrate with this enzyme was approximately 25 muM. A fourth, irregularly occurring, phosphodiesterase activity also showed mixed substrate specificity. The Km value registered for hydrolysis of either substrate with this fraction was approximately 0.4 muM. There was no evidence for a Ca2+-dependent specific activation by a boiled lung tissue supernatant of any of the purified enzymes.     

The effect of drugs which alter GABA-ergic function on cerebellar guanosine-3',5'-monophosphate content.

The actions of several classes of drugs, thought to be involved with gamma-amino-butyric acid (GABA) mechanisms, have been examined for effects on cerebellar cGMP content. Picrotoxin and TRH increased, while ethanol and diazepam decreased, cerebellar cGMP. Doses of gamma-hydroxybutyrate (GHB) and baclofen caused no significant effect at doses that caused behavioral changes. These cGMP actions were contrasted with those induced by the dopaminergic agents, apomorphine and haloperidol, which respectively, raised and lowered cerebellar cGMP. Apomorphine-induced increases in cGMP were blocked by haloperidol, but not by GHB or baclofen given eight min before sacrifice. However, baclofen given one hour before sacrifice caused effects similar to those of haloperidol. These results are discussed in terms of dopaminergic-GABAergic interactions.     

Sterically hindered cisplatin derivatives with multiple carboxylate auxiliary arms: synthesis and reactions with guanosine-5'-monophosphate and plasmid DNA.

Two novel sterically hindered cisplatin derivatives with the ligand L=NH(2)C(CH(2)CH(2)COOH)(3) were prepared: cis-PtCl(2)L(2) and cis-PtCl(2)L(NH(3)). The starting compound for the syntheses was NH(2)C(CH(2)CH(2)COOtBu)(3), also known as a building block for dendrimers. cis-PtCl(2)L(2) was prepared from K(2)PtCl(4) in an unusual two-phase reaction in water-chloroform, followed by deprotection of the tert-butyl protective groups with formic acid to yield a water-soluble complex. The mixed-ligand compound cis-PtCl(2)L(NH(3)) was prepared from [PPh(4)][PtCl(3)(NH(3))] in methanol, with subsequent deprotection in formic acid. DNA-binding properties of the two compounds were investigated using the model base guanosine-5'-monophosphate (5'-GMP) and pBR322 plasmid DNA. While cisplatin [cis-PtCl(2)(NH(3))(2)] induced an unwinding of 12 degrees in pBR322 plasmid DNA, cis-PtCl(2)L(NH(3)) induced only 3 degrees unwinding, which is indicative of a monofunctional binding mode. Remarkably, cis-PtCl(2)L(2) did not induce any distortion in plasmid DNA, which strongly suggests that the compound does not bind to DNA. Test reactions with 5'-GMP, monitored by 1H and 195Pt NMR, confirmed that cis-PtCl(2)L(2) is unable to bind to DNA, whereas cis-PtCl(2)L(NH(3)) binds only one nucleotide. Apparently, binding of platinum to nucleotides at the coordination site cis with respect to the ligand L is prevented by steric crowding. Thus, cis-PtCl(2)L(NH(3)) must bind DNA monofunctionally at the trans position. Besides, both compounds have a chloride replaced by one of the carboxylate arms, forming a a seven-membered chelate ring. In theory, cis-PtCl(2)L(2) could also form a second chelate ring, but this was not observed.