Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3',5'-bisphosphate. The bisphosphate was labeled in the 5'-position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [gamma-32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3'-phosphatase activity (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3',5'-[5'-32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3'-terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross-linked to the Escherichia coli ribosome.     

Microspectrofluorometric characterization of the fluorescent derivatives of biogenic amines produced by aqueous aldehyde (Faglu) fixation

Summary The fluorescent derivatives of the reaction between an aqueous aldehyde (Faglu) solution and the biogenic amines (5-hydroxytryptamine, dopamine and noradrenaline) have been examined in order to determine the conditions required for maximal fluorescence yield. The fluorescence intensity and spectra of the final reaction products have been characterized and found to be highly dependent on the pH of the reaction mixture. Fluorophores derived from catecholamines have maximal yield and are most easily characterized when the reaction is performed at pH 7.3, whilst those derived from 5-hydroxytryptamine have maximal yield and are most readily characterized when the reaction is performed at pH 10.0. The addition of potassium ferricyanide to the Faglu further enhances the fluorescence yield of 5-hydroxytryptamine-containing models and tissues at both pH 10.0 and pH 7.3. Using the modified Faglu reaction mixture, it has been possible to demonstrate 5-hydroxytryptamine in the central nervous system without the need for pharmacological manipulation.     

Immunochemical study on the pathway of electron flow in reduced nicotinamide adenine dinucleotide-dependent microsomal lipid peroxidation.

NADH could support the lipid peroxidation of rat liver microsomes in the presence of ferric ions chelated by ADP(ADP-Fe). The reaction had a broad pH optimum (pH 5.8--7.4) and was more active in the acidic pH range. Antibodies to NADH-cytochrome b5 reductase [EC 1.6.2.2] and cytochrome b5 inhibited NADH-dependent lipid peroxidation in the presence of ADP-Fe, whereas the antibody against NADPH-cytochrome c reductase [EC 1.6.2.4] showed no inhibition. These oberservations suggest that the electron from NADH was supplied to the lipid peroxidation reaction via NADH-cytochrome b5 reductase and cytochrome b5. On the other hand, NADPH-supported lipid peroxidation was strongly inhibited by the antibody against NADPH-cytochrome c reductase, confirming the participation of this this flavoprotein in the NADPH-dependent reaction. In the presence of both ADP-Fe and ferric ions chelated by EDTA(EDTA-Fe), NADH-dependent lipid peroxidation was highly stimulated up to the level of the NADPH-dependent reaction. In this case, the antibody against cytochrome b5 could not inhibit the reaction, while the antibody against NADH-cytochrome b5 reductase did inhibit it, suggesting the direct transfer of electrons from NADH-cytochrome b5 reductase to EDTA-Fe complex.     

The stereochemical course of the reaction catalyzed by soluble bovine lung guanylate cyclase

The stereochemical course of the reaction catalyzed by the soluble form of bovine lung guanylate cyclase has been investigated using [alpha-18O]guanosine 5'-triphosphate (Rp diastereomer) and guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) as substrates. The product from the 3-thiomorpholino-1',1'-dioxide sydnonimine-stimulated enzymatic cyclization of [alpha-18O] guanosine 5'-triphosphate was esterified with diazomethane. 31P NMR analysis of the triesters indicated that all of the 18O label was present in the axial position. Guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) was cyclized under stimulated and basal enzyme activities and, in both cases, the Rp diastereomer of guanosine 3',5'-cyclic phosphorothioate was formed. This was determined by direct comparison with material synthesized chemically from guanosine 5'-phosphorothioate. The results from these experiments show that the reaction catalyzed by guanylate cyclase proceeds with inversion of configuration at phosphorus and this indicates that the reaction proceeds by way of a single direct displacement reaction.     

One-substrate transketolase-catalyzed reaction

Apart from catalyzing the common two-substrate reaction with ketose as donor substrate and aldose as acceptor substrate, transketolase is also able to catalyze a one-substrate reaction utilizing only ketose (xylulose 5-phosphate) as substrate. The products of this one-substrate reaction were glyceraldehyde 3-phosphate and erythrulose. No free glycolaldehyde (a product of xylulose 5-phosphate splitting in the transketolase reaction) was revealed.     

Complement-induced histamine release from human basophils. II. Mechanism of the histamine release reaction.

The activation of human serum complement by incubation with zymosan generates C5a which releases histamine from autologous basophils. The characteristics of the C5a-induced histamine release were investigated. It is similar to IgE-mediated reactions in requiring Ca++ and in being inhibited by EDTA. However, it has marked differences from IgE-mediated reactions. C5a, at all concentrations, released histamine completely in less than 2 min. The C5a reaction has a narrow pH optimum that antigen-induced release and occurs well at 17 degrees to 37 degreesC but not at 0 degreesC. The optimal reaction temperature is 25 degrees to 30 degrees C. Unlike the antigen-induced release, no two-stage activation with C5a for the release of histamine could be demonstrated. There was additive release between C5a- IgE-mediated reactions. Leukocytes could be desensitized to the C5a-mediated reaction by 1) incubating the cells at 37 degrees C for 45 min, 2) pretreating the leukocytes with activated serum in the presence of EDTA, and 3) adding the activated serum to the leukocytes at 0 degrees C before transferring to the optimal reaction temperatures. Cells desensitized to the complement-induced release have normal reactions to IgE-mediated histamine release. In parallel experiments, cells from allergic donors desensitized for IgE-mediated reactions by incubation with antigen under sub-optimal conditions release histamine normally upon the addition of C5a. The results indicate that histamine release by C5a involves a mechanism of basophil activation that is different from the pathway involved in the IgE-induced reaction.     

ADP-ribosylation of diadenosine 5',5"-P1,P4-tetraphosphate by poly(ADP-ribose) polymerase in vitro

When the effect of diadenosine 5',5"'-P1,P4-tetraphosphate on a purified poly(ADP-ribose) polymerase reaction was examined, the compound strongly inhibited ADP-ribosylation reaction of histone, while the compound was much less inhibitory of the Mg2+-dependent automodification of this enzyme. In an attempt to study the mechanism of the inhibition, we analyzed the total reaction products, which were synthesized from NAD+ in the presence of diadenosine 5',5"'-P1,P4-tetraphosphate in a reaction mixture for ADP-ribosylation of histone, and found that a new, low molecular product was predominantly synthesized instead of ADP-ribosylated histone in the reaction. Approximately 90% of added NAD+ was converted into this low molecular product under an appropriate reaction condition. Further analysis revealed that the product was mono- and oligo(ADP-ribosyl)ated diadenosine nucleotide and that the bound oligo(ADP-ribose) is elongating at one end of the product during the reaction. Thus, the present study clearly demonstrated that diadenosine 5',5"'-P1,P4-tetraphosphate functions as an acceptor for ADP-ribose in a poly(ADP-ribose) polymerase reaction in vitro. The finding that histone H1 is required in the reaction mixture for the synthesis of this new product suggests that histone H1 and the diadenosine compound interact during this modification reaction.     

Reversible stepwise mechanism involving a carbanion intermediate in the elimination of ammonia from L-histidine catalyzed by histidine ammonia-lyase.

L-Histidine labeled with deuterium at the C-5' position of the imidazole ring, L-[5'-2H]histidine (His-5'-D), was used as a probe for investigating a stepwise reversible mechanism via a carbanion intermediate in the elimination of ammonia catalyzed by histidine ammonia-lyase (EC 4.3.1.3). The labeled L-histidine (His-5'-D) (2.45 mM) was incubated with histidine ammonia-lyase (200 units) from Pseudomonas fluorescens at pH 7.0 or 9.0 at 25.0 degrees C for 24 h. The time course of the reaction was examined to determine the rates of enzyme-catalyzed hydrogen exchange at C-5' of L-histidine and urocanic acid. The finding of the enzyme-catalyzed hydrogen exchange at C-5' of both L-histidine and urocanic acid in the presence of L-histidine provided a rational explanation for a stepwise reversible mechanism via a carbanion intermediate in the elimination reaction. The rate of increase in the concentration of urocanic acid exchanged with hydrogen (UA-5'-H) did not depend on the formation rate of urocanic acid and UA-5'-H was continuously formed at a constant rate (25.6 microM/h) even after the completion of urocanic acid formation. These observations suggested the presence of the reversible reaction of urocanic acid and a carbanion intermediate. Since there was only a minor contribution for the formation of UA-5'-H from L-histidine exchanged with solvent hydrogen (His-5'-H), the main pathway in the enzymatic reaction of His-5'-D must be the formation of UA-5'-D via a carbanion intermediate (carbanion-D). Regeneration of the carbanion-D from UA-5'-D by its reverse reaction and subsequent hydrogen incorporation at C-5' would contribute to a large extent for the formation of UA-5'-H. The stability of carbanion was also demonstrated to be approximately three times higher at pH 7.0 than at pH 9.0.     

Several dinucleoside polyphosphates are acceptor substrates in the T4 RNA ligase catalyzed reaction.

Several dinucleoside polyphosphates accept cytidine-3', 5'-bisphosphate from the adenylylated donor 5'-adenylylated cytidine 5',3'-bisphosphate in the T4 RNA ligase catalyzed reaction. The 5'-adenylylated cytidine 5',3'-bisphosphate synthesized in a first step, from ATP and cytidine-3',5'-bisphosphate, is used as a substrate to transfer the cytidine-3',5'-bisphosphate residue to the 3'-OH group(s) of diguanosine tetraphosphate (Gp4G) giving rise to Gp4GpCp and pCpGp4GpCp in a ratio of approximately 10 : 1, respectively. The synthesized Gp4GpCp was characterized by treatment with snake venom phosphodiesterase and alkaline phosphatase and analysis (chromatographic position and UV spectra) of the reaction products by HPLC. The apparent Km values measured for Gp4G and 5'-adenylylated cytidine 5',3'-bisphosphate in this reaction were approximately 4 mM and 0.4 mM, respectively. In the presence of 0.5 mM ATP and 0.5 mM cytidine-3',5'-bisphosphate, the relative efficiencies of the following nucleoside(5')oligophospho(5')nucleosides as acceptors of cytidine-3',5'-bisphosphate from 5'-adenylylated cytidine 5', 3'-bisphosphate are indicated in parentheses: Gp4G (100); Gp5G (101); Ap4G (47); Ap4A (39). Gp2G, Gp3G and Xp4X were not substrates of the reaction. Dinucleotides containing two guanines and at least four inner phosphates were the preferred acceptors of cytidine-3', 5'-bisphosphate at their 3'-OH group(s).     

Rhodium-catalyzed synthesis of 2(5H)-furanones from terminal alkynes and non-substituted alkynes under water-gas shift reaction conditions

Rhodium-catalyzed synthesis of 2(5H)-furanones from alkynes under water-gas shift reaction conditions was studied. By improving the reaction conditions for internal alkynes reported previously, the reaction could be extended to terminal alkynes. Terminal alkynes are selectively converted into 3- and 4-substituted 2(5H)-furanones (2 and 3). When acetylene itself is used, 2(5H)-furanone (2n) is obtained in a good yield. Examination of reaction solutions by IR spectroscopy and some other experimental findings suggest that the active species would be an alkyne-coordinated monomeric rhodium anion. A new reaction path is proposed.     

Resolution of 5-oxo-L-prolinase into a 5-oxo-L-proline-dependent ATPase and a coupling protein

5-Oxo-L-prolinase catalyzes a reaction in which the endergonic cleavage of 5-oxo-L-proline to form L-glutamate is coupled to the exergonic cleavage of ATP to ADP and Pi. In the present research, the enzyme present in a strain of Pseudomonas putida isolated from soil by enrichment culture was found to be composed of two protein components. Neither component alone could catalyze the 5-oxoprolinase reaction, but the reaction was effectively catalyzed when they were mixed. One component (A) exhibited 5-oxo-L-proline-dependent ATPase activity indicating that Component A can interact with both ATP and 5-oxo-L-proline. The other component (coupling protein; B) does not exhibit ATPase activity nor is there evidence that it binds 5-oxo-L-proline. The findings are consistent with (but do not prove) the hypothesis that the Component A catalyzes an initial step in the reaction which involves 5-oxoproline and ATP, such as phosphorylation of 5-oxoproline. The coupling protein (B) may function as a catalyst that converts a phosphorylated form of 5-oxoproline to glutamate, or it might alter the conformation of Component A so as to facilitate the reaction.     

Phosphate-stimulated breakdown of 5′-methylthioadenosine by rat ventral prostate

A soluble enzyme preparation catalysing the release of adenine from 5'-methylthioadenosine was purified some 30-fold from extracts of the rat ventral prostate. This reaction was completely dependent on addition of inorganic phosphate ions to the assay medium. This absolute requirement for phosphate ions suggests a phosphorolytic cleavage mechanism. After acid treatment, the other product of the reaction appeared to be 5-methylthioribose. The actions of some other well-characterized enzymes of nucleoside metabolism of 5'-methylthioadenosine were also investigated; purified purine nucleoside phosphorylases from calf spleen and human erythrocytes did not attack 5'-methylthioadenosine. The role of 5'-methylthioadenosine in mammalian tissues is discussed.     

Evidence for a novel metabolic pathway of (ADP-ribose)n: pyrophosphorolysis of ADP-ribose in HeLa S3 cell nuclei

ADP-ribose liberated from (ADP-ribose)n by the action of (ADP-ribose)n glycohydrolase was converted to ATP and ribose 5-phosphate (ribose 5-P) in the presence of pyrophosphate (PPi) in HeLa S3 cell nuclei. This reaction was reversible and dependent on the simultaneous presence of ADP-ribose, PPi, Mg2+, and nuclei. These results suggest the presence of a novel enzyme in the nuclei, designated as ADP-ribose pyrophosphorylase, which catalyzes the reaction shown in Equation 1. ADP-ribose + PPi in equilibrium ATP + Ribose 5-P (1) This reaction could represent a pathway for the biosynthesis of ATP from (ADP-ribose)n in eukaryotic cell nuclei.     

Different Activities of 5-Hydroxy-dUMP and 5-Hydroxymethyl-dUMP in Thymidylate Synthase-Catalyzed Reaction in View of Molecular Modeling and Structural Studies

In order to explain different activities shown by 5-hydroxy-dUMP (substrate) and its close analogue 5-hydroxymethyl-dUMP (slow-binding inhibitor) in the reaction catalyzed by thymidylate synthase, studies have been undertaken involving (i) ab initio RHF simulations, (ii) comparative analysis of crystallographic structures available from CSD, and (iii) QSAR analysis of experimental results describing thymidylate synthase interaction with various 5-substituted dUMP analogues. Assuming substrate activity of 5-hydroxy-dUMP to be associated with proton release from the C(5) hydroxyl in the enzyme-catalyzed reaction, acidities of 5-hydroxy and 5-hydroxymethyl substituents in dUMP molecule were compared. The results indicate the 5-hydroxyl deprotonation to be easier and supported by resonance electronic effect, pointing to a probable mechanism of different activities of the two dUMP analogues in thymidylate synthase reaction. The possibility is discussed that 5-mercapto-dUMP and 5-hydroseleno-dUMP, previously assumed to be inhibitors, could be also substrates for thymidylate synthase, as the 5-mercaptyl and 5-hydroselenidyl appear to be deprotonated even more easily than the 5-hydroxyl. Copyright 2000 Academic Press.     

Biosynthesis of vitamin B6 in Rhizobium: in vitro synthesis of pyridoxine from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine

Pyridoxine (vitamin B6) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD+, NADP+, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD+ for formation of pyridoxine 5'-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine.     

Structure-function relationship in Escherichia coli initiation factors. Environment of the Cys residue and evidence for a hydrophobic region in initiation factor IF3 by fluorescence and ESR spectroscopy

The reactions of rabbit muscle pyruvate kinase with 5′-p-fluorosulfonylbenzoyl adenosine (5′-FSBA) and 5′-p-fluorosulfonylbenzoyl guanosine (5′-FSBG) from pH 7.0 to 8.0 exhibit biphasic inactivation kinetics. These reactions are characterized by three events: a fast reaction yielding partially active enzyme (with 67% of its original activity for the 5′-FSBA reaction and 45% for the 5′-FSBG reaction) which is reactivated by dithiothreitol, and two slower reactions yielding fully inactive enzymes; the product of only one of the two slower reactions is reactivated by dithiothreitol. These reactions are termed fast dithiothreitol-sensitive, slow dithiothreitol-sensitive, and dithiothreitol-insensitive inactivations. The rates of all three phases of the reactions with 5′-FSBA and 5′-FSBG increase as the pH is raised. The 5′-FSBG reaction can be described in terms of initial reaction with a single ionizable group of pK_a 7.80, 8.60, and 7.94 for the fast dithiothreitol-sensitive, slow dithiothreitol-sensitive, and dithiothreitol-insensitive reactions, respectively; pH-independent rate constants of 0.173, 0.133, and 0.0165 min^?1 are calculated for these three phases of the overall reaction. The pH dependence of the dithiothreitol-insensitive inactivation by 5′-FSBA coincides with that for 5′-FSBG, but the data for the dithiothreitol-sensitive reactions with 5′-FSBA indicate that the reaction in each phase occurs at more than one site over the pH range tested. Differential protection by ligands against inactivation by 5′-FSBA and 5′-FSBG at pH 7.4 and 8.0 indicates that, for the fast dithiothreitol-sensitive reactions, the cysteine residues participating in the two reactions are not identical, although in both cases modification has been attributed to formation of a disulfide. For 5′-FSBA, the partial inactivation appears to result from modification of cysteine residues at the noncatalytic nucleotide site, whereas for 5′-FSBG the inactivation is due to modification within the catalytic metal-nucleotide site. Reaction with 5′-FSBG seems to occur at the same locus for both the fast and slow dithiothreitol-sensitive phases, with the rate difference being ascribable to negative cooperativity among subunits. For the slow dithiothreitol-sensitive inactivation by 5′-FSBA, protection by Mg²⁺ and by Mg²⁺ plus ADP suggests that the targets of modification include the active-site cysteine that is modified by 5′-FSBG. The dithiothreitol-insensitive inactivation, shown to be due to reaction of 5′-FSBA with a tyrosine, may result from reaction of both nucleotide analogs with the same residue, although differential protection by the natural ligands suggests that 5′-FSBA and 5′-FSBG bind to two subsites within the active site.     

Specific reactions between purified HIV-1 particles and CD4+ cell membrane fragments in a cell-free system of virus fusion or entry

The initial step of human immunodeficiency virus type 1 (HIV-1) infection has been studied by Env-mediated fusion or entry assays with appropriate cells expressing CD4 or CXCR4/CCR5 receptors in cultures, where many factors underlying cellular activities likely regulate the fusion/entry efficiency. Here we attempted to develop a more simplified in vitro cell-free fusion/entry reaction that mimics HIV-1 infection in cultures. Membrane fragments of target cells and intact infectious HIV-1 particles were purified, mixed and incubated. The core p24 protein was released from the purified virions and detected by ELISA without detergents in the supernatant of the reaction mixtures. This release reaction proceeded temperature-dependently and in a dose-dependent manner between the virion and membrane fractions, and was specific for HIV-1 Env and CD4. Env-deleted or VSV-G-pseudotyped HIV-1 released little p24, if any. Pretreatment of the membrane fragments with anti-CD4 antibodies inhibited the p24 induction from both X4-tropic and R5-tropic HIV-1. Furthermore, X4 but not R5 HIV-1 reacted with the membrane prepared from intrinsically CXCR4-positive HeLa-CD4 cells, whereas both viruses reacted with that prepared from CCR5-transduced HeLa-CD4 cells, indicating that this cell-free reaction mimics coreceptor usage of HIV-1 infection. Therefore, a potent entry inhibitor of X4 HIV-1, SDF-1alpha, blocked the release from X4 but not R5 HIV-1. Inversely, a weak entry inhibitor of R5 HIV-1, MIP-1beta, partially affected only the release from R5 HIV-1. These results suggest that this cell-free reaction system provides a useful tool to study biochemical fusion/entry mechanisms of HIV-1 and its inhibitors.     

Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners

A fungal cytochrome P450 monooxygenase (CYP5150A2) from the white-rot basidiomycete Phanerochaete chrysosporium was heterologously expressed in Escherichia coli and purified as an active form. The purified CYP5150A2 was capable of hydroxylating 4-propylbenzoic acid (PBA) with NADPH-dependent cytochrome P450 oxidoreductase (CPR) as the single redox partner; the reaction efficiency was improved by the addition of electron transfer protein cytochrome b5 (Cyt-b5). Furthermore, CYP5150A2 exhibited substantial activity with redox partners Cyt-b5 and NADH-dependent Cyt-b5 reductase (CB5R) even in the absence of CPR. These results indicated that a combination of CB5R and Cyt-b5 may be capable of donating both the first and the second electrons required for the monooxygenation reaction. Under reaction conditions in which the redox system was associated with the CB5R-dependent Cyt-b5 reduction system, the exogenous addition of CPR and NADPH had no effect on the PBA hydroxylation rate or on coupling efficiency, indicating that the transfer of the second electron from Cyt-b5 was the rate-limiting step in the monooxygenase system. In addition, the rate of PBA hydroxylation was significantly dependent on Cyt-b5 concentration, exhibiting Michaelis-Menten kinetics. This study provides indubitable evidence that the combination of CB5R and Cyt-b5 is an alternative redox partner facilitating the monooxygenase reaction catalyzed by CYP5150A2.     

Study of chemical stability of antivirally active 5-azacytosine acyclic nucleoside phosphonates using NMR spectroscopy

Hydrolytic decomposition of four 5-azacytosine acyclic nucleoside phosphonates was studied. Products of the decomposition are carbamoylguanidine derivatives. Stability and decomposition products of HPMP-5-azaC (a 5-azacytosine derivative with strong antiviral activity) differ from the other derivatives. The reaction pathway of HPMP-5-azaC involves a formyl derivative formed by intramolecular transformylation reaction.     

Carbamyl phosphate synthetase of Escherichia coli uses the same diastereomer of adenosine-5'-[2-thiotriphosphate] at both ATP sites.

Carbamyl phosphate synthetase from Escherichia coli has been shown to use only the A isomer of adenosine-5'-[2-thiotriphosphate] in both the ATPase reaction (MgATP HCO3- leads to MgADP + Pi) and the carbamyl phosphate synthesis reaction (2MgATP + HCO3- + L-glutamine leads to 2MgADP + Pi + carbamyl-P + L-glutamate). The B isomer was less than 5% as reactive. In the reverse reaction, only the A isomer of adenosine-5'-[2-thiotriphosphate] is synthesized from adenosine-5'-[2-thiodiphosphate] and carbamyl-P as determined by 31P NMR and a coupled enzymatic assay with Cd2+- hexokinase. It is therefore proposed that carbamyl phosphate synthetase uses the same diastereomer of MgATP at both ATP sites.