5′-Nucleotidase from Yeast,Having Special Affinity for 5′-AMP

Three kinds of diketopiperazines which have retarditive activity for the growth of plant seedlings and plant roots at concentrations ranging from 1 : 2,500 to 1 : 100,000, were isolated from the neutral fraction by extracting the cultured broth of Rosellinia necatrix. These three diketopiperazines have been proved to be l-prolyl-l-leucine anhydride, l-prolyl-l-valine anhydride and l-prolyl-l-phenylalanine anhydride respectively, and the last one seems to be a new diketo-piperazine.

Furthermore, a crystalline wax having m.p. 52°C, a physiologically inactive substance, was also isolated from the same neutral fraction and presumed to be the saturated hydrocarbon of n-pentacosane C₂₅H₅₂.     

Acyl Coenzyme A Synthetase from Pseudomonas fragi Catalyzes the Synthesis of Adenosine 5′-Polyphosphates and Dinucleoside Polyphosphates

Acyl coenzyme A (CoA) synthetase (EC 6.2.1.8) from Pseudomonas fragi catalyzes the synthesis of adenosine 5′-tetraphosphate (p₄A) and adenosine 5′-pentaphosphate (p₅A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5′-O-[γ-thiotriphosphate] (ATPγS), adenosine(5′)tetraphospho(5′)adenosine (Ap₄A), and adenosine(5′)pentaphospho(5′)adenosine (Ap₅A) are also substrates of the reaction yielding p₄(d)A in the presence of tripolyphosphate (P₃). UTP, CTP, and AMP are not substrates of the reaction. The K_m values for ATP and P₃ are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl₂ or CoCl₂ equimolecular with the sum of ATP and P₃. The relative rates of synthesis of p₄A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p₄A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p₄A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap₄A (from ATP), Ap₅A (from p₄A), and adenosine(5′)tetraphospho(5′)nucleoside (Ap₄N) from adequate adenylyl donors (ATP, ATPγS, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates).Dinucleoside polyphosphates have been detected in a wide variety of eukaryotic and prokaryotic organisms (13). In higher organisms, their concentrations are generally on the order of 0.01 to 1 μM. Human blood platelets and chromaffin cells of bovine adrenal medulla contain diadenosine polyphosphates located in the dense bodies (10, 26, 35) and chromaffin granules (32, 38), respectively, where they may reach higher local concentrations. The occurrence of dinucleoside polyphosphates has been described for lower eukaryotic (Saccharomyces cerevisiae, Dictyostelium discoideum, and Physarum polycephalum) and for prokaryotic (Salmonella typhimurium, Escherichia coli, and Clostridium acetobutylicum) organisms (13).Dinucleoside tetraphosphates participate in the control of purine nucleotide metabolism (36), where Ap₄A is an activator of both the IMP-GMP-specific cytosolic 5′-nucleotidase (EC 3.1.3.5) and AMP deaminase (EC 3.5.4.6) (K_a, micromolar range) and Gp₄G is an activator of GMP reductase (EC 1.6.6.8) (K_a, nanomolar range) (36). As the concentration of dinucleoside polyphosphates increases under unfavorable environmental conditions, they have been implicated in the cellular response to stress (31). A role of Ap₄A in DNA synthesis has been proposed elsewhere (14). Dinucleoside polyphosphates are also transition state analogs of some kinases (37). More recently, the dinucleoside triphosphatase activity of a putative tumor suppressor gene product has been described (3).The nucleoside 5′-polyphosphates (p_nN) are another family of related compounds, p₄A has been detected in rabbit and horse muscle (41), rat liver (44), S. cerevisiae spores (19), and chromaffin granules (38). As p₄A is a very strong inhibitor (K_i, nanomolar range) of asymmetrical dinucleoside tetraphosphatase (EC 3.6.1.17) (22), changes in the level of p₄A could affect the concentration and physiological roles of Ap₄A. Other enzymes known to be inhibited (K_i, micromolar range) by p₄N are guanylate cyclase (EC 4.6.1.2) (p₄A and p₄G) (18) and phosphodiesterase I (EC 3.1.4.1) (p₄G) (9). Effects of p₄A on the tone of the vascular system, mediated by P₂ receptors, have also been described elsewhere (21).The cellular level of dinucleoside polyphosphates results from their rate of degradation and synthesis. The following specific enzymes, implicated in the cleavage of dinucleoside polyphosphates, have been described (see reference 15 for a review): asymmetrical dinucleoside tetraphosphatase (EC 3.6.1.17), symmetrical dinucleoside tetraphosphatase (EC 3.6.1.41), dinucleoside tetraphosphate phosphorylase (EC 2.7.7.53), and dinucleoside triphosphatase (EC 3.6.1.29). In addition, there are other unspecific enzymes able to catalyze the hydrolysis of dinucleoside polyphosphates like E. coli 5′-nucleotidase (34) and phosphodiesterase I (9, 15, 26).This paper deals with the synthesis of (di)nucleoside polyphosphates. It has been known since 1966 that some aminoacyl tRNA synthetases (30, 45) catalyze the synthesis of Ap₄A through reactions 1 and 2: reaction 1 reaction 2 The possibility that other enzymes (mainly synthetases and some transferases) which catalyze the formation of AMP, via nucleotidyl-containing intermediates and by releasing PP_i, could catalyze the synthesis of dinucleoside polyphosphates was later raised (17). Luciferase (EC 1.13.12.7), considered as an oxidoreductase, catalyzes the synthesis of Ap₄A with ATP as substrate and luciferin as an essential activator (27, 40): reaction 3 reaction 4 Acetyl-CoA synthetase (EC 6.2.1.1) from S. cerevisiae also catalyzes the synthesis of p₄A and p₅A, from ATP and P₃ and P₄, respectively (16). In the reactions catalyzed by luciferase and acetyl-CoA synthetase, ATP is a very good substrate for the formation of the E · X-AMP complex (X = the appropriate acyl residue), whereas any NTP (or even P₃) is an acceptor (particularly in the case of luciferase) of the AMP moiety of the complex, provided that it has an intact terminal pyrophosphate (27, 40).Here we show that acyl-CoA synthetase from Pseudomonas fragi catalyzes the synthesis of p₄A, p₅A, Ap₄A, Ap₅A, and a variety of Ap₄Ns. In our view, these findings widen the knowledge of the mechanisms of synthesis of (di)nucleoside polyphosphates in prokaryotes and, by extrapolation, also in eukaryotes.     

An Improved Synthesis of 3′-Keto-5′-Tritylthymidine

Abstract

The title compound is prepared in consistently high yield and purity by molecular sieve catalyzed pyridinium dichromate oxidation of 5′-0-tritylthymidine. Shortcomings of other preparations are described, and properties of the title compound are reported.     

A method for the isolation of segments from the 5′ ends of retrovirus RNA

A method for the isolation of segments of any desired length from the 5′ end of retrovirus RNA has been tested. The method is based on selection of 5′-specific segments by hybridizing suitably fragmented genomic (35 S) RNA to mercurated strong stop cDNA followed by chromatography on sulfhydryl-agarose. The method has been shown to be effective for Akv viral RNA by observing the T1 oligonucleotide fingerprints of a 5′-enriched fraction. This fingerprint pattern is of lower complexity than that of total 35 S RNA, contains oligonucleotide spots that have previously been assigned as 5′ specific by conventional fingerprinting methods, and does not overlap with the pattern from 3′-specific RNA.     

A new method for separating cyclic AMP from 5′-AMP with application to the assay for cyclic AMP phosphodiesterase

A new method for assay of cyclic AMP phosphodiesterase (EC 3.1.4.17) has been developed based on the observation that a mixture of cyclic AMP and AMP can be resolved on a column of florisil (activated magnesium silicate) at pH 7.0. The cyclic nucleotide is retained by the silicate and the AMP which is not adsorbed is virtually quantitatively recovered. The adsorption of cyclic AMP by florisil is greatly influenced by the pH of the buffer but independent of its ionic strength. In the actual assay cyclic[³H]AMP is incubated with the enzyme source in the presence of Mg²⁺ and the reaction is stopped by the addition of CCl₃COOH (0.3 m). The mixture is then neutralized by dilution with 10 vol of 0.5 m sodium phosphate buffer, pH 7.0, and applied on a small (0.4 × 4.0-cm) florisil column equilibrated with the same buffer. The column is eluted with 3 vol of the buffer and the radioactivity of the eluate which contains only [³H]AMP is measured. The use of cyclic[³H]AMP of high specific activity in the assay allows a high degree of sensitivity while the addition of CCl₃COOH instantaneously terminates the reaction allowing for increased precision. The assay compares favorably in simplicity and speed with those currently employed for cyclic AMP phosphodiesterase.     

Application of Oxathiaphospholane Method for the Synthesis of Oligodeoxyribonucleotide 5′-O-Conjugates

Abstract

2-Thiono-1,3,2-oxathiaphospholane derivatives of lipophilic alcohols including borneol, cholesterol, menthol and heptadecanol were synthesized and reacted with support-bound oligodeoxyribonucleotides containing free 5′-hydroxyl groups. The reaction is catalyzed by DBU and leads to oligodeoxyribonucleotide conjugates possessing a lipophilic alcohol residue bound at the 5′-end via a phosphorothioate linkage.     

Production of Nucleic Acid-related Substances by Fermentative Processes: XIX. Accumulation of 5′-Inosinic Acid by a Mutant of Brevibacterium ammoniagenes

The accumulation of 5′-inosinic acid (IMP) by a mutant, KY 13102, induced from Brevibacterium ammoniagenes ATCC 6872 by ultraviolet light irradiation, was examined. Although growth was stimulated by adenine or adenosine, the microorganism showed fair growth in the medium containing amino acids but no adenine. Among six kinds of natural nutrients tested, meat extract and Casamino Acids were suitable for the accumulation of IMP. Manganese ion strongly affected growth, the accumulation of IMP and hypoxanthine, and cell morphology. Among amino acids tested, L-methionine, L-proline, and L-valine stimulated IMP accumulation. In the medium containing 1.0 g of L-proline per liter, 12.8 mg of IMP per ml was accumulated. The mechanism of IMP accumulation by the mutant is discussed.     

5′-AMP hydrolysis by suspensions and homogenates of pancreatic islet cells from normal and cortisone-treated rats

Summary Suspensions of endocrine pancreas cells were prepared by shaking collagenase-isolated rat islets of Langerhans in calcium-free buffer. When incubated with 1.0 mM substrate at pH 7.4, the cells split,P _i from 5-AMP at a rate of 87 nmol/h per g DNA, and from-glycerophosphate at a rate of 25 nmol/h per g DNAK _m for 5 AMP was about 54 M. Adenosine or theophylline inhibited the 5-AMP hydrolysis. Homogenization of the cells increased the activity toward 5-AMP by 23% and that toward-glycerophosphate by 115%. Injecting rats with cortisone had no effect on the 5-AMP hydrolysis by whole cells but significantly increased the activity in cell homogenates; the intracellular activity toward 5-AMP was more than doubled by the cortisone treatment. Staining whole islet cells for 5-AMP-splitting activity resulted in a demarcation of the cell periphery in control rats. Cells from cortisone-treated rats showed heavier deposits of reaction product, and their cell periphery did not stand out as clearly. It is suggested that 5-nucleotidase is largely an ectoenzyme in normal rat islet cells. The cells also contain an as yet unidentified intracellular phosphatase that seems to be solely responsible for the increased hydrolysis of 5-AMP in cortisone-treated rats.     

An Improved Protection-Free One-Pot Chemical Synthesis of 2′-Deoxynucleoside-5′-Triphosphates

A facile, straightforward, reliable, and an efficient method for the gram-scale chemical synthesis of both purine deoxynucleotides such as 2 ′-deoxyguanosine-5 ′-triphosphate (dGTP) and 2 ′-deoxyadenosine-5 ′-triphosphate (dATP) and pyrimidine deoxynucleotides such as 2 ′-deoxycytidine-5 ′-triphosphate (dCTP), thymidine-5 ′-triphosphate (TTP), and 2 ′-deoxyuridine-5 ′-triphosphate (dUTP) starting from the corresponding nucleoside is described. This improved “one-pot, three step” Ludwig synthetic strategy involves the monophosphorylation of nucleoside followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65%–70%).     

Production of Nucleic Acid-Related Substances by Fermentative Processes. XXVIII. Accumulation of 5′ Inosinic Acid by a Manganese-Insensitive Mutant of Brevibacterium ammoniagenes

A manganese-insensitive mutant, KY 13105, of Brevibacterium ammoniagenes which accumulates considerable amounts of 5' inosinic acid (IMP) in the presence of 100 to 1,000 mug of Mn(2+) per liter was obtained from an IMP-producing mutant of a manganese-sensitive strain, KY 13102. The effects of Mn(2+) at 0 to 30 mug/liter on IMP accumulation by KY 13105 were similar to those by KY 13102. However, the accumulation of IMP by KY 13105 was not affected by 100 to 1,000 mug of Mn(2+) per liter, showing a clear difference from KY 13102. The accumulation of IMP by KY 13105 was always accompanied by cellular morphological changes irrespective of Mn(2+) concentration. In the presence of Mn(2+), factors which affect IMP accumulation by KY 13105 were examined. Most of the nutrients tested stimulated IMP accumulation at a relatively low concentration (2 g/liter). Iron, calcium, and zinc were found to be essential for IMP accumulation and were independent of Mn(2+). Biotin regulated the growth but not the accumulation of IMP. Under limited or surplus amounts of Mn(2+), the dynamics of IMP fermentation were followed. Under both conditions, the fermentations proceeded in a similar way. The morphological changes were found to be closely related to IMP accumulation.     

A Simple Method for Preparation of 18α-Glycyrrhetinic Acid and Its Derivatives

Treatment of 18-glycyrrhizic acid with a methanolic solution of HCl resulted in 1 : 1 mixture of methyl esters of 18- and 18-glycyrrhetinic acids. Benzoylation of the mixture led to methyl esters of 3-benzoyl-18-glycyrrhetinic acid and 3-benzoyl-18-glycyrrhetinic acid, which were separated by chromatography on silica gel. 18-Glycyrrhetinic acid was prepared by alkaline hydrolysis of methyl 3-benzoyl-18-glycyrrhetinate and was further used for the syntheses of 3-keto-18-glycyrrhetinic acid and methyl esters of 18-glycyrrhetinic acid and 3-keto-18-glycyrrhetinic acid.     

VICMpred： An SVM-based Method for the Prediction of Functional Proteins of Gram-negative Bacteria Using Amino Acid Patterns and Composition

In this study, an attempt has been made to predict the major functions of gramnegative bacterial proteins from their amino acid sequences. The dataset used for training and testing consists of 670 non-redundant gram-negative bacterial proteins （255 of cellular process, 60 of information molecules, 285 of metabolism, and 70 of virulence factors）. First we developed an SVM-based method using amino acid and dipeptide composition and achieved the overall accuracy of 52.39% and 47.01%, respectively. We introduced a new concept for the classification of proteins based on tetrapeptides, in which we identified the unique tetrapeptides significantly found in a class of proteins. These tetrapeptides were used as the input feature for predicting the function of a protein and achieved the overall accuracy of 68.66%. We also developed a hybrid method in which the tetrapeptide information was used with amino acid composition and achieved the overall accuracy of 70.75%. A five-fold cross validation was used to evaluate the performance of these methods. The web server VICMpred has been developed for predicting the function of gram-negative bacterial proteins （http：//www.imtech.res.in/raghava/vicmpred/）.     

A Novel Method for Simultaneous Production of Two Ribosome-Inactivating Proteins, α-MMC and MAP30, from Momordica charantia L

Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) from Momordica charantia L. have been confirmed to possess anti-tumor and anti-virus activities. Traditional purification methods of these two ribosome-inactivating proteins (RIPs) were separate which was time consuming and cost effective as well as low efficient. In order to obtain sufficient samples for researches, a strategy combining ion-exchange and gel filtration chromatography was developed and optimized in this study. Using this novel purification method, averagely 1162 mg of α-MMC and 535 mg of MAP30 were obtained from 400 g of Momordica charantia L seeds. The homogeneities of them were assessed by electrophoresis analysis. Determination of molecular weights of α-MMC and MAP30 were 28.585 kDa and 29.094 kDa by MALDI-TOF/TOF and pI were 9.02 and 9.12, respectively. The single glycoproteins were identified by Periodate-Schiff''s base (PAS) and the saccharide content was tested to be 1.25% and 1.1% by anthrone-sulfuric acid method. Biological activities were evidenced by their ability to inhibit proliferation of lung adenocarcinoma A549 cell and to convert supercoiled plasmid pUC18 into relaxed forms. Finally, we also found that both two RIPs exhibited no superoxide dismutase (SOD) activity.     

Strand-Length Measurements of Normal and 5-Iodo-2′-Deoxyuridine-treated Vaccinia Virus Deoxyribonucleic Acid Released by the Kleinschmidt Method

Purified vaccinia virus, which had been grown on chick-embryo chorioallantoic membranes in the presence or in the absence of 5-iodo-2′-deoxyuridine (IUdR), was suspended in 5 m ammonium acetate and subjected to the one-step Kleinschmidt procedure on surfaces of distilled water or salt solutions. Deoxyribonucleic acid (DNA) molecules were clearly revealed, and in many instances accurate length measurements could be made. The longest continuous molecules from normal virus measured 78, 77, and 65 μ. The most frequent length was approximately 30 μ, which corresponds to only one-third to one-half of the total DNA per virus particle predicted from various chemical analyses. These data provide direct evidence that normal vaccinia DNA may occur as a linear molecule of approximately 150 × 10⁶ molecular weight units, but, for reasons still unknown, the majority of these molecules appears to break into segments of equal length during release from the virion. There is no evidence for the presence of cyclic DNA. The DNA molecules are typically double-stranded. DNA from IUdR-treated vaccinia presents a markedly different picture: the molecules are mostly fragmented into small pieces, and rosettes or tangled masses equivalent to even one-quarter the length of normal molecules occur very rarely. The possibility is discussed that at least part of the virus-inhibitory effect of IUdR on vaccinia is due to extensive fragmentation of the DNA molecules into which IUdR has been incorporated in place of thymidine.     

Facile Synthesis of Base-Labile 2′-Deoxyribonucleosides: An Improved Synthesis of 2′-Deoxy-5-aza-Cytidine

Abstract

The use of the Fmoc group for the protection of the hydroxy functions of the sugar moiety gave an improved overall yield of 2′-deoxy-5-azacytidine (6β), due to the mildly-basic conditions required for its removal from the protected nucleoside.     

Novel Method for the Synthesis of 2′ -Phosphorylated Oligonucleotides

We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2′-O-methylribonucleotides) that contain a 2′-phosphorylated ribonucleoside residue, and optimized it to avoid 2′ -3′ -isomerization and chain cleavage. Structures of the 2′ -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2′-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.     

Mutants of Serratia marcescens lacking cyclic nucleotide phosphodiesterase activity and requiring cyclic 3′,5′-AMP for the utilization of various carbohydrates

Adenine requiring mutants of Serratia marcescens SM-6-F'lac ⁺ have been found to grow well in minimal-glucose medium solely supplemented with cAMP. From one of these ade strains double mutants (called ade cpd) were isolated which could no longer utilize cAMP but which still grew on 5′AMP. Dialyzed cell extracts (soluble fraction) of the double mutants, assayed for cAMP phosphodiesterase, were unable to hydrolyze cAMP whereas cell extracts of the parental strains yielded 5′AMP at a rate of 1.6–2.0 μmoles min⁻¹ mg⁻¹ protein. The loss of the phosphodiesterase activity in S. marcescens cpd W1181 did not cause an accumulation of large amounts of cAMP as was found for the diesterase-negative mutant AB257^pc-1 of Escherichia coli. The induced synthesis of β-galactosidase in mutant cpd W 1181 showed about the same sensitivity to transient and permanent catabolite (glucose) repression as the corresponding cpd ⁺ strain. Starting from S. marcescens cpd W1181 three independent double mutants (called cpd cya) were isolated which required exogenous cAMP for utilizing various carbohydrates as carbon source, for motility and for the formation of extracellular lipase and the red pigment prodigiosine. The intracellular concentration of cAMP in these mutants, grown in nutrient broth, was 40–60% of that of the parental strain which is about 4×10⁻⁴ M. However, the adenylate cyclase in cell extracts of the mutants W1237 and W1270 was like that of the corresponding cya ⁺ strain (about 2×10⁻² μmoles min⁻¹ mg⁻¹ protein).     

An improved method for the enzymatic transformation of nucleosides into 5′-monophosphates

An improved method to transform nucleosides into 5-monophosphates using nucleoside phosphotransferase from Erwinia herbicola is reported. The method is based on the shift in the equilibrium state of the reaction to the formation of desired product due to its precipitation by Zn²⁺. Under optimal conditions, the extent of nucleoside transformations into nucleoside-5-monophosphates were 41–91% (mol).Revisions requested 22 September 2004; Revisions received 11 October 2004