Identification of cytidine 2',3'-cyclic monophosphate and uridine 2',3'-cyclic monophosphate in Pseudomonas fluorescens pfo-1 culture.

Cytidine 2',3'-cyclic monophosphate (2',3'-cCMP) and uridine 2',3'-cyclic monophosphate (2',3'-cUMP) were isolated from Pseudomonas fluorescens pfo-1 cell extracts by semi-preparative reverse phase HPLC. The structures of the two compounds were confirmed by NMR and mass spectroscopy against commercially available authentic samples. Concentrations of both intracellular and extracellular 2',3'-cCMP and 2',3'-cUMP were determined. Addition of 2',3'-cCMP and 2',3'-cUMP to P. fluorescens pfo-1 culture did not significantly affect the level of biofilm formation in static liquid cultures.

Quantitative separation of nucleotides on mercurated dextran.

Dextran gels of varying porosites (Sephadex G series) were chemically modified so as to contain covalently bound monofunctional mercury. Mercurated Sephadex of the porosity G-25 was then used to fractionate mixtures of mono- and dinucleotides into the constituent components. Separation is based on the affinity of the nitrogen binding sites of the purine and pyrimidine derivatives for organomercurial Hg+. Thus, a mixture composed of the four mononucleotides Cyd-5'-P, Ado-3'-P, Guo-2'(3')-P, dThd-5'-P and of the four dinucleotides Cyd-P-Cyd, Ado-P-Ado, Guo-P-Urd, and Urd-P-Urd could be separated into eight well-resolved fractions by using a combination Tris-bicarbonate/carbonate buffer system of increasing pH as an eluent. Complete separation was also achieved when a mixture of Ado 3:5'-P, Ado 5'-P, Ado-5'-PP, and Ado-5'-PPP was fractionated on mercurated Sephadex G-25. Again, Tris-bicarbonate/carbonate buffer served as an eluent. Lastly, fractionation can also be performed at a constant pH by offering other suitable ligands, for instance Cl-, that will compete with nucleotides for monofunctional Hg+. The fractionation behavior of mercurated Sephadex G-25 can be fully understood on the basis of the complexing properties of monofunctional Hg+. This has been shown by calculating the net retention volume ratios of several nucleotides with the help of the known interaction parameters of corresponding nucleosides with CH3 HgOH and by comparing the predicted ratios with the experimentally measured ones. Finally, the acid-base properties of mercurated Sephadex G-25 as well as its affinity for chloride and iodide ions have been determined. The data agree quite well with those known for CH3 HgOH.

Isolation and characterization of a second molybdopterin dinucleotide: molybdopterin cytosine dinucleotide.

The pterin cofactor (bactopterin) in the molybdoenzyme CO dehydrogenase isolated from Pseudomonas carboxydoflava has previously been shown to differ from molybdopterin in molecular mass, phosphate content, stability, and other properties, implying a novel structure. The structure of the CO dehydrogenase pterin has been investigated in the present studies by alkylation and isolation of the carboxamidomethyl derivative. The alkylated pterin was identified as [di-(carboxamidomethyl)]molybdopterin cytosine dinucleotide on the basis of its absorption properties and by degradation with nucleotide pyrophosphatase yielding carboxamidomethylmolybdopterin and CMP. Further treatment of these products with alkaline phosphatase produced species with absorption and chromatographic properties identical to those of the corresponding dephospho compounds. Molybdopterin cytosine dinucleotide is the second molybdopterin variant to be structurally characterized. The fact that molybdopterin cytosine dinucleotide and molybdopterin guanine dinucleotide contain molybdopterin in their structure shows that the pterin moiety, with its unique dithiolene-containing sidechain, is a structural element which is common to the organic portion of the molybdenum cofactors of many molybdoenzymes.

Presence of cytidine 5'-tetraphosphate in commerical samples of cytidine 5'-triphosphate.

A contaminant compound has been isolated from commercial samples of CTP by ion-exchange chromatography on a Dowex-1 column. It has been characterized as cytidine 5'-tetraphosphate from its ultraviolet spectrum, labile and total phosphate content, and periodate consumption. It is present in proportions from 0.3 to 3.9%, apparently regardless of the method of preparation, age of sample, or commericial source of CTP.

A modified procedure for the preparation of di- and triribonucleotides from pancreatic ribonuclease digest of RNA.

A novel procedure for the separation of oligonucleotides from pancreatic RNase-digest of RNA is described. The method involves a group-separation of uracil-containing and of cytosine-containing nucleotides on Dowex 50W. The obtained groups are further separated on DEAE-Sephadex A-25 by a linear gradient of NH4HCO3.

Introducting of the 3-deoxy group in 3,6-dideoxyhexoses: a novel cofactor function for pyridoxamine-5'-phosphate.

A cofactor needed for the reduction of CDP-4-keto-6-deoxy-D-glucose to CDP-4-keto-3,6-dideoxy D-glucose has been detected and purified. Its spectra in neutral, acid, and basic solutions, its behavior on thin-layer chromatography, and other properties led to its identification as pyridoxamine-5'-phosphate. Moreover, authentic pyridoxamine-5'-phosphate substituted for the purified cofactor with about the same specific activity. Other vitamin B(6) derivatives did not stimulate the reaction, and inhibition was observed with some of them.

[Preparation and evaluation of new ion-exchange chromatographic stationary phase for the use in high performance liquid chromatography].

A new method for the bonding of diethylamine(DEA) on the surface of silica to prepare novel hydrophilic packings for HPLC has been studied. After allyl glycidyl ether being synthesized, the Si-DEA anion-exchange bonded phase was prepared by the reaction of the double bond in allyl group with Si-H silica. The bonded phases obtained were characterized by elemental analysis, diffuse reflectance infrared Fourier transform(DRIFT) spectroscopy and HPLC evaluation. The methods were used for both porous silica and monodisperse non-porous silica. The contents of carbon, hydrogen and nitrogen of porous Si-DEA packing (MPS-DEA) were 3.31%, 0.95% and 1.34% respectively and those of monodisperse non-porous Si-DEA packing (NPS-DEA) were 2.55%, 0.97% and 0.96% respectively. The diethylamine absorption peak can be observed at 2970 cm-1 from the Si-DEA silica DRIFT spectrum. These data revealed that the diethylamine had been bonded on MPS-DEA and NPS-DEA packings. In HPLC tests, nucleotides and nucleosides such as cytosine, uracil, cytidine-5'-monophosphate, adenosine-5'-monophosphate, inosine-5'-monophosphate and guanosine-5'-monophosphate were satisfactorily separated on the porous anion-exchange packing (MPS-DEA), and a group of proteins (lysozyme, ribonuclease, ovalbumin, bovine serum albumin, insulin and gamma-globulin) were separated within 15 minutes successfuly. All test results indicated that the new method for preparing better anion-exchange silica packings is effective for both porous silica and monodiperse non-porous silica.

Cytidylate cyclase: development of assay and determination of kinetic properties of a cytidine 3',5'-cyclic monophosphate-synthesizing enzyme.

A method is described for the separation of cytidine 3',5'-cyclic monophosphate (cyclic CMP) from cytidine tri-, di- and mono-phosphates and from cytidine 3',5'-cyclic pyrophosphate, cytidine 2'-monophosphate-3',5'-cyclic monophosphate, cytidine 2'-O-aspartyl-3',5'-cyclic monophosphate and cytidine monophosphate, compounds previously shown to be the result of putative cytidylate cyclase activity. This separation, involving elution of a novel bilayer column of QAE-Sephadex and alumina with 0.03 M-HCl, has been incorporated into an assay protocol to determine the enzyme-catalysed conversion of radiolabelled CTP to cyclic CMP. By this assay, cytidylate cyclase activity has been shown to be present in rat lung, spleen, ovary, testes, brain, stomach, liver, heart and kidney preparations; the activity was of a similar order in each tissue and had a sharp pH optimum of 7.0-7.5. The liver preparation had a Vmax. of 1.2 nmol of cyclic CMP formed/min per mg, and a Km of 220 microM-CTP, and although active in the absence of added cations, it was stimulated by Fe2+ and Mn2+ ions. In several of the tissues examined, the cytidylate cyclase activity was inversely proportional to age of the animals.

Extraction, purification and identification of cytidine 3',5'-cyclic monophosphate from rat tissues.

The large-scale extraction and purification to homogeneity of cyclic CMP and its unequivocal identification are described. Rat liver, kidney, heart, spleen and lung tissues were subjected to a sequential purification procedure involving freeze-clamping, perchlorate extraction, alumina and boronate column chromatography, polyacrylamide-gel column electrophoresis and high-voltage paper electrophoresis. The purified sample co-chromatographed with authentic cyclic CMP on t.l.c. and high-pressure liquid chromatography and was positive in a cyclic CMP radio-immunoassay. The u.v., i.r. and p.m.r. spectra were each essentially identical with those of authentic cyclic CMP. Fast-atom bombardment of authentic cyclic CMP yielded a mass spectrum containing a molecular protonated ion: mass-ion-kinetic-energy scanning of this ion produced a spectrum unique to 3',5'-cyclic CMP. The extracted nucleotide produced an identical mass-ion-kinetic-energy spectrum.

Electrophoretic behaviour of oligonucleotides and mono-, di- and triphosphate nucleotides by capillary zone electrophoresis.

A systematic investigation has been made into the mechanisms of the capillary zone electrophoresis (CZE) separation of 12 common nucleotides (mono-, di- and triphosphorylated) and polydeoxythymidylic acid oligonucleotides (pd(T)5-18) using electrophoretic mobility values calculated from migration time data. Relationships between electrophoretic mobility and the physicochemical characteristics of the analytes (charge, dissociation constants, charge-to-mass ratio) and the background electrolyte conditions (buffer strength, percentage organic modifier and buffer pH) were characterised. Nucleotide migration was dominated by the negatively charged phosphate groups. Additionally, there were important contributions to migration behaviour from the ionised amide groups of the nucleobases guanine and uracil at higher buffer pH values or with the presence of methanol in the electrolyte. Calculated electrophoretic mobility values for the nucleotides showed a substantially improved (5-fold) inter-run repeatability compared with migration time data. These studies show the value of representing nucleotide migration data as electrophoretic mobility in CZE for obtaining a more thorough analysis of separation mechanisms and to compensate for variation in migration time data caused by small changes in electrosmotic flow. Oligonucleotides pd(T)5-11 could be adequately resolved from their nearest neighbour, but the limit of single-base separation was pd(T)10 from pd(T)11 under the conditions used. It was calculated that a difference in charge-to-mass ratio of 2.64 x 10(-5) was required for resolution under the CZE conditions used.

Isolation of Escherichia coli RNA polymerase binding sites on T5 and T7 DNA: further evidence for sigma-dependent recognition of A-T-rich DNA sequences.

Previous isolation and analysis of E. coli RNA polymerase (EC 2.7.7.6) binding sites on lambda DNA had demonstrated the existence of a sigma-dependent process of recognition of A-T-rich DNA sequences. We have now extended this finding to T5 and T7 DNA and häve provided evidence for the double-strandedness of the isolated binding sites. The possible equation of these sites to the genetically defined promoters is discussed.