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F Eckstein

Publications and source records attributed to F Eckstein.

At least 289 records · Page 16Linked to original sources
PubMed1985

Interaction of hydrolysis-resistant analogs of cyclic GMP with the phosphodiesterase and light-sensitive channel of retinal rod outer segments.

cGMP opens cation-selective channels when applied to the cytoplasmic side of excised patches of membrane from retinal rod outer segments (ROS). If the light-sensitive channel in intact rods is gated only by cGMP, it should be possible to find a hydrolysis-resistant analog of cGMP that blocks the normal response to light by holding the channel open independent of the degree of illumination. We have studied the interaction of 8-bromo-cGMP (8-Br-cGMP) and the SP and RP phosphorothioate derivatives of cGMP [(Sp)-cGMP[S] and (RP)-cGMP[S]) with the cGMP phosphodiesterase (PDEase) of ROS, the cGMP-sensitive channel of excised ROS patches, and the light-sensitive channel of intact rods. All three analogs were hydrolyzed by PDEase much more slowly than was cGMP. The maximal rates of hydrolysis of 8-Br-cGMP, (SP)-cGMP[S], and (RP)-cGMP[S] were 7.3, 3.7, and less than 0.2 s-1, respectively, compared with 4000 s-1 for cGMP. These analogs are effective competitive inhibitors of the PDEase, with Ki values of 48, 25, and 90 microM, respectively. The nucleotide-activated conductances of excised patches were half-maximal at concentrations of 1.6, 210, and 1200 microM, respectively, compared with 17 microM for cGMP. Thus, 8-Br-cGMP is a highly potent channel agonist. The effects of these analogs on the dark current and photoresponses of intact rod cells were also measured. A suction electrode monitored membrane current across the ROS, while a patch electrode sealed on the inner segment was used to introduce a cGMP analog and to control membrane potential. All three analogs increased the dark current and markedly slowed the response to light flashes. 8-Br-cGMP increased the dark current of the outer segment as much as 48-fold. After the concentration of this analog had risen sufficiently, little of the current could be shut off by light, as expected of a direct effect on the light-sensitive channel of the plasma membrane. These results are consistent with the notions that (i) the light-sensitive channel of rods is controlled solely by the instantaneous concentration of cGMP and (ii) the cGMP-sensitive channel of excised patches is identical to the light-sensitive channel of intact rods.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1984

Cleavage of phosphorothioate-substituted DNA by restriction endonucleases.

M13 RF DNA was synthesized in vitro in the presence of various single deoxynucleoside 5'-O-(1-thiotriphosphate) phosphorothioate analogues, and the three other appropriate deoxynucleoside triphosphates using a M13 (+)-single-stranded template, Escherichia coli DNA polymerase I and T4 DNA ligase. The resulting DNAs contained various restriction endonuclease recognition sequences which had been modified at their cleavage points in the (-)-strand by phosphorothioate substitution. The behavior of the restriction enzymes AvaI, BamHI, EcoRI, HindIII, and SalI towards these substituted DNAs was investigated. EcoRI, BamHI, and HindIII were found to cleave appropriate phosphorothioate-substituted DNA at a reduced rate compared to normal M13 RF DNA, and by a two-step process in which all of the DNA is converted to an isolable intermediate nicked molecule containing a specific discontinuity at the respective recognition site presumably in the (+)-strand. By contrast, SalI cleaved substituted DNA effectively without the intermediacy of a nicked form. AvaI, however, is only capable of cleaving the unsubstituted (+)-strand in appropriately modified DNA.

Bacteriophage phi X 174↗
PubMed1984

The stereochemical course of the ribulose-5-phosphate kinase-catalyzed reaction.

Spinach-leaf ribulose-5-phosphate kinase catalyzes the reaction of (Rp)-[beta, gamma-18O, gamma-18O]adenosine 5'-(3-thiotriphosphate) with ribulose 5-phosphate to form ribulose 1-[18O]phosphorothioate 5-phosphate. This product is incubated with CO2, Mg2+, and ribulose-bisphosphate carboxylase to form the [18O]phosphorothioate of D-glycerate. Reduction of this material using phosphoglycerate kinase/ATP, glyceraldehyde-3-phosphate dehydrogenase/NADH, triose-phosphate isomerase, and glycerol-phosphate dehydrogenase/NADH produces glycerol 3-[18O]phosphorothioate, which is subjected to ring closure using diethylphosphorochloridate. This in-line reaction produces a diastereoisomeric mixture of glycerol 2,3-cyclic phosphorothioates. 31P NMR spectroscopy was used to analyze the 18O content of the products. The anti-diastereoisomer, which is the major isomer formed and corresponds to the downfield 31P NMR signal (Pliura, D.H., Schomburg, D., Richard, J.P., Frey, P.A., and Knowles, J.R. (1980) Biochemistry 19, 325-329), retains the 18O label. This observation indicates that the ribulose-5-phosphate kinase reaction proceeds with inversion of configuration at phosphorus. The reaction is, therefore, unlikely to involve the participation of a covalent phosphoryl-enzyme intermediate.

Glycerophosphates↗
PubMed1984

Assignment of resonances in the 31P NMR spectrum of d(GGAATTCC) by regiospecific labeling with oxygen-17.

The chemical synthesis of the octanucleotide d(GGAATTCC) in which each of the phosphate groups is sequentially replaced by an 17O-containing phosphate group using a polymer-supported phosphoramidite method is described. All seven phosphorus resonances in the 31P spectrum of d(GGAATTCC) can be resolved. Assignment of these resonances to a particular phosphate group in the chain is possible because labeling of a phosphate with 17O causes its particular signal to disappear from the spectrum. Phosphate residues toward the middle of the octamer have 31P NMR shifts similar to those found in polydeoxynucleotides, whereas those toward the ends resemble those of dinucleoside phosphates. These data are interpreted in terms of less flexibility of the phosphate groups in the center of the molecule as compared to those at the ends.

Amides↗
PubMed1984

The stereochemical course of the restriction endonuclease EcoRI-catalyzed reaction.

The restriction endonuclease EcoRI hydrolyzes the Rp diastereomer of d(pGGsAATTCC), an analogue of d(pGGAATTCC) containing a chiral phosphorothioate group at the cleavage site between the deoxyguanosine and the deoxyadenosine residues (Connolly, B.A., Potter, B.V.L., Eckstein, F., Pingoud, A., and Grotjahn, L. (1984) Biochemistry 23, 3343-3453). Performing the reaction in H2(18)O leads to d(pGG) and the hexanucleotide d([18O, S]pAATTCC) which has an 18O-containing phosphorothioate group at the 5' terminus. Further hydrolysis of this hexamer with nuclease P1 yields deoxyadenosine 5'-O-[18O]phosphorothioate which can be stereospecifically phosphorylated with adenylate kinase and pyruvate kinase to give Sp-[18O] deoxyadenosine 5'-O-(1-thiotriphosphate). 31P NMR spectroscopy shows the oxygen-18 in this compound to be in a bridging position between the alpha- and beta-phosphorus atoms. Thus, the hydrolysis reaction catalyzed by EcoRI proceeds with inversion of configuration at phosphorus. This result is compatible with a direct enzyme-catalyzed nucleophilic attack of H2O at phosphorus without involvement of a covalent enzyme intermediate.

Chromatography, High Pressure Liquid↗
PubMed1984

Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage site.

The synthesis and characterization of an octanucleotide, d(GGsAATTCC), containing the recognition sequence of the EcoRI restriction endonuclease with a phosphorothioate internucleotidic linkage at the cleavage site are described. Two approaches for the synthesis of the RP and SP diastereomers of this octamer by the phosphite method are presented. The first consists of the addition of sulfur instead of H2O to the phosphite at the appropriate position during chain elongation. This method results in a mixture of diastereomers that can be separated by high-performance liquid chromatography after 5'-terminal phosphorylation. The second uses the presynthesized and diastereomerically pure dinucleoside phosphorothioate d[Gp(S)A] for the addition to the growing oligonucleotide chain as a block. The products are characterized by digestion with nuclease P1, fast atom bombardment mass spectrometry, 31P NMR spectroscopy, and conversion to d(GGAATTCC) by desulfurization with iodine. Only the RP diastereomers of d(GGsAATTCC) and its 5'-phosphorylated derivative are cleaved by EcoRI endonuclease. The rate of hydrolysis is slower than that of the unmodified octamer. The phosphorothioate octamer will be useful for the determination of the stereochemical course of the EcoRI-catalyzed reaction.

Chromatography, High Pressure Liquid↗
PubMed1984

Direct mass spectroscopic method for determination of oxygen isotope position in adenosine 5'-O-(1-thiotriphosphate). Determination of the stereochemical course of the yeast phenylalanyl-tRNA synthetase reaction.

Negative ion fast atom bombardment mass spectrometry has been used to distinguish between (Sp)-adenosine 5'-O-(1-thiotriphosphate) containing either an alpha- nonbridging or an alpha-beta-bridging 18O label. The method does not require any nucleotide derivatization and so avoids the excessive manipulations and purifications necessary to distinguish between the above two species using conventional mass spectroscopy. Furthermore, it is between 50 and 200 times more sensitive than other direct methods based on 31P nuclear magnetic resonance spectroscopy. Routinely, 100 nmol of nucleoside phosphorothioate is ample to establish the 18O isotope position by normal as well as linked scan mass spectrometry. In cases where normal mass spectrometry is considered adequate, 10 nmol of material suffices. This technique should be useful in determining the stereochemical course of enzymatic nucleotidyl transfer and nuclease-catalyzed hydrolysis reactions under conditions of limiting availability of enzyme or substrate. Yeast phenylalanyl-tRNA synthetase was used to prepare the 18O-labeled adenosine 5'-O-(1-thiotriphosphate) species, and this enzyme was concomitantly shown to catalyze adenylyl transfer with inversion of configuration at phosphorus.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Fluorescent labelling of tRNA and oligodeoxynucleotides using T4 RNA ligase.

3'-O-(5'-phosphoryldeoxycytidyl) phosphorothioate and fluorescent 3'-O-(5'-phosphoryldeoxycytidyl) S-bimane phosphorothioate can be ligated to tRNA by T4 RNA ligase. They are also efficient donors for the enzymatic ligation to oligodeoxynucleotides bearing a 3'-cytidine terminus. Cytidine 3',5'-bisphosphate is also a substrate for the ligation reaction with DNA restriction fragments with a 3'-terminate cytidylic acid residue. Oligo- and polynucleotides with a 3'-phosphorothioate group react readily with electrophiles as exemplified by the reaction with monobromobimane.

Bridged Bicyclo Compounds↗
PubMed1983

A stereospecifically 18O-labelled deoxydinucleoside phosphate block for incorporation into an oligonucleotide.

Fully protected diastereoisomers of deoxyguanylyl (3' leads to 5') deoxyadenosine stereospecifically labelled on phosphorus with oxygen-18 have been synthesized by oxidation of phosphite triester intermediates in the presence of 18O-labelled water. The diastereoisomers have been chromatographically separated and their absolute configuration at phosphorus determined. (Rp)-[18O]deoxyguanylyl (3' leads to 5')deoxyadenosine has been prepared by complete deprotection of the parent diastereoisomer of the Sp configuration. Methylation of the former compound permits assignment of the absolute configurations of the methyl esters of N1-methyldeoxyguanylyl (3' leads to 5') N1-methyldeoxyadenosine.

Dimethyl Sulfoxide↗
PubMed1983

Assignment of resonances in the phosphorus-31 nuclear magnetic resonance spectrum of poly[d(A-T)] from phosphorothioate substitution.

Two phosphorothioate analogues of poly[d(A$-T)] have been synthesized enzymatically. In one, poly[d(A$-T)], dTMP is replaced by thymidine 5'-O-phosphorothioate; in the other, poly[d(T$-A)], dAMP is replaced by 2'-deoxyadenosine 5'-O-phosphorothioate. The 31P NMR spectrum of poly[d-(A-T)] in solutions at low salt concentration shows two resonances at 51.80 and -4.25 ppm relative to trimethyl phosphate. The corresponding values for poly[d(T$-A)] are 51.51 and -4.43 ppm. These data allow the assignment of the downfield resonance at -4.23 ppm in poly[d(A-T)] to the phosphate group of d(TpA) and the resonance at -4.41 ppm to that of d(ApT). Thus, strong evidence is provided for a repeating dinucleotide structure. A comparison of the 31P NMR spectra of the various polymers in solutions of 2 M CsF reveals that both resonances are shifted upfield by approximately 0.9 ppm in the case of the phosphorothioates and by 0.2 or 0.4 ppm in the case of the phosphates. An upfield shift of about 0.18 ppm can also be observed for the two corresponding dinucleoside monophosphates. Thus, the upfield shift induced by high concentrations of CsF is not specific for the polymer backbone.

Alkaline Phosphatase↗
PubMed1983

A substrate radical intermediate in the reaction between ribonucleotide reductase from Escherichia coli and 2'-azido-2'-deoxynucleoside diphosphates.

The B2 subunit of ribonucleotide reductase from Escherichia coli contains a tyrosine radical which is essential for enzyme activity. In the reaction between ribonucleotide reductase and the substrate analogue 2'-azido-2'-deoxycytidine 5'-diphosphate a new transient radical is formed. The EPR characteristics of this new radical species are consistent with a localization of the unpaired electron at the sugar moiety of the nucleotide. The radical shows hyperfine couplings to a hydrogen and a nitrogen nucleus, the latter probably being part of the azide substituent. The formation of the nucleotide radical in this suicidal reaction is concomitant with the decay of the tyrosine radical of the B2 subunit. Kinetic data argue for a first (pseudosecond) order decay of the B2 radical via generation of the nucleotide radical followed by a slower first order decay of the nucleotide radical. End products in the reaction are cytosine and radical-free protein B2. In the reaction between bacteriophage T4 ribonucleotide reductase and 2'-azido-2'-deoxycytidine 5'-diphosphate an identical nucleotide radical is formed. The present results are consistent with the hypothesis that the appearance and structure of the transient radical mimic stages in the normal reaction pathway of ribonucleotide reductase, postulated to proceed via 3'-hydrogen abstraction and cation radical formation of the substrate nucleotide (Stubbe, J., and Ackles, D. (1980) J. Biol. Chem. 255, 8027-8030). The nucleotide radical described here might be equivalent to such a cation radical intermediate.

Cytidine Diphosphate↗
PubMed1983

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.

Animals↗
PubMed1983

Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction.

(Rp)- and (Sp)-5'-O-thymidyl 3'-O-thymidyl [18O]phosphates have been synthesized by reaction of the respective (Sp)- and (Rp)-phosphorothioate precursors with N-bromosuccinimide in dioxane and H218O. Stereochemical analysis of the product derived from the (Rp)-phosphorothioate by digestion with snake venom phosphodiesterase in H217O and examination of the isotopic chirality of the resulting thymidine 5'-[16O,17O,18O]phosphate demonstrate that the replacement reaction has proceeded with inversion of configuration at phosphorus. Inspection of the 31P NMR spectrum of the methyl esters prepared from (Sp)-5'-O-thymidyl 3'-O-thymidyl [18O]phosphate confirms that the replacement reaction has proceeded with very little if any racemization. This spectrum also allows the assignment of the absolute configuration of these methyl triesters. (Rp)-5'-O-Thymidyl 3'-O-thymidyl [18O]phosphate has been used to demonstrate that the stereochemical course of the hydrolytic reaction catalyzed by nuclease P1 from Penicillium citrum proceeds with inversion of configuration at phosphorus and therefore probably does not involve the participation of a covalent enzyme intermediate.

Animals↗
PubMed1983

Stereochemical course of DNA hydrolysis by nuclease S1.

Nuclease S1 hydrolyzes the Sp-diastereomer of 5'-O-(2'-deoxyadenosyl)-3'-O-thymidyl phosphorothioate in H2(18)O to [18O]deoxyadenosine 5'-O-phosphorothioate which can be phosphorylated enzymatically to the Sp-diastereomer of [alpha-18O]deoxyadenosine 5'-O-(1-thiotriphosphate). 31P nmr spectroscopy shows the oxygen-18 in this compound to be in a nonbridging position at the alpha-phosphorus, indicating that the hydrolysis reaction catalyzed by nuclease S1 proceeds with inversion of configuration at phosphorus. This result is compatible with a direct nucleophilic attack of H2O at phosphorus without the involvement of a covalent enzyme intermediate.

Aspergillus oryzae↗
PubMed1983

Left-handed DNA: from synthetic polymers to chromosomes.

The interconversions between right-handed (R) and left-handed (L) helical conformations of DNA have been assessed by spectroscopic, electrophoretic, immunochemical, and enzymatic techniques. We have screened salt and solvent conditions which facilitate these transitions, as well as certain chemical modifications of the bases and backbone of defined synthetic polynucleotides. These include major and minor groove substituents as well as phosphorothioate analogues of selected phosphodiester bonds. We have established: R-L transitions in poly[d(G-C)] with iodo, bromo, methyl, and aza substitutions at the C5 position of cytosine, or phosphorothioate modification of the dGpC linkage. R-L transitions in the [d(A-C).d(G-T)]n sequence family using polymers modified as in the case of poly[d(G-C)]. The isomerizations are highly salt and temperature dependent. a possible L form of poly[d(A-T)] substituted with 2-amino adenine. the immunogenicities of constitutive and facultative Z-DNAs. the recognition specificities of different anti-Z-DNA IgGs for the spectrum of available polynucleotide probes. Some IgGs are sequence-specific. stabilization by IgG of otherwise transient left-handed conformations. anti-Z-DNA IgG binding to acid-fixed polytene chromosomes from the Diptera Drosophila, Chironomus, and Glyptotendipes. Laser scanning microscopy shows a maximal binding of 1 IgG per 3000-15,000 basepairs in acid fixed preparations. anti-Z-DNA IgG binding to negatively supercoiled plasmid, viral, phage, and recombinant closed circular DNAs. transcription from Z and Z* (associated) left-handed templates. From these and other results we propose that Z*-DNA may have important structural-functional roles in the cell.

Animals↗