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Biomedical subjects

F Eckstein

Publications and source records attributed to F Eckstein.

At least 325 records · Page 18Linked to original sources
PubMed1979

Affinity labeling of the 3'-OH terminal binding site of the ribonucleic acid chain on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli.

Nucleoside triphosphates modified at the 3'-OH are chain terminators for RNA polymerase. They form inactive ternary complexes with the enzyme, poly(dT), and oligoadenylate, the stabilities of which depend upon the length of the oligonucleotide. Employing [5'-32P]p(Ap)10A, together with the reactive analogues 3'-(bromoacetamide)-3'-deoxyadenosine triphosphate or 3'-(isothiocyanato)-2',3'-dideoxyadenosine triphosphate, as well as 3'-amino-3'-deoxyadenosine triphosphate, followed by cross-linking with glyoxal, we labeled RNA polymerase primarily at the beta' subunit. The latter therefore appears to contain at least in part the 3'-OH terminus of the nascent RNA chain when the enzyme is in the form of the ternary complex.

Affinity Labels↗
PubMed1979

Stereochemistry of hydrolysis of adenosine 3':5'-cyclic phosphorothioate by the cyclic phosphodiesterase from beef heart.

Adenosine 3':5'-cyclic phosphorothioate, Sp-diastereomer was hydrolyzed by cyclic phosphodiesterase from beef heart in the presence of [18O]water to [18O]adenosine 5'-phosphorothioate. This was phosphorylated by myokinase and pyruvate kinase to [18O]adenosine 5'-(1-thiotriphosphate),Sp-diastereomer. The position of 18O was determined to be in a nonbridging position. This result indicates that the hydrolysis proceeded with inversion of configuration at phosphorus.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Crystal and molecular structure of adenosine 5'-O-phosphorothioate O-p-nitrophenyl ester (Sp diastereomer). Substrate stereospecificity of snake venom phosphodiesterase.

Phosphorylation of 2',3'-O-methoxymethylidene adenosine with bis(p-nitrophenyl) O,O-phosphorochloridothioate, followed by alkaline and acidic deprotection, afforded a mixture of the Rp and Sp diastereomers of adenosine 5'-O-phosphorothioate O-p-nitrophenyl ester in good yield. Only one of these diastereomers is a substrate for snake venom phosphodiesterase. The other diastereomer, remaining after complete enzymatic digestion of the substrate, was crystallized as the triethylammonium salt in the triclinic space group P1. The structure was solved from three-dimensional X-ray data and refined to an R value of 5.2%. The asymmetric cell unit contains two independent molecules with almost identical conformations. The ribose is puckered C(2')-endo, the heterocycle is in anti position and the C(5')-O(5') bond gauche, gauche. The triethylammonium cation is coordinated to the free oxygen of the phosphorothioate group and the P-S bond has double bond character. The absolute configuration of the phosphorous is Sp. This is in agreement with an earlier stereochemical study of the hydrolysis of the diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate by snake venom phosphodiesterase.

Adenine Nucleotides↗
PubMed1979

Synthesis of sugar-modified nucleoside 5'-triphosphates with partially purified nucleotide kinases from calf thymus.

The partial purification of some nucleoside monophosphate kinases (ATP:nucleosidemonophosphate phosphotransferases, EC 2.7.4.4) from calf thymus by chromatography on Blue Sepharose to remove interfering phosphatase activity is described. Their specificities towards nucleoside monophosphates modified in the sugar are investigated. Pyrimidine nucleoside monophosphate kinase is not very much affected by such modifications, whereas GMP kinase does not tolerate such alteration. The effect on AMP kinase is intermediate.

Animals↗
PubMed1979

Determination of the turn-off reaction for the hormone-activated adenylate cyclase.

Previous work suggested that hormonal activation of adenylate cyclase involves the introduction of GTP to the regulatory site, and subsequent hydrolysis of the bound GTP terminates the activation. In many tissues the turn-off GTPase reaction cannot be readily measured because of a high background of nonspecific GTP hydrolysis. To circumvent this problem a general assay for the turn-off reaction has now been developed. The adenylate cyclase is first activated by hormone and GTP and the introduction of GTP is then stopped either by addition of an excess of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) or by addition of a receptor blocking agent. The decay of adenylate cyclase activity brought on by these inhibitors is used to calculate the rate constant of the turn-off reaction. In turkey erythrocyte and rat parotid membranes the rate constant of the decay process as determined with GDP beta S is similar to that determined with the beta-adrenergic blocker propranolol. The rate constants (min-1 at 30 degrees C) for various adenylate cyclase preparations are 10 for turkey erythrocyte, 7.5 for rat parotid, and 6.2 for the rat liver enzyme. The finding of similar rate constants in the various preparations indicates that GTP hydrolysis at the regulatory site is a general mechanism for terminating the activation of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1979

Mechanistic studies on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli using phosphorothioate analogues. 1. Initiation and pyrophosphate exchange reactions.

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) can replace adenosine triphosphate (ATP) in the initiation reaction catalyzed by deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase from Escherichia coli. In both cases, the Sp diastereomer is a better initiator than the Rp isomer. The diasteromers of 3'-uridyl 5'-adenosyl ,O-phosphorothioate [Up(S)A] can replace UpA in the primed initiation reaction catalyzed by RNA polymerase; however, the Rp diastereomer is a better initiator than the Sp isomer. By using ATP or CpA as initiator and UTP alpha S, isomer A, as substrate, we determined the stereochemical courses of both the initiation and primed initiation reactions, respectively, with T7 DNA template and found them to proceed with inversion of configuration. Determination of the stereochemical course of the pyrophosphate exchange reaction catalyzed by RNA polymerase provides evidence that this reaction is the reverse of the phosphodiester bond-forming reaction.

DNA-Directed RNA Polymerases↗
PubMed1979

Stereochemistry of hydrolysis by snake venom phosphodiesterase.

[18O]Adenosine 5'-O-phosphorothioate-O-p-nitrophenyl ester was prepared by saponification of the bis (-O,O-p-nitrophenyl ester) with K18OH. Only the diastereoisomer with the Rp configuration si a substrate for snake venom phosphodiesterase. The asymmetrically labeled [18O]adenosine 5'-O-phosphorothioate formed in this reaction was converted enzymatically to [18O]adenosine 5'-(1-thiodiphosphate) with the Sp configuration. The position of the 18O label, either bridging [1,2-mu-18O] or nonbridging [1-18O] was then determined. The results show that the reaction catalyzed by snake venom phosphodiesterase takes place with retention of configuration at phosphorus. This indicates that the hydrolysis proceeds via a covalent nucleotide enzyme intermediate.

Crotalid Venoms↗
PubMed1979

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation.

Various analogs of adenosine 5'-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles. It is shown that the fluorophosphate analog ATP(gamma F) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(gamma F) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions. The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(gamma F), it is strong inhibitor of both soluble and membrane-bound mitochondrial ATPases. The biological implication of the complementary effects of ATP(gamma F) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.

Adenosine Triphosphatases↗
PubMed1979

Diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate: chemical synthesis and enzymatic properties.

A procedure is described for the synthesis of the title compounds via phosphotriester intermediates. The 2-cyanoethyl group is used to protect the P-SH function during the course of the synthesis. Resolution of the phosphorus diastereomers is accomplished at the phosphotriester stage. Removal of the 2-cyanoethyl group without racemization, followed by removal of the other protective groups, affords the optically pure diastereomers of 5'-O-adenosyl 3'-O-uridyl phosphorothioate. Their designation as Rp and Sp follows from the stereospecificity in the hydrolysis catalyzed by RNase A. These diastereomers are useful for the investigation of the stereospecificity as well as of the stereochemical course of action of nucleases. Snake venom exonuclease hydrolyses only the Rp diastereomer, whereas both diastereomers are substrates for RNases A and T2. The results with the latter indicate that RNase T2 also operates by an in-line mechanism.

Adenosine↗
PubMed1979

Stereochemistry of internucleotide bond formation by polynucleotide phosphorylase from Micrococcus luteus.

Polynucleotide phosphorylase catalyzes the formation of polynucleotides from the Sp diastereomer of adenosine 5'-O-(l-thiodiphosphate) ADPalphaS), whereas the Rp diastereomer is a competitive inhibitor. The absolute configuration of the phosphorothioate diester bond in the polymer was determined by copolymerizing ADPalpha S, Sp isomer with UDP and degrading the resulting copolymer with R Nase A and spleen phosphodiesterase to give, inter alia, uridine 2',-3'-cyclic phosphorothioate. The latter product was shown to be the endo isomer by high-performance liquid chromatography. No evidence for the presence of the exo isomer was obtained. It can thus be concluded that the Sp diastereomer of ADPalphaS polymerizes with inversion of configuration at phosphorus without racemization to give a phosphorothioate diester bond with the Rp configuration.

Kinetics↗
PubMed1979

Specific heavy metal labeling of the 3-'terminus of phosphorothioate modified yeast tRNAPhe.

Yeast tRNAPhe containing a phosphorothioate modified -CS-CS-A terminus binds two moles of chloroterpyridineplatinum(II). This result was determined by titrating the tRNA with [3H](terpy)PtCl] Cl, removing excess platinum by cation exchange chromatography, and determining the amount of bound platinum by radiocounting techniques. It has thus been established that adjacent phosphorothioate modified nucleotides can be labeled with an electron dense stain, a necessary requirement for electronmicroscopic sequencing of polynucleotides to become practical.

Kinetics↗
PubMed1978

Inhibition of primase, the dnaG protein of Escherichia coli by 2'-deoxy-2'-azidocytidine triphosphate.

2'-Deoxy-2'-azidocytidine-5'-triphosphate was investigated as an inhibitor in two reconstructed enzyme systems which catalyze the replication of two viral DNAs. During replication of the duplex replicative form of phiX174 DNA, DNA polymerase III holoenzyme was weakly inhibited and inhibition was reversed by dCTP. A more pronounced inhibition, not reversed by either dCTP or CTP, was observed during replication of the single-stranded DNA of the bacteriophage G4, a close relative of phiX174. This effect depended on the incorporation of 2'-deoxy-2'-azidocytidine-5'-triphosphate by primase (dnaG protein) which synthesizes a 29-residue RNA primer at the unique origin of bacteriophage G4 DNA replication. Extension of the primer strand, terminated by 2'-deoxy-2'-azidocytidine-5'-triphosphate is then severely inhibited. Primase was also inhibited by the 2'-deoxy-2'-azido derivatives of ATP, GTP, and UTP.

Coliphages↗