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E Holler

Publications and source records attributed to E Holler.

At least 163 records · Page 9Linked to original sources
PubMed1985

Non-disruptive detection of DNA polymerases in nondenaturing polyacrylamide gels.

A non-disruptive method is described with which DNA polymerases can be detected in homogeneous preparations and unfractionated cell extracts after electrophoresis in nondenaturing polyacrylamide gradient gels. The technique involves diffusion of DNA polymerase activity into an overlay assay agarose gel, the synthesis of radioactive DNA, removal of excess substrates and autoradiography. Cell extracts from a variety of organisms were studied using this method. The activity from Escherichia coli crude extracts migrated in a position corresponding to a higher molecular mass than did purified preparations of DNA polymerase I. DNA polymerases of higher organisms generally migrated in positions corresponding to 400--900 kDa, in some cases, close to 200 kDA.

Animals↗
PubMed1985

Interaction of DNA polymerase I of Escherichia coli with nucleotides. Antagonistic effects of single-stranded polynucleotide homopolymers.

Binding of deoxyribonucleoside 5'-triphosphates to DNA polymerase I of Escherichia coli was measured by using a microscale nonequilibrium dialysis method. It allowed rapid and economic measurement of dissociation constants, with negligible interfering side reactions. A stoichiometry of 1 mol of nucleoside 5'-triphosphate/mol of DNA polymerase was measured, and the occurrence of a single binding site was established, for which the nucleotides competed in the binary complex with the polymerase. Binding affinities decreased in the order dGTP greater than or equal to dATP greater than dCTP congruent to dTTP. These results are in agreement with previous findings [Englund, P. T., Huberman, J. A., Jovin, T. M., & Kornberg, A. (1969) J. Biol. Chem. 244, 3038-3044] except that, in a few cases, values of dissociation constants were smaller by factors of 2-3. The cations Mg2+ and Mn2+, as well as spermine, slightly enhanced complex stability at low levels and decreased it at high concentrations, while NaCl and Hg2+ had only destabilizing effects. Recognition between nucleoside 5'-triphosphates and nucleotide templates was studied by titration of the polymerase-[3H]dGTP complex with polynucleotide homopolymers. Complementary poly(dC) did not affect binding of dGTP, and non-complementary templates caused rejection of the nucleotide. Rejection of dGTP followed a saturation dependence with an equivalence of 110 +/- 10 monomer units of polynucleotides bound per molecule of DNA polymerase. The results favor a model by which recognition arises chiefly from the stereogeometrical fit of complementary template and nucleoside 5'-triphosphate into a rigid binding site.

DNA Polymerase I↗
PubMed1984

Noncovalent complexes of diadenosine 5',5"'-P1,P4-tetraphosphate with divalent metal ions, biogenic amines, proteins and poly(dT).

Diadenosine 5',5'''-P1,P4-tetraphosphate was shown by circular dichroic measurements to bind to metal ions (Mg2+, Ca2+, Mn2+, Co2+, Zn2+), to biogenic amines (cadaverine, putrescine, spermidine, spermine), to L-arginine, to proteins (lysozyme, bovine serum albumin, Arg-rich histone f3, Lys-rich histone), and to poly(dT). Most cations effect destacking of the intramolecular adenine rings. Poly(dT) bound to the dinucleotide with a stoichiometry of 2 residues TMP per molecule of adenosine 5',5'''-P1,P4-tetraphosphate.

Adenine Nucleotides↗
PubMed1984

Phenylalanyl-tRNA synthetase of Escherichia coli K10. Effects of zinc(II) on partial reactions of diadenosine 5',5"'-P1,P4-tetraphosphate synthesis, conformation, and protein aggregation.

The synthesis of diadenosine 5',5"'-P1-,P4-tetraphosphate (Ap4A) catalyzed by phenylalanyl-tRNA synthetase in the presence of Zn2+ involves the same partial reactions (synthesis of phenylalanyladenylate and transfer of the adenylate moiety to ATP) as occur in the absence of this metal ion. However, transfer is strongly stimulated while adenylate synthesis is depressed. Also inhibited are pyrophosphorolysis of phenylalanyladenylate and transfer of phenylalanine from the adenylate to cognate tRNA, because overall tRNA phenylalanylation is depressed [Mayaux, J.-F., & Blanquet, S. (1981) Biochemistry 20, 4647-4654], whereas binding of tRNA to the synthetase is not. At moderate concentrations of Zn2+, and in the presence of 5 microM phenylalanine and 0.5 mM ATP, transfer of AMP is rate limiting, while at higher concentrations of Zn2+ synthesis of adenylate is rate determining. The Zn2+ concentration optimum for stimulation depends on the concentration of phenylalanine and ATP. The effects of Zn2+ are mediated through two classes of binding site(s) on the synthetase, the half-saturations of which are 1-4 and 20-30 microM Zn2+, respectively. Binding of Zn2+ to the second class of site(s) causes inhibition of the synthetase, whereas binding to the first class is responsible for activation and inhibition, which may be caused by a conformational change. Evidence for the latter is the observed decrease in protein intrinsic fluorescence intensity and the decrease in fluorescence intensity of 6-(p-toluidinyl)naphthalene-2-sulfonate, which is used as a reporter group. The kinetics of the binding reaction show a saturation dependence on Zn2+, also suggesting that a conformational change occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1984

Serological identification and separation of canine lymphocyte subpopulations.

In vitro treatment of bone marrow grafts with absorbed rabbit-antidog thymocyte globulin (ATG) prevented graft-versus-host disease in a substantial number of the dogs differing by one DLA haplotype. Absorbed ATG has now been used for serological identification of canine lymphocyte populations. Specific labeling of canine T-lymphocytes by absorbed ATG could be demonstrated by (a) a distribution of ATG-positive cells in suspensions of canine lymphoid organs similar to that of T cells observed in other species and by specific staining of paracortical thymus-dependent lymph node areas in immunohistochemistry, (b) complementary labeling of nylon-wool-separated cells by ATG and antiimmunoglobulin sera, and (c) correlation of ATG surface labeling with responder activities in mixed lymphocyte cultures.

Animals↗
PubMed1983

Catabolism of diadenosine 5',5"'-P1,P4-tetraphosphate in procaryotes. Purification and properties of diadenosine 5',5"'-P1,P4-tetraphosphate (symmetrical) pyrophosphohydrolase from Escherichia coli K12.

Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum. Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold. The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase. The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000. Activity maximum is at pH 8.3. The Km value computed for Ap4A is 25 +/- 3 microM. The sulfhydryl group(s) is essential for enzyme activity. Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively. Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis. Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively. Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations. Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations. Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold. The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine. Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.

Acid Anhydride Hydrolases↗
PubMed1983

Circular dichroism and ordered structure of bisnucleoside oligophosphates and their Zn2+ and Mg2+ complexes.

Circular dichroism, absorbance, hypochromicity, and the formation of Mg2+ and Zn2+ complexes have been measured for a series of bisnucleoside oligophosphates that contain adenosine, guanosine, and mixed guanosine/adenosine, guanosine/cytidine, and guanosine/uridine, as well as 7-methylguanosine and ribose-methylated purine nucleosides. All of the metal complex ions have stacking interactions at 2 degrees C, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 8.0. There is a measurable degree of base stacking for all unsubstituted purine nucleotides that differs, however, from that of bases in nucleic acids. The degree of base stacking varies with the length of oligophosphate chains and the state of methylation. The effect of 7-methylation of guanosine is interpreted as causing a switch of nucleic acid base stacking from an atypical to a typical mode, which could be important for cap function in mRNA. The Mg2+ and Zn2+ complexes give rise to characteristic circular dichroism. In all instances excepting 7-methylated bisguanosine oligophosphates, the active secondary structures are disrupted, and in this regard, Zn2+ is more effective than Mg2+. At least two sets of binding sites are involved. A single metal ion is bound tightly. Stability, in terms of equilibrium constants, increases by more than 1000-fold as a function of chain length varying from two to six phosphates. The consequences of methylation are only minor. Electrostatic attraction between metal ions and phosphates is the most likely mechanism of these phenomena as judged by the effect of high ionic strength.

Circular Dichroism↗
PubMed1982

Kinetics of acyl transfer ribonucleic acid complexes of Escherichia coli phenylalanyl-tRNA synthetase. A conformational change is rate limiting in catalysis.

Kinetics of complex formation between phenylalanyl-tRNA synthetase and Phe-tRNAPhe have been measured by the stopped-flow technique. Either the protein intrinsic fluorescence or the fluorescence of the added indicator 6-(p-toluidinyl)naphthalene-2-sulfonate was observed. Identical results were obtained with each method. Acyl-tRNAs with variable structures of the acyl and tRNA moieties were examined. Kinetics were measured as a function of pH and at different ionic strengths. Kinetic constants were compared with those of enzymatic phenylalanylation of tRNAPhe. The results are as follows. (1) Phe-tRNAPhe binds to phenylalanyl-tRNA synthetase in two, mutually exclusive types of complexes, one at the tRNA-specific binding site of the enzyme and the other in a region which involves the Phe-specific binding site of the enzyme [Holler, E. (1980) Biochemistry 19, 1397-1402]. The Phe site directed association includes a conformational change of the complex that is rate limiting. (2) The conformational change and catalytic tRNA aminoacylation follow similar values of rate constants irrespectively of pH and ionic strength. It is concluded that aminoacylation is limited by the kinetics of a conformational change of the nascent enzyme-Phe-tRNAPhe complex. (3) The nature of Phe site directed binding was probed by variation of the structure of Phe-tRNAPhe. Both the acyl and the tRNA moieties are recognized by the enzyme. Of the acyl moiety, only the phenyl ring but not the amino group is essential for binding. The amino group can be acetylated or replaced by a hydroxyl group. Protonation of the amino group results in loss of Phe site directed binding. It gives a pKa = 6.9, which is close to that for protonation of a phenylalanine ester. (4) Rate constants were only slightly affected by addition of 200 mM NaCl at pH 7.5, indicating that the contribution by electrostatic forces was probably minimal. Mg2+ ions were essential for Phe site directed binding. Complexation of enzyme, Phe-tRNAPhe, and Mg2+ either was random or was at preequilibrium with the conformational change. (5) Binding of Phe-tRNAPhe at the tRNA-specific site of the enzyme was studied in the presence of in situ synthesized phenylalanyl adenylate. The reaction was bimolecular with rate constants of 50 microM-1 s-1 and 15 s-1 for association and dissociation, respectively.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Catalytic mechanism of phenylalanyl-tRNA synthetase of Escherichia coli K10. Conformational change and tRNAPhe phenylalanylation are concerted.

Catalytic phenylalanylation of tRNAPhe and the reverse reaction, AMP-dependent deacylation of Phe-tRNAPhe, have been measured by steady-state and pre-steady-state techniques, including rapid sampling and fluorescence stopped-flow methods. (1) Stoichiometry of adenylate synthesis under steady-state phenylalanylation of tRNAPhe indicates half-of-the-sites reactivity. (2) Identity of values of rate constants under pre-steady- and steady-state conditions demonstrates that the rate-limiting steps in catalysis are bond making for phenylalanylation and bond breaking for AMP-dependent deacylation, respectively. (3) Values of catalytic rate constants are the same as those for the conformational change of the Phe site directed enzyme-Phe-tRNAPhe complex [Baltzinger, M., & Holler, E. (1982) Biochemistry (preceding paper in this issue)]. (4) A model is developed that accounts for the observed concert of chemical and geometrical reactions as well as for experimental evidence that nascent Phe-tRNAPhe may not be the same as in solution. In this model, nascent Phe-tRNAPhe is thought to be the tetrahedral intermediate that is formed by nucleophilic attack of the adenylate by the tRNA. It awaits the conformational change in order to break down into Phe-tRNAPhe and AMP. The model can serve as a unifying basis for an interpretation of discrimination against noncognate amino acids and tRNAs and also gives an explanation why severe product inhibition is not observed [Güntner, C., & Holler, E. (1979) Biochemistry 18, 2028-2038].

Adenosine Monophosphate↗
PubMed1982

[Adriamycin and autologous bone marrow transplantation. Preclinical studies in dogs (author's transl)].

We investigated the toxicity of adriamycin (ADM) in dogs with regard to autologous bone marrow transplantation (BMT). Gastrointestinal toxicity limited the dose of ADM in single administration (3.0 mg/kg) and chronic cardiotoxicity in repeated administration (9 X 1.5 mg/kg every two weeks). The recovery of hemopoiesis was complete within 25 days after single and repeated doses of ADM as indicated by the restoration of blood counts and concentration of hemopoietic precursors (CFUc) as well as proliferate activity of bone marrow. At this time bone marrow was obtained and reinfused after total body irradiation (TBI) with 10 Gy. Single and repeated doses of 1.5 mg/kg ADM did not delay recovery of blood counts after autologous BMT as compared to transplantation of untreated marrow - in contrast a single dose of 3.0 mg/kg ADM did. Our results indicate that toxicity of ADM is not influenced by autologous BMT. Previous shorter therapy with conventional doses of ADM does not preclude autologous BMT.

Animals↗
PubMed1981

Phenylalanyl-tRNA synthetase of baker's yeast. Modulation of adenosine triphosphate-pyrophosphate exchange by transfer ribonucleic acid.

Native and modified phenylalanine transfer ribonucleic acid (tRNAPhe) can modulate phenylalanine-dependent adenosine triphosphate--inorganic [32P]pyrophosphate (ATP--[32P]PPi) exchange activity via inhibition of adenylate synthesis. Inhibition is visualized if concentrations of L-phenylalanine, ATP, and pyrophosphate are subsaturating. In the proposed mechanism, tRNAPhe is a noncompetitive inhibitor at conditions where only one of the two active sites per molecule of enzyme is occupied by L-phenylalanine, ATP, and pyrophosphate. At saturating concentrations of these reactants, both active sites are occupied and, according to the model, inhibition is eliminated. Occupation by these reactants is assumed to follow homotropic negative cooperativity. The type of effects depends on modification of tRNAPhe. Native tRNAPhe, tRNA2'-dAPhe, and tRNAoxi-redPhe are inhibitors, tRNAPhepCpC has no effect, and tRNAoxPhe is an activator. Kinetics of activation by tRNAoxPhe are slow, following the time course of Schiff base formation and subsequent reduction by added cyanoborohydride. Besides showing that a putative enzyme amino group is nonessential for substrate binding and adenylate synthesis, this result may suggest that an enzyme amino group could interact with the 3'-terminal adenyl group of cognate tRNA. In the case of asymmetrical occupation of the enzyme active sites by all of the small reactants ATP, L-phenylalanine, and pyrophosphate, the interaction with the amino group might trigger the observed noncompetitive inhibition of the pyrophosphate exchange by tRNAPhe.

Adenosine Triphosphate↗
PubMed1981

Detection of ligand-induced conformational changes in phenylalanyl-tRNA synthetase of Escherichia coli K10 by laser light scattering.

The diffusion constant of phenylalanyl-tRNA synthetase has been measured by laser light scattering under conditions of complex formation with Mg2+, L-phenylalanine, MgATP, tRNAPhe, modified tRNAPhe, tRNAPhe (yeast), and noncognate tRNA. The diffusion constant (pH 7.5, 20 degrees C) of the free enzyme is (2.85 +/- 0.005) x 10(-7) cm2 s-1, of the enzyme . Mg2+ complex (2.40 +/- 0.05) x 10(-7) cm2 s-1 and of the enzyme . Mg2+ . tRNAPhe complex (2.95 +/- 0.06) x 10(-7) cm2 s-1. The effect of tRNAPhe is only seen when the enzyme is saturated with Mg2+. The smaller substrates exhibit no effect besides a small increase of the value of the diffusion constant under conditions where the enzyme-phenylalanyladenylate is synthesized. Of the noncognate tRNATyr and tRNAIle, the latter is able to associate with the enzyme, causing the value of the diffusion constant to increase. tRNAPhe (yeast) and tRNAhvPhe (photo-cross-linked tRNAPhe) exhibit similar effects. The observed variation of the diffusion constant is attributed to conformational changes of the enzyme. The opposite effects of Mg2+ and tRNAPhe are interpreted as an expansion and recontraction, respectively, of the enzyme molecule. In several cases, the effects were used to follow a titration of the enzyme with a ligand. Dissociation constants were calculated from the resulting titration curves, yielding values which are in agreement with those obtained by other techniques. It is established by comparison that of the two possible binding sites for each Mg2+ and tRNAPhe the diffusion constant reflects occupation of only a single class of sites.

Adenosine Triphosphate↗
PubMed1981

[Radiobiologic considerations about further development of whole-body irradiation with subsequent bone marrow transplantation applied during the treatment of acute leukemia (author's transl)].

The results achieved hitherto with whole-body irradiation in case of acute leukemia are examined with respect to the optimization criteria of a radiotherapy. The most important failure risk is the recurrence which occurs in more than 50% of all cases, then follows the interstitial pneumonia. The authors think that an increase of the total dose, the adaption of the dose distribution to the distribution of leukemia cells in the body, and a higher fractionation of the total dose are approaches for improving the therapy results.

Acute Disease↗