Search PubMed⌕ Search

Biomedical subjects

D Porschke

Publications and source records attributed to D Porschke.

44 records · Page 3Linked to original sources
PubMed1984

Selective binding of amino acid residues to tRNAPhe.

The interaction of amino acid amides with tRNAPhe was studied by measurements of the Wye base fluorescence. Binding of phenylalanine-, tyrosine- and tryptophan-amides leads to considerable quenching, whereas the amides of e.g. glycine and leucine do not induce quenching under the same conditions. Binding constants at 0.13 M salt - 100 M-1 for Phe-, 110 M-1 for Tyr- and 300 M-1 for Trp-amide - are about a factor of 6 higher than those evaluated from independent measurements for binding to simple single-stranded polynucleotides; the corresponding factor is 10 for double-stranded polynucleotides. Since the apparent enthalpy changes derived from measurements at different temperatures remains relatively low (-9 to -20 kJ/mol), the increased affinity appears to be mainly due to an increase of the entropy changes. Titration experiments performed in the presence of Mg2+ indicate cooperative interactions of the aromatic residues with the anticodon loop that are consistent with preferential binding to one of two loop conformations. Measurements of binding constants at different pH-values indicate the protonation of a tRNA residue in the tryptophanamide-tRNAPhe complex characterised by a pK value of about 7.0.

Allosteric Regulation↗
PubMed1984

Dynamics of DNA condensation.

The condensation of DNA induced by spermine and spermidine is investigated by equilibrium titrations and stopped-flow and field-jump experiments using scattered light detection. The spermine concentration required for the cooperative condensation process is measured at different DNA concentrations; these data are used to evaluate both the condensation threshold degree of spermine binding and the binding constant of spermine according to an excluded-site model. Stopped-flow measurements of the spermine-induced condensation demonstrate the existence of two processes: (1) A "fast" reaction is observed in the millisecond time range, when the reactant concentrations are around 1 microM; it is associated with a characteristic induction period and is assigned to the intramolecular condensation reaction. (2) A slow reaction with time constants of, e.g., 100 s strongly dependent upon both spermine and DNA concentrations is assigned to an intermolecular DNA association. The unusual time course of the intramolecular condensation reaction with the induction period provides evidence for a "threshold kinetics". During the induction period, spermine molecules are bound to DNA, but the degree of binding remains below the threshold value. As soon as the degree of ligand binding arrives at the threshold, the DNA is condensed in a relatively fast reaction. Model calculations of the spermine binding kinetics according to an excluded-site model demonstrate that the spermine molecules bound to DNA are mobile along the double helix. A comparison of the experimental data with the results of Monte Carlo simulations suggests a rate constant of approximately 200 s-1 for spermine movement by one nucleotide residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗
PubMed1984

Mechanism of codon recognition by transfer RNA and codon-induced tRNA association.

The steps of UUC recognition by tRNAPhe were analysed by temperature-jump measurements. At ion concentrations close to physiological conditions we found three relaxation processes, which we assigned to (1) formation of codon-anticodon complexes, (2) a conformational change of the anticodon loop coupled with Mg2+ binding, and (3) codon-induced association of tRNA. The relaxation data were evaluated both by the usual procedure (fitting the exponentials evaluated from the individual experiments of a set to a reaction model) and by "global fitting", i.e. fitting a set of relaxation curves obtained at various concentrations directly to a reaction model, thus leaving out the intermediate exponential fitting step. The data can be represented quantitatively by a three-step model: the codon binds to the anticodon at a rate of 4 X 10(6) to 6 X 10(6) M-1S-1 as is usual for the formation of oligomer helices; the conformation change of the anticodon loop is associated with inner sphere complexation of Mg2+ at a rate of 10(3) S-1; the codon-tRNA complexes form dimers at a rate of 5 X 10(6) to 15 X 10(6) M-1S-1. A similar mechanism is found for the binding of the wobble codon UUU to tRNAPhe at increased concentrations of Mg2+. Measurements at different Mg2+ concentrations demonstrate the distinct role of this ion in the codon recognition and the codon-induced tRNA dimerization. We propose a simple mechanism, based upon the special properties of magnesium ions, for long-distance transfer of reaction signals along nucleic acid chains.

Anticodon↗
PubMed1984

Interactions of nucleic acid double helices induced by electric field pulses.

Electric field pulses induce a substantial increase of the light scattering intensity of double-helical DNA. The relative change of light scattering and also the reciprocal relaxation time constants under electric field pulses increase with increasing nucleotide concentration. These observations, together with a large difference between dichroism orientation time constants and light scattering time constants under electric field pulses, demonstrate that the main part of the light scattering effect is due not to field-induced orientation but to interactions between DNA helices. From the concentration dependence of the light scattering time constants we obtain, according to an isodesmic reaction model, association rate constants in the range 3 X 10(10) M-1 helices s-1 for DNA with approx. 300 base-pairs. These values are at the limit of a diffusion-controlled DNA association and do not show any dependence upon the field strength. The dissociation rate constants kd decrease strongly with increasing field strength E and thus demonstrate that the interactions between the helices are induced by the electric field. This conclusion is consistent with independent measurements which do not reveal any DNA association at zero field strength. The observed linear relation between log(kd) and E2 suggests a field-induced reaction driven by dipole changes. According to this interpretation the change of dipole moment should be in the range of approx. 1400 debye. The dissociation rates for DNA helices with approx. 300 to approx. 800 base-pairs strongly increase with increasing salt concentration (measured in the range 1-5 mM ionic strength), whereas the association rate constants remain virtually unchanged. Measurements of the linear dichroism in the same range of DNA chain length demonstrate that for long field pulses of e.g., 40 microseconds, the amplitude approaches a maximum value and then decreases. The dichroism relaxation curves observed after long field pulses exhibit a component with a positive dichroism and an increased decay time. These observations suggest the formation of a DNA aggregate with an unusual arrangement of the bases.

Animals↗
PubMed1979

The binding of Arg- and Lys-peptides to single stranded polyribonucleotides and its effect on the polymer conformation.

The interactions between basic oligopeptides (Lys2, Lys3, Arg2 and Arg3) and single stranded polynucleotides (poly(A), poly(C), poly(I) and poly(U) were investigated at low ion concentration by UV spectroscopy, circular dichroism and field jump relaxation. Various domains of binding were detected: 1) High concentrations (up to 1 mM) of some peptides induce opalescence followed by coacervation. Arg3 causes coacervation in all polynucleotides used, yet Lys3 only in poly(I). In the case of poly(I) the threshold concentration for coacervation is much lower for Arg3 (150 muM) than for Lys3 (500 muM). 2) Medium concentrations (greater than 10 muM) of Arg3 and Lys3 induce helix formation in poly(U). In the case of poly(I) cooperative helix formation is only induced by Lys3, but not by Arg3. 3) The onset of peptide association is observed at very low peptide concentrations (greater than or equal to 1muM) already by using the field jump method. The association is reflected by a relaxation process, that can be described by a single exponential within experimental accuracy. Measurements of relaxation time constants as a function of the peptide concentration provide information on the association constants K, the number of nucleotide residues per binding place n and the rate constants kR and kD. Using a simple model with independent and "separate" binding sites, K for Arg3 and Lys3 is found to be in the range of 10(6) to 10(7) M-1. In the case of Arg2 and Lys2 K is lower by a factor of about 10. For various polynucleotides KArg3 is only slightly higher than KLys3, except in the case of poly(I), where KArg3/KLys3 approximately 5. Similar data are obtained by application of a "sphere model" (see below). These results provide quantitative evidence for specific hydrogen bonding between the guanidino group of Arg and inosine. They also explain the absence of helix formation for poly(I) + Arg3: Arg blocks the hydrogen bonding sites of inosine. Thus cooperative coupling leads in this case to a considerable amplification of specificity in the peptide-polynucleotide interation. Both field jump and stopped flow data demonstrate a high mobility of the peptide ligands along the polymer, resulting in a redistribution being fast compared with the overall binding step. Based on this result the relaxation data are analysed by a "sphere" model, which considers a) excluded binding under the condition of fast ligand distribtuion along the lattice and b) the connection of sites into a polymer sphere. The rate constants obtained by this model are in the range of 4 X 10(11) M-1 s-1. These high values reflect the large reaction distance for polymers of chain lengths around 1000. A comparison with rate constants obtained previously for oligomer complexes indicates that the recombination rate is approximately a function of the square root of the nucleotide chain length, which is directly related to the mean radius of coiled polymers.

Circular Dichroism↗
PubMedDate not supplied

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code.

The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (tm) for poly(A).poly(U) and poly(I).poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of tm at low concentrations (approximately 1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala greater than Gly greater than Ser greater than Asn greater than Pro greater than Met, Val greater than Ile, Leu for poly(A).poly(U) and Ala, Gly, Ser greater than Asn greater than Pro greater than Val greater than Ile, Met, Leu for poly(I).poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.

Amino Acid Sequence↗
PubMedDate not supplied

Selective binding of amino acid residues to tRNA molecules detected by anticodon-anticodon interactions.

Anticodon-anticodon pairing of complementary tRNA's has been studied by fluorescence temperature jump measurements in the presence of different ligands as an approach for the evaluation of ligand binding to tRNA. This procedure is particularly useful for ligands which do not show spectroscopic changes upon binding, but affect the pairing potential of anticodons. Addition of phenylalanine-, tyrosine- and tryptophan-amide leads to a substantial decrease of the tRNAPhe.tRNAGlu pairing constant Kp, whereas Kp remains almost unaffected by addition of leucine amide and increases upon addition of glycine amide. The effects observed for the aromatic amino acid amides can be described quantitatively by a site binding model with preferential binding of the amides to tRNAPhe. The binding constants evaluated according to this model (Phe-amide 120 M-1, Tyr-amide 160 M-1 and Trp-amide 580 M-1) are consistent with values obtained independently by fluorescence titrations with tRNAPhe. Selective binding of these amino acid residues to tRNAPhe is deduced from the observed concentration dependence which is not compatible with a corresponding binding process to tRNAGlu. Addition of glutamic acid diamide induces an increase of the tRNAPhe.tRNAGlu pairing constant, which is however equivalent to that observed for tRNAPhe.tRNALys pairing and thus does not demonstrate a selective binding to tRNAGlu. The pairing of tRNAPhe with tRNAGlu is strongly enhanced by addition of Mg2+ or spermine. Evaluation of the Mg2+ data by a site model leads to constants of 360 M-1 for the binding of Mg2+ to monomer tRNA and 3000 M-1 for the binding of Mg2+ to the tRNAPhe.tRNAGlu dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗