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Biomedical subjects

R F Steiner

Publications and source records attributed to R F Steiner.

At least 55 records · Page 3Linked to original sources
PubMed1983

The dependence of the molecular dynamics of calmodulin upon pH and ionic strength.

The mobilities of several fluorescent probes placed at different locations on calmodulin in the absence of Ca2+ have been found to depend upon the charge, ionic strength, and temperature. In general, the time decay of fluorescence anisotropy could be fitted with two rotational correlation times. The shorter of these reflects primarily the motion of the probe itself, while the longer corresponds to the motion of a major portion of the molecule. An increase in ionic strength or a decrease in net charge results in a decrease in the relative amplitude of the shorter correlation time, while an increase in temperature produces an increase in its amplitude. These results are consistent with, and suggest, that an increase in probe mobility accompanies an expansion of the calmodulin molecule under conditions of high electrostatic stress.

Animals↗
PubMed1983

Fluorescence decay studies of modified dinucleoside monophosphates containing 1-N6-ethenoadenosine.

Five dinucleoside monophosphates containing 1-N6-ethenoadenosine (epsilon A) have been studied using fluorescence measurements. The fluorescence spectra of these dinucleoside monophosphates are almost the same as the fluorescence spectrum of epsilon AMP. Fluorescence quantum yields of these dimers are greatly reduced compared to that of epsilon AMP. Intramolecular base-base interactions may be responsible for fluorescence quenching. It is found that the fluorescence decay kinetics does not obey a simple decay law but that the decay data can be well described as a sum of three exponentials. This implies that these dimers cannot be characterized as a two-state system, but can be described as systems consisting of three or more conformational states. Sequence effects upon the fluorescence behavior are observed. The fluorescence quenching and decay parameters of Gp epsilon A and Up epsilon A indicate a higher degree of base-base interaction than in their epsilon ApG and epsilon ApU counterparts.

Adenine↗
PubMed1983

Fluorescence decay studies of poly(riboadenylic acid) containing 1-N6-ethenoadenosine.

The fluorescence properties of the 1-N6-etheno derivatives of poly(riboadenylic acid) (poly(rA, epsilon rA)) have been examined. The fluorescence quantum yield of poly(rA, epsilon rA) decreases with an increase in the degree of the epsilon A substitution and is much smaller than that for epsilon AMP even for low degrees of epsilon A substitution. The nearest-neighbor interactions such as epsilon-adenine-adenine and epsilon-adenine-epsilon-adenine may be responsible for this behavior. It is found that the fluorescence decay kinetics obeys a three-exponential decay law for poly(rA, epsilon rA), suggesting that there exist at least three different stacked conformational states.

Adenine↗
PubMed1982

Synergistic effect of Ca2+ and Mg2+ upon the interaction of phosphorylase kinase with glycogen.

Phosphorylase kinase (ATP: phosphorylase-b phosphotransferase, EC 2.7.1.38) forms molecular complexes with glycogen over a range of conditions. The extent of interaction at pH 7.0, 20 degrees C, is greatly enhanced in the simultaneous presence of Ca2+ and Mg2+. If either cation is absent, the extent of complex formation is substantially reduced. Significant changes in the physical properties of phosphorylase kinase are observed in the presence of Ca2+ plus Mg2+, suggesting that a conformational change may occur under these conditions.

Animals↗
PubMed1982

Librational modes in liganded and unliganded hemoglobin as seen by fluorescence spectroscopy.

Hemoglobin labeled with N-iodoacetylaminoethyl-5-naphthalene-1-sulfonate at the beta-93 cysteine shows normal allosteric properties including the Bohr effect and heme-heme-interaction, and the oxygen affinity is somewhat increased. Viscosity-resolved correlation times obtained from direct measurement of the decay of fluorescence anisotropy show values of 4.15 +/- 0.23 and 9.13 +/- 0.46 ns for the liganded and unliganded derivatives, respectively, in 0.05 M tris buffer, pH 7.5 at 15 degrees C. Addition of 1 mM inositol hexaphosphate to the liganded and unliganded derivatives results in correlation times of 9.31 +/- 0.87 and 43.69 +/- 16.04 ns, respectively. Similar values of correlation times are obtained from Perrin plots. Considering that the correlation times expected for the single subunits, the dimers, and the tetramers of the system are approximately 10-15, 20-30, and above 40 ns, respectively, it can be concluded that the molecule of hemoglobin becomes increasingly rigid upon removal of ligands and addition of inositol hexaphosphate. These observations support the hypothesis of a functional relevance of the internal flexibility of hemoglobin.

Fluorescent Dyes↗
PubMed1981

Molecular dynamics of hemoglobin subunits as seen by fluorescence spectroscopy.

Fluorescent conjugates of beta A subunits and their respective heme-free derivatives have been prepared in which a 1,5-N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonate probe has been specifically placed at the beta-93 or beta-112 cysteine. The fluorescence anisotropy decay and static fluorescence polarization of these conjugates have been examined. Fluorescence measurements have also been made using 1-anilino-8-naphthalenesulfonate complexes, as well as the intrinsic fluorescence of the tryptophan groups. For the cases of the beta-93 and beta-112 conjugates there is substantial evidence for internal rotational freedom of the subunits. The internal mobility of the polypeptide is especially pronounced for the beta-112 conjugate. In contrast, the 1-anilino-8-naphthalenesulfonate probe placed within the heme pocket shows no indication of any rotation, other than that associated with the entire beta-subunit. Tryptophan fluorescence has been measured for the apo-beta subunits and for the peptides beta (1-55) from hemoglobins A and S. Perrin-Weber plots show the presence of multiple rotational modes suggesting mobility of the tryptophan groups.

Anilino Naphthalenesulfonates↗
PubMed1980

Ligand binding and self-association of proteins.

This review deals with the quantitative analysis of protein self-association and ligand binding when there is a signficant mutual influence of the two processes. The particular points of interest are the evaluation of the pertinent equilibrium constants and the prediction and interpretation of concentration profiles in transport experiments, including gel elution and sedimentation velocity. The case of dimertetramer equilibrium with four binding sites for ligand is considered in detail. Three representative experimental studies are described which deal with hemoglobin, phosphorylase b, and tubulin.

Animals↗
PubMed1980

Structural changes induced in glycogen phosphorylase b by the binding of glucose and caffeine.

The allosteric inhibitors glucose and caffeine cause significant structural alterations in glycogen phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1). Both cause a masking of two sulfhydryl groups and a reduction of binding affinity for AMP. Caffeine produces an alteration in the microenvironment of the binding site for 1-anilin-naphthalene-8-sulfonate, resulting in a decrease of quantum yield of fluorescence and a change in spectral distribution. The binding of glucose is exothermic with an enthalpy of binding of -6.0 kcal/mol. Glucose causes a change in the molecular ellipticity in the pyridoxal-5'-phosphate region. The implications of these results are discussed.

Adenosine Monophosphate↗
PubMed1979

Ligand binding and self-association of phosphorylase b.

The mutual influence of ligand binding and self-association has been examined for phosphorylase b in the presence of a series of small ligands. The stepwise equilibrium constants describing the mutual dependence have been evaluated and discussed in terms of possible molecular mechanisms.

Adenosine Monophosphate↗
PubMed1978

Physical properties of poly (3,N4-ethenocytidylic acid).

Homopolymers of etheno CMP have been prepared by the action of polyribonucleotide phosphorylase upon etheno CDP. At alkaline pH the optical properties are consistent with a structure consisting of partially helical single-stranded chains whose helical regions are stabilized by base stacking. At acid pH the degree of helicity increases markedly. The degree of cooperativity displayed by the helix leads to coil transition induced by pH or temperature is less than for the case of polyribocytidylic acid. In the presence of acridine orange the alkaline form develops a strong extrinsic CD spectrum.

Acridines↗
PubMed1977

Structural changes accompanying the removal of pyridoxal 5'-phosphate from phosphorylase b.

The changes in physical properties accompanying the removal of pyridoxal 5'-phosphate from glycogen phosphorylase b have been examined. The apoenzyme retains a high degree of structural rigidity, as determined from the time decay of anisotropy. The bulk of the secondary structure remains intact, although a significant change in circular dichroism indicates some degree of alteration. The mobility of a sulfhydryl-linked spin label increases. The restoration of pyridoxal 5'-phosphate reverses this effect, with indication of interaction between subunits. One or more new binding sites for 1-anilinonaphthalene-8-sulfonate appear for the apoenzyme. The kinetics of the recombination of pyridoxal 5'-phosphate with the apoenzyme, as monitored by difference spectra, indicate a high activation energy for the process. The apoenzyme is a reversibly associating system at 20-30 degrees C, pH 7.0.

Animals↗
PubMed1977

The interactive binding of two ligands by an allosteric protein.

The equilibrium constants describing the simultaneous binding of the ligands AMP and 1-anilinonapthalene-8-sulfonate (ansyl) by phosphorylase b have been evaluated. The binding of a single molecule of AMP per dimer unit causes only a moderate reduction in the binding constant for a single molecule of ansyl, but reduces that for a second molecule of ansyl by 10-fold. The binding of two molecules of AMP per dimer unit almost abolishes the binding of a single molecule of ansyl.

Adenosine Monophosphate↗