PubMed Health⌕ Search

Biomedical subjects

A Ehrenberg

Publications and source records attributed to A Ehrenberg.

At least 19 recordsLinked to original sources
PubMed1992

The structure of the mammalian antibacterial peptide cecropin P1 in solution, determined by proton-NMR.

Cecropins are peptides with antibacterial activity originally found in insects. Recently a cecropin-type peptide was isolated from pig intestine. This peptide, porcine cecropin P1, which has 31 amino acid residues and is not amidated in the C-terminus, has been synthesized, purified, and investigated by CD and two-dimensional 1H-NMR at pH 5.0 in aqueous solution with 30% (by vol.) 1,1,1,3,3,3-hexafluoro-2-propanol. All proton resonances have been assigned except for the N-terminal serine. Using constraints derived from NOE connectivities and 3JNH alpha-coupling constants, three-dimensional structures have been calculated by means of a distance-geometry program. Some of these structures have been refined by energy minimization and restrained molecular dynamics. The structures reveal an alpha-helix of approximately seven turns along nearly the full length of the peptide. The central part of the helix is very well defined by the NMR constraints. Also the chemical shifts of the alpha protons and the results of CD measurements are in accord with this structure, which is different from the helix-hinge-helix structure earlier found in cecropin A and related peptides. In the alpha-helix of cecropin P1 there is a long amphipathic section, of 4-5 turns, and a short hydrophobic section of one to two turns, with an intervening Glu-Gly sequence, which is a potential bend-forming section. The helix can easily span a lipid membrane.

Amino Acid Sequence↗
PubMed1992

Studies on ribonucleoside-diphosphate reductase from Escherichia coli. The product dCDP is a competitive inhibitor and functions as a spectroscopic probe for the substrate binding site; demonstration by enzyme kinetics and 1H NMR.

Ribonucleoside-diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two protein subunits, R1 of 171.5 kDa and R2 of 86.8 kDa, and catalyzes the reduction of all four common ribonucleoside diphosphates. In a search for ligands that bind weakly to the enzyme active site and may be in fast exchange suitable for NMR studies, we have found that the product dCDP is a competitive inhibitor. Kinetics with CDP as substrate shows Km = 4.8 x 10(-5) M and dCDP inhibits with Ki = 1.6 x 10(-4) M. With an assumed diffusion limited binding rate approximately less than 10(9) M-1s-1, the dissociation rate of dCDP would be approximately less than 10(5) s-1. In 1H-NMR experiments studying linewidths, i.e. spin-spin relaxation, dCDP is indeed demonstrated to be in fast exchange. Enzyme subunit R1 causes a line broadening of dCDP resonances. Unexpectedly less broadening was observed when subunit R2 combined with R1. No paramagnetic interaction from the tyrosyl radical of R2 could be detected. It is concluded that dCDP is a promising NMR probe for studies of active-site properties of the enzyme.

Binding Sites↗
PubMed1992

Binding of the competitive inhibitor dCDP to ribonucleoside-diphosphate reductase from Escherichia coli studied by 1H NMR. Different properties of the large protein subunit and the holoenzyme.

Ribonucleoside-diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two nonidentical subunits, proteins R1 and R2. The binding of the product dCDP to protein R1 and to the holoenzyme R1R2 has been studied by means of 1H-NMR spectroscopy. In presence of the effector dTTP at 25 degrees C, dCDP was found to be in rapid exchange between the binding sites and the solvent which results in a broadening of the dCDP resonances. When both proteins R1 and R2 are present, so that the complex R1R2 is formed, a smaller broadening is observed than with protein R1 alone. No further linewidth decrease was observed when the [R2]/[R1] ratio exceeded 1. The binding constant of dCDP to R1 or R1R2 is the same, Kd = 0.9 mM. The smaller broadening of the dCDP resonances observed with the complex R1R2 as compared with R1 may be explained by the combination of two effects: (a) the overall tumbling time of the protein will increase when going from R1 to R1R2, which will cause the broadening to increase correspondingly, and (b) a twofold decrease of the number of binding sites in rapid exchange, which will decrease the broadening by a factor of 0.5. The effect of R2 without iron (apoR2) is reduced compared with native R2, probably because of some denatured proteins, while a C-terminal peptide from R2 did not cause any narrowing at all.

Binding Sites↗
PubMed1978

Magnetic susceptibility of laccases and ceruloplasmin.

1. Recent magnetic susceptibility measurements on laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) from the lacquer tree Rhus vernicifera showed a deviation from Curie behaviour above 50 K, which was taken as evidence for an antiferromagnetically coupled Cu(II)-Cu(II) pair in the oxidized enzyme. The magnetic susceptibility of this protein has been reinvestigated. Further measurements on laccase from the fungus Polyporus versicolor and human ceruloplasmin (iron(II):oxygen oxidoreductase, EC 1.16.3.1) are presented. 2. The magnetic susceptibility of fungal laccase and lacquer tree laccase can be accounted for by the EPR detectable copper ions in the temperature range 40--300 K. 3. If an antiferromagnetically coupled Cu(II)-Cu(II) pair exists in the laccases, then the coupling, expressed as --J, should be at least of the order of 300 cm-1, as deduced from the Curie dependence of the susceptibility and the sensitivity in our measurements. 4. If an analogy with the laccases is assumed for the EPR invisible copper in ceruloplasmin then a limiting value of the coupling may be deduced also in this case, with --J at least of the order of 200 cm-1.

Catechol Oxidase↗
PubMed1978

The tyrosine free radical in ribonucleotide reductase from Escherichia coli.

One of the two nonidentical subunits of ribonucleotide reductase from Escherichia coli, protein B2, contains an organic free radical required for enzyme activity. Earlier isotope subtitution experiments (Sjöberg, B.-M., Reichard, P. Gräslund, A., and Ehrenberg, A. (1977) J. Biol. Chem. 252, 536-541) demonstrated that the radical was localized to a tyrosine residue of the enzyme and suggested that the spin density of the radical was centered at the methylene carbon of tyrosine. However, additional isotope substitution experiments now show that the spin density of the radical must be delocalized over the aromatic ring of the tyrosine residue.

Electron Spin Resonance Spectroscopy↗
PubMed1978

Formation of microsomal cytochrome P-450 complexes studied by the NMR relaxation of water.

Cytochrome P-450 in microsomes from liver of phenobarbital treated and control rats has been studied by light absorption and by magnetic resonance methods (EPR and NMR). The nuclear relaxation rate of water protons was measured for microsomal suspensions in the presence of various reactants of Type I and II. The change of relaxation rates correlates well with the spin state conversion of the heme iron. No competition between eventual inner-sphere water molecules and the reactants seems to occur. The temperature dependence of the low spin to high spin equilibrium was studied by light absorption and was accounted for in the temperature variation of the molar relaxation rates of the two spin states.

Animals↗
PubMed1978

Characterization of the iron-sulfur protein of the mitochondrial outer membrane partially purified from beef kidney cortex.

The iron-sulfur protein present in the mitochondrial outer membrane has been partially purified from beef kidney cortex mitochondria by means of selective solubilization followed by DEAE-cellulose chromatography. The EPR spectrum of the iron-sulfur protein with g-values at 2.01, 1.94 and 1.89 was well resolved up to 200 K which is unusual for an iron-sulfur protein. Analyses confirmed a center with two iron and two labile sulfur atoms in the protein. By measuring the effect of oxidation-reduction potential on the EPR signal amplitude, midpoint potentials at pH 7.2 were determined both for the purified iron-sulfur protein, +75 (+/- 5) mV, and in prepared mitochondrial outer membrane, +62 (+/- 6) mV. At pH 8.2 slightly lower values were indicated, +62 and 52 mV, respectively. The oxidation-reduction equilibrium involved a one electron transfer. A functional relationship to the rotenone-insensitive NADH-cytochrome c oxidoreductase in the mitochondrial outer membrane is suggested. Both this activity and the iron-sulfur center were sensitive to acidities slightly below pH 7 in contrast to the iron-sulfur centers of the inner membrane.

Animals↗
PubMed1978

Molecular motion and order in oriented lipid multibilayer membranes evaluated by simulations of spin label ESR spectra. Effects of temperature, cholesterol and magnetic field.

A simulation method to interpret electron spin resonance (ESR) of spin labelled amphiphilic molecules in oriented phosphatidylcholine multibilayers in terms of a restricted motional model is presented. Order and motion of the cholestane spin label (3-spiro-doxyl-5alpha-cholestane) incorporated into egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine, pure and in mixture with cholesterol, were studied at various temperatures. With egg yolk phosphatidylcholine identical sets of motional parameters were obtained from simulations of ESR spectra obtained at three microwave frequencies (X-, K- and Q-band). With dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine analyses of the spectra show that phase transitions occur in samples containing up to 30 mol % cholesterol. The activation energy for the motion of the spin label is about three times larger above than below the phase transition, indicating a more collective motion in the lipid crystalline state than in the gel state. In the liquid crystalline state the activation energy is larger in the pure phosphatidylcholines than with cholesterol added. Additions of cholesterol to egg phosphatidylcholine induces a higher molecular order but does not appreciably affect correlation times. This is in contrast to dipalmitoylphosphatidylcholine where both order and correlation times are affected by the presence of cholesterol. The activation energies follow the same order as the transition temperatures: dipalmitoylphosphatidylcholine greater than dimyristoylphosphatidylcholine greater than egg yokd phosphatidylcholine, suggesting a similar order of the cooperativity of the motion of the lipid molecules. Magnetic field-induced effects on egg phosphatidylcholine multibilayers were found at Q-band measurements above 40 degrees C. The cholestane spin label mimics order and motion of cholesterol molecule incorporated into the lipid bilayers. This reflects order and motion of the portions of the lipid molecules on the same depth of the bilayer as the rigid steroid portions of the intercalated molecules.

Chemical Phenomena↗
PubMed1978

A molecular mechanism of the energetic coupling of a sequence of electron transfer reactions to endergonic reactions.

A molecular mechanism of the energetic coupling of a sequence of electron transfer reactions to endergonic reactions is proposed and discussed from a physical point of view. The scheme represents a synthesis of concepts of electron transfer by tunneling and the conformational and chemiosmotic aspects of energy coupling processes. Its relation to existing experimental information and theoretical models is discussed, and further experimental tests are suggested.

Energy Transfer↗
PubMed1978

Fluorescence study of DNA-binding metabolites of benzo(a)pyrene formed in hepatocytes isolated from 3-methylcholanthrene-treated rats.

Hepatocytes and liver microsomes isolated from 3-methylcholanthrene-treated rats metabolize benzo(a)pyrene to products that bind to endogenous DNA and exogenously added calf thymus DNA, respectively. By using a sensitive fluorescence technique, it has been possible to characterize the major DNA-binding metabolite in hepatocytes as being produced by further metabolism of 9-hydroxybenzo(a)pyrene. In microsomes, two products binding to calf thymus DNA were recovered, a major species formed by activation of 9-hydroxybenzo(a)pyrene and a minor fraction formed by further metabolism of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene. The available evidence indicates that the ultimate products responsible for binding to DNA were identical to 9-hydroxybenzo(a)pyrene 4,5-oxide and 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide, respectively. Our data further suggest that metabolic activation of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene and 9-hydroxybenzo(a)pyrene results in quite different DNA:metabolite complexes. The former product(s) seems to be strongly associated with hydrophobic regions in DNA, whereas the latter metabolite(s) appears to be more exposed to the exterior.

Animals↗
PubMed1977

Surface potential effects on metal ion binding to phosphatidylcholine membranes 31P NMR study of lanthanide and calcium ion binding to egg-yolk lecithin vesicles.

31P NMR of phosphatidylcholine (lecithin) from egg-yolk in sonicated vesicles has been measured in the presence of various ions. Addition of Ln3+ or Ca2+ shifted the 31P resonance of the phosphate groups of the outer surface of the vesicles. These shifts were measured at varied lanthanide or Ca2+ concentration at different ionic strengths obtained by addition of NaCl. The shifts induced by Tb3+ and Ca2+ have been analyzed using the theory of the diffuse double layer. Corrections were introduced for the effect of the ionic strength on the activities of the ions. The binding efficiency is shown to be controlled by the electrostatic potential produced by the bound cations at the membrane surface. This potential is slightly modified due to weak chloride binding. Binding constants have been derived.

Binding Sites↗
PubMed1977

Free-radical formation in gamma-irradiated oriented DNA containing electron-affinic radiosensitizers.

Electron paramagnetic resonance (e.p.r.) was used to study the free radicals induced by gamma-irradiation at 77 K in oriented DNA with incorporated electronaffinic radiosensitizing compounds (4-nitroacetophenone, metronidazole, and Ro-07-0582). The observed e.p.r. spectra were compared with those obtained from pure oriented DNA, which had previously been analysed in detail and found to consist mainly of two components, arising from anion redicals on thymine, and cation radicals on guanine. The major spectral changes caused by the radiosensitizers could be explained as a considerable increase in the formation of cationic free radicals on guanine. There were also indications of the formation of anion radicals on the radiosensitizer molecules. No hydrogen-addition free radicals on thymine were observed when the radiosensitized samples were annealed, in contrast to the pure DNA samples.

Acetophenones↗