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Biomedical subjects

L H Piette

Publications and source records attributed to L H Piette.

At least 37 records · Page 2Linked to original sources
PubMed1980

Electron spin resonance studies of bovine plasma amine oxidase. Probing of the environment about the substrate-liberated sulfhydryl groups in the active site.

A series of nitroxide spin-labeled reagents have been employed to explore the environment of the cysteine residues in bovine plasma amine oxidase. When the enzyme was reduced by substrate or phenylhydrazine, 1 essential sulfhydryl residue/subunit was liberated. This cysteine residue was reacted then with the spin label 3-(maleimido-methyl)-2,2,5,5-tetramethyl-1-pyrrolinyloxyl. The ESR spectra of the labeled enzyme derivatives suggested that this essential sulfhydryl residue is located in a pocket, whereas the nonessential sulfhydryl residues are probably located near the surface. By varying the length of the nitroxide spin-labeled N-ethylmaleimide derivatives, it was determined that the liberated essential cysteine residues are in a restricted environment. The ESR spectral data also suggested that the nitroxide radical and the essential copper in the enzyme do not interact with one another. The effect of ionic strength, pH, and urea denaturation on the environment of the essential sulfhydryl residue were also investigated.

Amine Oxidase (Copper-Containing)↗
PubMed1980

ESR spin label studies of the nucleosome core particle and histone core.

An imidazole spin label has been used to study the accessibility and conformational state of tyrosines in both the nucleosome core particles and histone core extracted from chicken erythrocytes. About 40% of the tyrosyl residues in the histone core can be labeled under nondenaturing conditions. However, less than 15% of the tryosyls in the nucleosome core particle can be labeled even at 200- to 300-fold M excess of label. The effect of urea on the conformational state of the spin-labeled tyrosyls in both the nuclesome core particles and the histone core has been studied. Ionic effects on the spin-labeled nucleosome core have been investigated. Several conformational transitions are observed in the range of 1 mM NaCl to 2.5 M NaCl. Three major transitions are found at 0.1 M to 0.6 M, 0.7 M to 1.8 M and 2 M to 2.5 M NaCl, respectively. The observed changes can be interpreted as swelling and conformational change of the inner histone core, gradual separation of DNA from the histone core, and tightening of the histone core.

Animals↗
PubMed1980

Spin-label study of histone H1-DNA interaction. Comparative properties of the central part of the molecule and the N and C-amino tails.

Covalent spin labelling of lysine and arginine residues in histone H1 under specified conditions allows the study of histone-DNA interaction. When the labelled histone is free in solution, the electron spin resonance spectrum is characteristic of a label moving in a viscous medium while the label becomes fully immobilized upon the interaction of histone and DNA. This property is used to study histone-DNA interactions under various conditions of ionic strength and thermal denaturation. On the other hand, spin label probes in the globular part of the molecule shows no strong immobilization upon binding to linear DNA, suggesting a special mechanism for the binding of histone H1 to chromatin.

Animals↗
PubMed1976

Conformational state of DNA in chromatin subunits. Circular dichroism, melting, and ethidium bromide binding analysis.

This study compares some physical properties of DNA in native chromatin and mono-, di-, trinucleosomes obtained after mild micrococcal nuclease digestion. Melting curves and derivatives are shown to be very similar from one sample to another although a shift from 79 to 82 degrees C is observed between the mainly monophasic peak of multimers and chromatin. Careful analysis of the positive band of the circular dichroism spectra shows the appearance of a shoulder at 275nm, the intensity of which increases from the mono- to the di- and trinucleosome. This shoulder is maximum for native chromatin. At the same time binding isotherms of ethidium - bromide are characterized by two highly fluorescent binding sites for all the samples but the product KN of the apparent binding constant of the higher affinity binding sites by the apparent number of those sites increases from the mono- to the di- and trinucleosome. There again the valus is maximum for native chromatin. Such results strongly suggest that the native state of chromatin requires something more than the indefinite repeat of an elementary subunit.

Animals↗
PubMed1976

Electron spin resonance spin-label studies of intercalation of ethidium bromide and aromatic amine carcinogens in DNA.

A spin-labeled technique has been developed for the study of carcinogen-DNA intercalation mechanisms utilizing a modified ethidium bromide as a model ligand and specifically synthesized spin labels of carcinogenic aromatic amines such as 2-aminofluorene, 2-aminoanthracene, and 6-aminochrysene. The technique takes advantage of the characteristic electron spin resonance anisotropy of the corresponding spin labels complexed with DNA. Analysis of the orientation-dependent information thus obtained has clearly shown that the lignad moieties in all 4 respective complexes are accommodated in adjacent base-pair layers of the DNA with the nitroxide reporter oriented in 1 preferential direction. The geometry of the nitroxide rings with respect to their respective constraining ligands in a particular complex appeared to be largely dependent upon the size of the ligand moiety and the position of the reporter attachment on the ligand molecule. A coplanar configuration is most favored for the spin labels in which the ligand moiety per se is a 3-fused ring with the nitroxide substituted at a position on or close to the longitudinal end of the ligand molecule. Temperature studies in which the heat-induced release of the bound label is monitored by electron spin resonance gives an accurate measure of the complex dissociation characteristics that appear to parallel the helical coil cooperative melting transition monitored by absorbance measurements. The extent of dissociation of the bound spin labels parallels almost exactly the melting, i.e., unzipping of the DNA double helix, suggesting that the spin labels are immobilized by intercalation at the site on the DNA directly involved in the maintenance of the double helical structure. Analysis of the electron spin resonance spectra of the complexes in terms of the extent of immobilization indicates that, for aromatic amine spin labels, two different binding species exist, one strongly immobilized presumably at a guanine-cytosine base-pair region and the other partially immobilized in a region other than guanine-cytosine base pair. Competitive binding of the labels with respect to their parent ligand and binding isotherm results show that nitroxide substitution does not affect the type of binding but does affect the extent of binding, i.e.. binding constant with the labeled compound having a lower affinity.

Amines↗
PubMed1975

In vivo electron spin resonance in rats.

In vivo ESR studies have been made on Wistar and Sprague-Dawley rats using travelling wave helices implanted in the left lobe of the liver. Tissue regenerates completely around the Teflon-enclosed turns of the coil and histological studies have shown that it is composed of liver cells and connective tissue. Electron spin resonance studies of this tissue in vitro reported by Commoner and Ternberg in 1961 have demonstrated that it exhibits the 'tissue radical' signal. The implanted coils have detected, in vivo, a three-line spectrum due to a strong concentration of 4-hydroxy- 2,2,6,6-tetramethylpiperidine-1-oxyl free radical ('tempol'), a nitroxide spin label, injected either intramuscularly, intraperitoneally or intravenously into the experimental animal immediately prior to the measurement. They have also detected, in vivo, a similar three-line spectrum due to 'spin-labelled chlorpromazine', a tranquillizing drug to which this nitroxide spin label has been attached, injected intramuscularly or intraperitoneally into the animal immediately prior o the measurement. Work is currently in progress to increase the in vivo sensitivity of the implanted helices by menas of improved coupling techniques and the use of a time-averaging computer.

Animals↗