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Biomedical subjects

C von Holt

Publications and source records attributed to C von Holt.

At least 37 records · Page 2Linked to original sources
PubMed1986

Rat liver glucocorticoid receptor isolated by affinity chromatography is not a Mg2+- or Ca2+-dependent protein kinase.

The glucocorticoid hormone receptor (92 kDa), purified 9000-fold from rat liver cytosol by steroid affinity chromatography and DEAE-Sephacel chromatography, was assayed for the presence of protein kinase activity by incubations with [gamma-32P]ATP and the photoaffinity label 8-azido-[gamma-32P]ATP. Control preparations isolated by affinity chromatography in the presence of excess steroid to prevent the receptor from binding to the affinity matrix were assayed for kinase activity in parallel. The receptor was not labeled by the photoaffinity label under photoactivation conditions in the presence of Ca2+ or Mg2+. A Mg2+-dependent protein kinase (48 kDa) that could be photoaffinity labeled with 8-azido-ATP copurified with the receptor. This kinase was also present in control preparations. The kinase could phosphorylate several minor contaminants present in the receptor preparation, including a protein (or proteins) of similar molecular weight to the receptor. The phosphorylation of 90-92-kDa proteins was independent of the state of transformation or steroid-binding activity of the receptor. These experiments provide direct evidence that neither the glucocorticoid receptor nor the 90-92-kDa non-steroid-binding protein associated with the molybdate-stabilized glucocorticoid receptor possesses intrinsic Ca2+- or Mg2+-dependent protein kinase activity.

Animals↗
PubMed1986

Covalent labelling of histones with aurothiomalate. Gold-labelled H2A-H2B dimers assemble to octamers, which form crystalline helical tubes.

Histone octamers were covalently labelled with aurothiomalate at amino groups by the method of carbodiimide activation. The labelling procedure was demonstrated to result in the specific covalent coupling through a single bond of the heavy metal atom label to protein amino groups. Such octamers were dissociated to yield soluble H2A-H2B dimers containing three gold atoms per dimer. The dimers were reconstituted with native H3-H4 tetramers to form labelled octamers, which were crystallized to form helical tubes. This strongly suggests that this procedure resulted in minimal changes of protein conformation.

Affinity Labels↗
PubMed1985

The amino acid sequence of wheat histone H2A(1). A core histone with a C-terminal extension.

The complete amino acid sequence (145 residues) of histone variant H2A(1) from wheat germ Triticum aestivum cultivar T4 has been established from Edman degradation of large overlapping fragments. The sequence of histone variant H2A(1) differs from the homologous calf histone in 61 amino acid positions. These differences include an extension of H2A(1) by 19 amino acids at its carboxyl end.

Amino Acid Sequence↗
PubMed1984

Formaldehyde and glutaraldehyde in the fixation of chromatin for electron microscopy.

Glutaraldehyde and formaldehyde have been used to fix chromatin core particles for electron microscopy. Glutaraldehyde crosslinks protein only, whereas formaldehyde crosslinks protein and DNA. This is confirmed by the observation that the detergents sodium dodecyl sulphate, Sarkosyl NL 35 and benzylalkyldimethyl ammonium chloride separate the DNA from the protein in the case of glutaraldehyde fixed core particles but not in the case of formaldehyde fixed core particles. The fixative used in the preparation must therefore be considered as a further variable when evaluating electron microscopic images of chromatin.

Aldehydes↗
PubMed1983

The identity of conformational states of reconstituted and native histone octamers.

Histone octamers were reconstituted from the following preparations: (a) natural histone H3-H4 tetramer and histone H2A-H2B dimer, either selectively extracted from chromatin with solutions chloride or prepared by dissociation of the natural octamer; (b) acid-denatured core histones, either an unfractionated mixture or individually purified proteins. Complexes assembled from these histones elute from exclusion chromatography columns with octamer size as verified by cross-linking with dimethylsuberimidate. The reconstituted octamers all crystallize in the same form of helical tubes as the natural octamer.

Animals↗
PubMed1983

Octamer reconstitution from acid-extracted chicken erythrocyte histones.

Histone octamers have been reconstituted from acid-extracted chicken erythrocyte histones. By the criteria of molecular size on exclusion chromatography as well as sedimentation velocity and conformational properties established by circular dichroism, fluorescence spectroscopy and imido-ester cross-linking, the reconstituted octamers have a structure identical to that of salt-extracted octamers.

Animals↗
PubMed1982

The effect of Ca2+ on the stability of chicken erythrocyte histone octamers.

Histones can be extracted from chicken erythrocyte chromatin with 2 M CaCl2 10 mM Tris (pH 7.4). The core histones so extracted exist as H3-H4 tetramers and H2A-H2B dimers since calcium at greater than 0.5 M result in dissociation of the histone octamer. Reconstitution of octamers occurs on removal of the Ca2+ by dialysis. Although less than 0.5 M calcium do not result in octamer dissociation, perturbations in the structure can be detected by CD and tyrosine fluorescence spectroscopy.

Animals↗
PubMed1982

The reconstitution of histone H3-H4 tetramer from acid extracted histones in the absence of urea.

Simple mixing of acid purified histones H3 and H4 in equimolar quantities at low ionic strength near pH 7 does not yield the tetramer but rather a high Mr aggregate. Dialysis of acid extracted total or core histones into 2 M NaCl 150 mM phosphate (pH 7.4) followed by fractionation of the histone complexes at lower ionic strength (150 mM NaCl) results in an H3-H4 tetramer of a structure identical to that derived from salt-extracted histones. Dialysis of acid extracted total or core histones directly into the lower ionic strength buffer with subsequent fractionation, results in H3-H4 tetramer of closely similar structure.

Animals↗
PubMed1981

Interaction of the activated cytoplasmic glucocorticoid hormone receptor complex with the nuclear envelope.

Highly purified activated cytoplasmic glucocorticoid hormone receptor binds with high affinity to sites in the nuclear envelope. Nuclear envelope fragments can be isolated from purified chromatin. They bind activated cytoplasmic glucocorticoid receptor with the same equilibrium constant as nuclear envelopes. The presence of envelope components in chromatin is confirmed by the virtual identity of the gel electrophoretic glycoprotein pattern of nuclear envelope, chromatin nonhistones, and nuclear envelope fragments from chromatin.

Animals↗
PubMed1980

The histone H2B from the sperm cell of the starfish Marthasterias glacialis.

A single species of histone H2B with a blocked N-terminus has been isolated from sperm of the starfish Marthasterias glacialis. The amino-terminal region of the histone H2BMarthasterias shows no similarity to the multiple forms of histones H2B found in the sperm of sea urchins, but there are distinct homologies to the histone of Patella granatina and that of the embryo of Parechinus angulosus. The sequence of the 90 carboxy terminal amino acids is highly conserved as in the other histones H2B.

Amino Acid Sequence↗