Isolation of a high-molecular-weight high-mobility-group-type non-histone protein from hen oviduct.
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ABSTRACT:
SUBMITTER: Teng CS
PROVIDER: S-EPMC1186326 | biostudies-other | 1978 Dec
REPOSITORIES: biostudies-other
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Project description:Nuclear high-mobility-group (HMG) proteins were isolated from hen oviduct. These were proteins HMG-1, -2, -3, -14 and -17, which are equivalent to the classification of calf thymus HMG proteins. Hen oviduct proteins HMG-1 and -2 were individually isolated by HCIO4.extraction and CM-Sephadex chromatographic separation. Their mol.wts. were determined as 28 000 and 27 000, respectively. The proteins have a high content of acidic and basic amino acids. The association of proteins HMG-1 and -2 with the genome of hen oviduct nuclei was probed by a limited digestion with nucleases. Hen oviduct nuclei were incubated with deoxyribonuclease I or micrococcal nuclease until 10% of the DNA was digested. The nuclear suspension was centrifuged and the contents of proteins HMG-1 and -2 in the supernatant and sediment fractions were analysed by polyacrylamide-gel electrophoresis. HMG proteins were found to be preferentially released by micrococcal-nuclease digestion rather than by deoxyribonuclease I.
| S-EPMC1161198 | biostudies-other
Project description:Peptides produced by CNBr cleavage of non-histone chromosomal protein HMG 2 (CNBr peptides) were isolated and characterized, and their partial sequences were determined. The present sequence data account for over half of the sequence of the protein HMG (high-mobility-group) 2 molecule, and, together with previously published results, provide interesting information on the charge distribution within the molecule. Comparison of the CNBr-peptide-sequence data for protein HMG 2 with the previously published data on the CNBr peptides from protein HMG 1 reveals extensive sequence homology between the two proteins. Detailed evidence for the amino acid-sequence data has been deposited as Supplementary Publication SUP 50095 (6 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1978) 169, 5.
| S-EPMC1161205 | biostudies-other
Project description:The interaction of the non-histone chromosomal protein HMG (high-mobility group) 1 with histone H1 subfractions was investigated by equilibrium sedimentation and n.m.r. sectroscopy. In contrast with a previous report [Smerdon & Isenberg (1976) Biochemistry 15, 4242--4247], it was found, by using equilibrium-sedimentation analysis, that protein HMG 1 binds to all three histone H1 subfractions CTL1, CTL2, and CTL3, arguing against there being a specific interaction between protein HMG 1 and only two of the subfractions, CTL1 and CTL2. Raising the ionic strength of the solutions prevents binding of protein HMG 1 to total histone H1 and the three subfractions, suggesting that the binding in vitro is simply a non-specific ionic interaction between acidic regions of the non-histone protein and the basic regions of the histone. Protein HMG 1 binds to histone H5 also, supporting this view. The above conclusions are supported by n.m.r. studies of protein HMG 1/histone H1 subfraction mixtures. When the two proteins were mixed, there was little perturbation of the n.m.r. spectra and there was no evidence for specific interaction of protein HMG 1 with any of the subfractions. It therefore remains an open question as to whether protein HMG 1 and histone H1 are complexed together in chromatin.
| S-EPMC1161647 | biostudies-other
Project description:Total chromosomal HMG (high-mobility-group) proteins have been isolated from oestrogen-stimulated chick oviduct. The antibodies against these proteins were induced in mice and subsequently their spleen cells were fused with myeloma cells to form hybridomas. A highly purified HMG protein, 17, was used to select for the hybridomas that produce antibody against HMG protein 17. The hybridomas were cultured and injected into mice to produce ascites. The antibody against HMG protein 17 in the IgG (immunoglobulin G) fraction of the ascites fluid was obtained by Protein A-Sepharose column chromatography. We have devised a solid-phase radioimmunoassay and enzyme-linked serological assay for the detection and characterization of this antibody directed against HMG protein 17. This anti-(HMG protein 17) IgG interacted only with HMG protein 17, but not with other chromosomal proteins, e.g. histone H1, "95K protein' (a chick oviduct-specific chromosomal protein) and HMG proteins 1, 2 and 14. The monospecific nature of this anti-(HMG protein 17) IgG fraction is confirmed.
| S-EPMC1158252 | biostudies-other
Project description:The high-mobility-group (HMG) non-histone chromosomal proteins from calf thymus, liver, spleen and kidney were extracted, and fractionated by CM-Sephadex chromatography and trichloroacetic acid precipitation. The isolated proteins HMG 1, HMG 2 and HMG 17 from the tissues were compared by polyacrylamide-gel electrophoresis, isoelectric focusing and amino acid analysis. The results show that the three proteins are very similar in the tissues studied, implying a lack of tissue specificity.
| S-EPMC1185803 | biostudies-other
Project description:Antiserum against chick oviduct high-mobility-group protein 1 (HMG 1) has been induced in the rabbit. With this antiserum, immunobiochemical techniques have been used to probe the quantitative change of HMG 1 in the cellular fractions of chick oviduct before or after oestrogen stimulation. HMG 1 is detectable in the cytosol, microsomal and nuclear fraction of the chick oviduct cell. After administration of oestrogen to young chicks in vivo for 5 days, the quantity of HMG 1 is increased 4-fold in the cytosol, 3.5-fold in the microsomal fraction and 1.6-fold in the nuclear fraction. The finding of large amounts of HMG 1 in cytoplasm of oviduct cell is not likely due to its leakage from the nucleus. We anticipate that HMG 1 is synthesized in the cytoplasm and then transported into the nucleus. The synthesis and transportation of HMG proteins is probably regulated by oestrogen.
| S-EPMC1163213 | biostudies-other
Project description:[(3)H]Triamcinolone acetonide (15nm) was incubated with cytosol (150000g fraction) prepared from oviducts of egg-laying hens. The extent of steroid binding, as determined by charcoal assays, was greatest between 2-4h at 4 degrees C. A similar time curve was obtained when cytosol preparations were first fractionated with (NH(4))(2)SO(4) before labelling. The addition of 10mm-Na(2)MoO(4) or 10mm-ATP during the incubation of hen oviduct cytosol with [(3)H]triamcinolone acetonide lowered the extent of steroid binding. The presence of glycerol (20%), however, increased the extent of [(3)H]triamcinolone acetonide binding in cytosol fractions from chick (330%) and hen (160%) oviducts. The [(3)H]triamcinolone acetonide-receptor complex was stable for over 4h at 4 degrees C, but dissociated rapidly at 37 degrees C, exhibiting a half-life of about 10min. The presence of 10mm-Na(2)MoO(4) and 10mm-ATP or both had a small protective effect on the dissociation of [(3)H]triamcinolone acetonide-receptor complex. The receptor from hen oviduct showed significant affinity for unlabelled triamcinolone acetonide, cortisol, compound R5020 and dihydrotestosterone and, to a lesser extent, for oestradiol, oestrone and progesterone. Diethylstilboestrol treatment of immature chicks appeared to induce a more specific binder, which showed affinity for unlabelled triamcinolone acetonide, cortisol and compound R5020 only. Scatchard analysis of [(3)H]triamcinolone acetonide binding in hen oviduct cytosol revealed a K(d) value of 6.4nm. The steroid-receptor complex sedimented as a 7-8S and a 4S entity on low-salt (0.01m-KCl)- and high-salt (0.3m-KCl)-containing sucrose gradients respectively. The cytosol [(3)H]triamcinolone acetonide-receptor complex showed no affinity for ATP-Sepharose or DNA-cellulose, but acquired this ability on heat activation (23 degrees C, 40min). The data indicate the avian oviduct possesses a high-affinity binding molecule that fulfils the criteria of a glucocorticoid receptor.
| S-EPMC1163214 | biostudies-other
Project description:After labelling of mouse liver nuclei with [gamma-32P]ATP in vitro, 10-20% of the radioactivity incorporated into the saline-soluble nuclear and HAP2 chromatin fractions was located in a low-molecular-weight component (component 10) with pI near 4.5 in urea. By using combinations of ion-exchange chromatography, preparative thin-layer isoelectric focusing and gel filtration, this component was isolated from both nuclear fractions. Recovery from the saline-soluble fraction was poor under conditions that allow endogenous phosphatases to be active. Component 10 was shown to be a phosphoprotein on the basis of enzyme-digestion experiments and the detection of phosphoserine and phosphothreonine. The 32P radioactivity did not appear to be associated with phosphorylated basic amino acids. Its molecular weight was determined by gel chromatography and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels as approx. 10000, and tryptic digestion of the reduced carboxymethylated protein in urea yielded two 32P-labelled peptides. It has not been possible as yet to assign a function to component 10, though its similarity to other low-molecular-weight acidic proteins is discussed.
| S-EPMC1186038 | biostudies-other
Project description:Detergent-solubilized hen oviduct signal peptidase has been characterized previously as an apparent complex of a 19 kDa protein and a 23 kDa glycoprotein (GP23) [Baker & Lively (1987) Biochemistry 26, 8561-8567]. A cDNA clone encoding GP23 from a chicken oviduct lambda gt11 cDNA library has now been characterized. The cDNA encodes a protein of 180 amino acid residues with a single site for asparagine-linked glycosylation that has been directly identified by amino acid sequence analysis of a tryptic-digest peptide containing the glycosylated site. Immunoblot analysis reveals cross-reactivity with a dog pancreas protein. Comparison of the deduced amino acid sequence of GP23 with the 22/23 kDa glycoprotein of dog microsomal signal peptidase [Shelness, Kanwar & Blobel (1988) J. Biol. Chem. 263, 17063-17070], one of five proteins associated with this enzyme, reveals that the amino acid sequences are 90% identical. Thus the signal peptidase glycoprotein is as highly conserved as the sequences of cytochromes c and b from these same species and is likely to be found in a similar form in many, if not all, vertebrate species. The data also show conclusively that the dog and avian signal peptidases have at least one protein subunit in common.
| S-EPMC1130799 | biostudies-other
Project description:Two new, closely similar, acidic proteins were extracted and purified from calf thymus and designated AP-X and AP-Y (acidic proteins X and Y). They contain about 33% acidic residues, mostly non-amidated, and 20% lysine, but no arginine, tyrosine, histidine or tryptophan. There is a single phenylalanine residue in a molecular weight of approx. 5000. Circular dichroism and proton nuclear magnetic resonance show that they do not take up secondary or tertiary structure in free solution, as expected from the low content of hydrophobic amino acids. They appear structurally related to the high-mobility-group proteins HMG 14 and 17. Controlled extraction experiments indicate that proteins AP-X and AP-Y are at least partially located in the calf thymus nucleus.
| S-EPMC1153892 | biostudies-other