Two Forms of Histone–Acetyltransferase in High Salt Extracts of Rat Liver Nuclei

1973 ◽  
Vol 51 (8) ◽  
pp. 1177-1194 ◽  
Author(s):  
Peter F. Lue ◽  
A. G. Gornall ◽  
C. C. Liew
Keyword(s):  
Rat Liver ◽  
Histone Acetyltransferase ◽  
Extraction Procedure ◽  
Gel Filtration ◽  
Electrophoretic Analysis ◽  
High Salt ◽  
Sephadex Column ◽  
Nuclear Extracts ◽  
Calf Thymus ◽  
Liver Nuclei

Two forms of 'histone' acetyltransferase (AT I and AT II) were separated by DEAE-Sephadex chromatography of rat liver nuclear extracts prepared by sonication in 0.3 M (NH4)2SO4. A 12-fold greater amount of AT I was not adsorbed on the DEAE-Sephadex column, whereas AT II was adsorbed and was eluted with 0.15–0.2 M (NH4)2SO4. Chromatography of AT I on Sephadex G-200 and cellulose phosphate indicated the presence of additional forms of the enzyme. Optimal conditions for the assay of the enzyme have been determined.In vitro acetylation of calf thymus histone fractions by AT I or AT II showed that at low histone concentration (313 μg/ml), the "arginine-rich" fractions (f2a1 and f3) were acetylated to a greater extent than were the "lysine-rich" fractions (f1 and f2b). However, at a higher concentration of histone (1250 μg/ml) f2a1 acetylation decreased as a result of coprecipitation with the enzyme. Electrophoretic analysis of the acetylated fractions indicated that multiple sites were probably acetylated on f3, f2a2, and f2a1, whereas only one residue was reactive in f1 and f2b.Interconversion of AT I and AT II has been demonstrated and it was shown that [Formula: see text]. AT I contained mainly histones f2a2 and f3, and was converted to AT II by treatment with high salt and gel filtration chromatography. After hypotonic lysis of nuclei and DEAE chromatographic analysis of extracts obtained by washing the nuclear pellet with 0.3 M sodium citrate of NaCl, most of the acetylating activity in these extracts was identified as AT II. Thus the greater amount of AT I observed initially in (NH4)2SO4 extracts was probably a consequence of the sonication step used in this extraction procedure.

A novel class of inactive steroid-binding sites in female rat liver nuclei

1986 ◽  
Vol 110 (1) ◽  
pp. 27-36 ◽  
Author(s):  
D. M. Bechet ◽  
B. N. Perry
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Specific Binding ◽  
Gel Filtration ◽  
Nuclear Extract ◽  
Female Rat ◽  
Rat Liver Nuclei ◽  
Heat Stable ◽  
Nuclear Extracts ◽  
Liver Nuclei

ABSTRACT Nuclear salt extracts from intact female rat liver showed insignificant levels of progesterone-, oestradiol-, testosterone- or dexamethasone-specific binding. However, brief exposure of nuclear extracts to dextran-coated charcoal (DCC) induced binding for all the above classes of steroids. This 'DCC-effect', which was reproduced neither by gel filtration nor by extensive dialysis of the nuclear extract, could not be ascribed to removal of endogenous free or loosely bound steroids. We show that rat liver nuclei contain a class of secondary binding sites (BsII), which exhibit moderate or low affinity for steroid ligands, positive co-operativity, and cross-reaction between classes of steroids. The capacity of BsII sites to bind steroids depends strictly on prior neutralization by DCC of endogenous heat-stable non-dialysable inhibitor(s). The putative roles of these BsII binding sites are discussed in relation to component(s) probably responsible for inhibitory activity. J. Endocr. (1986) 110, 27–36

scholarly journals Effects of polyamines and methylglyoxal bis(guanylhydrazone) on hepatic nuclear structure and deoxyribonucleic acid template activity

1975 ◽  
Vol 151 (3) ◽  
pp. 505-512 ◽  
Author(s):  
K B Brown ◽  
N F Nelson ◽  
D G Brown
Keyword(s):  
Dna Polymerase ◽  
Rat Liver ◽  
Template Activity ◽  
Isolated Rat Liver ◽  
E Coli ◽  
Rat Liver Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei ◽  
Dna Template ◽  
Isolated Rat

1. The interaction of polyamines and methylglyoxal bis(guanythydrazone) (1, 1′-[(methylethanediylidene)-dinitrilo]diguanidine) with isolated rat liver nuclei was investigated by electron microscopy. 2. At 4mM, putrescine was without effect; however, spermidine, spermine or methylglyoxal bis(guanythydrazone) resulted in dispersed chromatin and alterations in nucleolar structure. In addition, spermidine or methylglyoxal bis(guanylhydrazone) caused marked aggregation of interchromatin granules. 3. The DNA template property of calf thymus DNA was examined by using DNA polymerases from Escherichia coli, Micrococcus lysodeikticus and calf thymus in the presence of 0-5 mM-amine. 4. In the presence of DNA polymerase, spermine or methylglyoxal bis(guanylhydrazone) inhibited activity, whereas putrescine or spermidine had much less effect or in some cases stimulated [3H]dTMP incorporation. 5. Template activity which was inhibited by spermine or methylglyoxal bis(guanylhydrazone) could be partially restored by additional DNA or enzyme. 6. When mixed with calf thymus DNA, calf thymus histone inhibited template activity as measured with E. coli DNA polymerase. The template activity of such a ‘histone-nucleate’ could not be restored by putrescine, spermidine, spermine or methylglyoxal bis(guanylhydrazone). 7. DNA template activity of isolated rat liver nuclei was tested by using E. coli DNA polymerase. None of the amines was able to increase the template activity of the nuclear DNA in vitro.

scholarly journals SOLUBLE ENZYMES OF NUCLEI ISOLATED IN SUCROSE AND NON-AQUEOUS MEDIA

1953 ◽  
Vol 37 (2) ◽  
pp. 177-187 ◽  
Author(s):  
Herbert Stern ◽  
A. E. Mirsky
Keyword(s):  
Adenosine Deaminase ◽  
Organic Solvents ◽  
Rat Liver ◽  
Nuclear Membrane ◽  
Aqueous Media ◽  
Isolated Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei ◽  
Soluble Enzymes ◽  
Soluble Components

Nuclei of calf thymus and liver and of rat liver were isolated in sucrose media and a number of their properties studied in relation to those of corresponding nuclei isolated in non-aqueous media with a view to determining their capacity to retain soluble components. The best preparations of sucrose nuclei were obtained from calf thymus. Cytochrome oxidase measurements and DNA/N ratios were far less sensitive than microscopic examination as indicators of purity when rat liver and calf thymus nuclei were compared. No satisfactory preparation of calf liver nuclei was obtained, contamination with whole cells having been appreciable; such preparations, nevertheless, could be used to advantage in the tests undertaken. DNA content of thymus nuclei isolated in sucrose was much the same as that of non-aqueous ones, pointing to a retention of soluble protein under aqueous conditions of isolation. That this net retention of protein was not due to the impermeability of the nuclear membrane was shown by the hydrolysis of the DNA upon addition of some crystalline DNAase to a sucrose suspension of nuclei. A comparative study of liver and thymus nuclei isolated in aqueous and non-aqueous media with respect to the soluble enzymes glucose-6-phosphate dehydrogenase, adenosine deaminase, and nucleoside phosphorylase yielded the following results: 1. Lyophilization of sucrose-isolated nuclei and their extraction with the organic solvents used in the non-aqueous procedure did not inactivate any of the enzymes tested. In the case of thymus the reverse was true, there being a marked increase in activity of all the enzymes studied. 2. In thymus, nucleoside phosphorylase and adenosine deaminase were active to approximately the same extent in nuclei isolated by either procedure. Glucose phosphate dehydrogenase alone was more active in sucrose-isolated nuclei, pointing to the possibility of an adsorption of this enzyme. 3. In rat liver nuclei isolated in sucrose, lyophilization and treatment with organic solvents revealed only the presence of some dehydrogenase. 4. The washing out of soluble enzymes was most markedly demonstrated in the case of calf liver. Only traces of the nucleoside enzymes were found in the sucrose-isolated nuclei, and in the case of the dehydrogenase only a half of that present in the non-aqueous nucleus remained. The main conclusions drawn were as follows:— 1. In sucrose media the nuclear membrane is ineffectual in preventing the inward or outward diffusion of protein. 2. The extent to which soluble proteins are retained by a nucleus isolated in sucrose appears to depend upon internal structural factors, such as the concentration of DNA in the nucleus. 3. With respect to determining the composition of nuclei in terms of soluble components, the sucrose isolation procedure is considered to be of indifferent merit and hence invalid for such a type of analysis.

Detection of 5-Hydroxymethyl-2′-Deoxyuridine in Dna from Calf Thymus, Hela Cells, and Rat Liver Nuclei

1990 ◽  
Vol 9 (2) ◽  
pp. 179-187 ◽  
Author(s):  
Russell C. Cattley ◽  
Susan R. Dietze ◽  
Frank C. Richardson ◽  
James A. Popp
Keyword(s):  
Rat Liver ◽  
Hela Cells ◽  
Rat Liver Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei

scholarly journals Nuclear polyadenylate-binding protein.

1985 ◽  
Vol 5 (8) ◽  
pp. 1993-1996 ◽  
Author(s):  
A B Sachs ◽  
R D Kornberg
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Rat Liver ◽  
Specific Binding ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Single Peak ◽  
Nuclear Extracts ◽  
Dodecyl Sulfate ◽  
Liver Nuclei ◽  
High Degree

Polyadenylate-binding activity can be detected in eluates from sodium dodecyl sulfate gels by a nitrocellulose filter-binding assay. Nuclear extracts from rat liver show a single peak of binding activity at 50 to 55 kilodaltons; cytoplasmic extracts show a single peak at 70 to 80 kilodaltons, corresponding to a 75-kilodalton protein previously described. Similar results are obtained with yeast and mouse fibroblasts, indicating a high degree of conservation of both nuclear and cytoplasmic polyadenylate-binding proteins. The activity from rat liver nuclei has been purified 125-fold on the basis of specific binding to polyadenylate and shows two main bands in sodium dodecyl sulfate gels at 53 and 55 kilodaltons.

Nuclear polyadenylate-binding protein

1985 ◽  
Vol 5 (8) ◽  
pp. 1993-1996
Author(s):  
A B Sachs ◽  
R D Kornberg
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Rat Liver ◽  
Specific Binding ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Single Peak ◽  
Nuclear Extracts ◽  
Dodecyl Sulfate ◽  
Liver Nuclei ◽  
High Degree

Polyadenylate-binding activity can be detected in eluates from sodium dodecyl sulfate gels by a nitrocellulose filter-binding assay. Nuclear extracts from rat liver show a single peak of binding activity at 50 to 55 kilodaltons; cytoplasmic extracts show a single peak at 70 to 80 kilodaltons, corresponding to a 75-kilodalton protein previously described. Similar results are obtained with yeast and mouse fibroblasts, indicating a high degree of conservation of both nuclear and cytoplasmic polyadenylate-binding proteins. The activity from rat liver nuclei has been purified 125-fold on the basis of specific binding to polyadenylate and shows two main bands in sodium dodecyl sulfate gels at 53 and 55 kilodaltons.

scholarly journals Two species of histone acetyltransferase in rat liver nuclei.

1984 ◽  
Vol 259 (13) ◽  
pp. 8372-8377
Author(s):  
M Yukioka ◽  
S Sasaki ◽  
S L Qi ◽  
A Inoue
Keyword(s):  
Rat Liver ◽  
Histone Acetyltransferase ◽  
Rat Liver Nuclei ◽  
Liver Nuclei

scholarly journals High total histone/deoxyribonucleic acid ratios for rat liver nuclei

1973 ◽  
Vol 135 (1) ◽  
pp. 115-123 ◽  
Author(s):  
Subir K. Chanda ◽  
Regina Ickowicz ◽  
Alexander L. Dounce
Keyword(s):  
Amino Acid ◽  
Direct Measurement ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Deoxyribonucleic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Total Histone ◽  
Rat Liver Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei

The ratios of total histone to DNA for rat liver nuclei isolated by four methods as well as for calf liver nuclei isolated by one method were determined by obtaining the ratios of the total areas of the electrophoretic histone peaks for the liver nuclei to the corresponding total area given by a known amount of standard calf thymus histone. Ratios of total histone to DNA of approx. 2 for rat liver nuclei isolated at pH3.8 or 5.8 and for calf liver nuclei isolated at pH3.8 were confirmed twice by the above procedure and also by direct measurement, by the method of Lowry et al. (1951), of histone extracted in 0.2m-H2SO4. The histones of calf thymus, calf liver and rat liver were characterized by their amino acid compositions and by polyacrylamide-gel electrophoresis.

scholarly journals Two Different Drosophila ADA2 Homologues Are Present in Distinct GCN5 Histone Acetyltransferase-Containing Complexes

2003 ◽  
Vol 23 (1) ◽  
pp. 306-321 ◽  
Author(s):  
Selen Muratoglu ◽  
Sofia Georgieva ◽  
Gábor Pápai ◽  
Elisabeth Scheer ◽  
Izzet Enünlü ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Histone Acetyltransferase ◽  
Gel Filtration ◽  
Adaptor Protein ◽  
X Chromosomes ◽  
Nuclear Extracts ◽  
Gene Coding

ABSTRACT We have isolated a novel Drosophila (d) gene coding for two distinct proteins via alternative splicing: a homologue of the yeast adaptor protein ADA2, dADA2a, and a subunit of RNA polymerase II (Pol II), dRPB4. Moreover, we have identified another gene in the Drosophila genome encoding a second ADA2 homologue (dADA2b). The two dADA2 homologues, as well as many putative ADA2 homologues from different species, all contain, in addition to the ZZ and SANT domains, several evolutionarily conserved domains. The dada2a/rpb4 and dada2b genes are differentially expressed at various stages of Drosophila development. Both dADA2a and dADA2b interacted with the GCN5 histone acetyltransferase (HAT) in a yeast two-hybrid assay, and dADA2b, but not dADA2a, also interacted with Drosophila ADA3. Both dADA2s further potentiate transcriptional activation in insect and mammalian cells. Antibodies raised either against dADA2a or dADA2b both immunoprecipitated GCN5 as well as several Drosophila TATA binding protein-associated factors (TAFs). Moreover, following glycerol gradient sedimentation or chromatographic purification combined with gel filtration of Drosophila nuclear extracts, dADA2a and dGCN5 were detected in fractions with an apparent molecular mass of about 0.8 MDa whereas dADA2b was found in fractions corresponding to masses of at least 2 MDa, together with GCN5 and several Drosophila TAFs. Furthermore, in vivo the two dADA2 proteins showed different localizations on polytene X chromosomes. These results, taken together, suggest that the two Drosophila ADA2 homologues are present in distinct GCN5-containing HAT complexes.

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