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1.
Association of adenovirus type 2 early proteins with a soluble complex that synthesizes adenovirus DNA in vitro 总被引:3,自引:0,他引:3
H M Rho Y H Jeng W S Wold M Green 《Biochemical and biophysical research communications》1977,79(2):422-428
A soluble Ad2 DNA synthesizing complex was prepared from Ad2-infected KB cell nuclei and purified by exclusion chromatography on a BioGel A-50m column. The purified complex was able to synthesize DNA from all regions of the virus genome, as indicated by RI restriction endonuclease analysis of labeled DNA. Experiments were performed to identify Ad2-induced early polypeptides present in the complex. Ad2-infected and mock-infected cells were labeled with [35S]methionine 7–10 h postinfection, then incubated for 8 h to allow the 35S-labeled early polypeptides to become associated with the complex. The polypeptides in the purified complex and each of the cell fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major components of the purified complex were the 73K DNA binding phosphoprotein and 11K, two adenovirus 2-induced early polypeptides. The 11K has a preferred nuclear location. Small quantities of other Ad2-induced early proteins, 21K, 15K, and possibly 8.3K were also associated with the complex. 相似文献
2.
A sensitive immunological technique is described that detects proteins in sodium dodecylsulfate gels of crude cell extracts. The method is based on the binding of 125I-protein A to gels that have been incubated with antibody to a specific protein. Using antibody to the yeast protein, single polypeptides can be detected in mutant and wild-type extracts. The size of these polypeptides correlates both with the type of mutation and with its location in the region. 相似文献
3.
Analysis of the products of the RNA-directed DNA polymerase reaction using glycerol-substituted polyacrylamide gels has proven superior to the glycerol gradient method for quantitative assay of a series of nucleic acid samples. To overcome the phenomenon of a smearing of background radioactive counts in the gels caused by unincorporated 3H-thymidine triphosphate, an automatic washing procedure making use of activated charcoal has been devised for the gels. This washing virtually eliminates all background while leaving the RNA hybrid as well as the molecules fixed in situ on the gels. 相似文献
4.
5.
The purification of protease IV of E.coli and the demonstration that it is an endoproteolytic enzyme
Philippe Régnier 《Biochemical and biophysical research communications》1981,99(4):1369-1376
A strong proteolytic activity is unmasked and solubilized when outer membrane fragments are preincubated with 0.083% sodium dodecyl sulfate. This proteolytic activity cleaves αS1 casein into the same degradation products as protease IV, a recently described protease of located in the outer membrane (Ph. Régnier, preceding paper), it is concluded that sodium dodecyl sulfate solubilizes the same protease. Protease IV has been purified 11,200 fold, probably to homogenetiy, by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by elution of the protein from gel slices. The purified enzyme is fully active, its molecular weight, determined from its migration in denaturating gels is 23,500. αS1 casein is cleaved by protease IV into two large polypeptides which are not further degraded and some small peptides of about 5,000 daltons. The production of discrete polypeptide species suggests that protease IV is an endoproteolytic enzyme. 相似文献
6.
James E. Fleming Paula S. Melnikoff Klaus G. Bensch 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,802(2):340-345
Several hundred proteins have been resolved on two-dimensional gels of extracts of [35S]methionine-labeled adult Drosophila melanogaster. 27 of these polypeptides disappear from the gel pattern after feeding the K+ ionophore nonactin. These proteins have been identified as mitochondrial, since the two-dimensional gel pattern of extracts of isolated mitochondria correlates well with the pattern of the proteins missing from that of nonactin-treated flies. Nine new proteins also appear on the two-dimensional gels of the extracts from the nonactin-treated flies. Apparently, these nine proteins are precursors of the mature mitochondrial forms. These particular data support the concept that processing of many of the cytoplasmically synthesized mitochondrial proteins requires a specific membrane potential, and that some of these proteins are modified intramitochondrially. However, using [35S]methionine incorporation techniques, not all labeled polypeptides disappear from mitochondria during such treatment. Feeding similarly radiolabeled flies with chloramphenicol, an inhibitor of mitochondrial protein synthesis, results in the disappearance of only one protein from the gel pattern with the concurrent appearance of a ‘new’ high-molecular-weight polypeptide. Collectively, these data show that a specific group of [35S]methionine-labeled mitochondrial proteins can be identified by selective inhibition of mitochondrial function in whole cell protein maps of adult D. melanogaster. 相似文献
7.
J F Habener B Kemper J T Potts A Rich 《Biochemical and biophysical research communications》1975,67(3):1114-1121
Translation in a cell-free extract of Krebs II ascites cells of a mRNA fraction prepared from bovine parathyroid glands results in the synthesis of two radioactive products that appear identical to pre-proparathyroid hormone (Pre-ProPTH) (M.W. ~ 14,000), the suspected earliest biosynthetic precursor of parathyroid hormone (PTH) (M.W. 9,500), and to proparathyroid hormone (ProPTH) (M.W. 10,200), the immediate biosynthetic precursor of PTH. The two products of synthesis in the ascites extract co-electrophoresed on both urea-acetate and urea-SDS acrylamide gels with Pre-ProPTH obtained from cell-free translation of parathyroid RNA in extracts of wheat-germ and with ProPTH isolated from parathyroid slices. Both products were precipitated with an antiserum to PTH. Partial analysis of the amino acid sequence of [35S]methionine-labeled Pre-ProPTH synthesized by the ascites extract indicates that a substantial fraction of the product is lacking the two N-terminal methionines present in the Pre-ProPTH synthesized by the wheat-germ system. The results indicate that, (), unlike the wheat-germ, ascites extracts contain enzymes that remove the initiator methionine from Pre-ProPTH and convert Pre-ProPTH into ProPTH (no ProPTH was observed in the wheat-germ system) and () the cleavage processes appear to occur in association with synthesis, inasmuch as neither removal of NH2-terminal methionine nor formation of ProPTH was observed upon incubation of Pre-ProPTH isolated from either the wheat-germ system or from the ascites system when put back into the ascites system. 相似文献
8.
Indentification of two somatomedin A active polypeptides and in vivo effects of a somatomedin A concentrate 总被引:5,自引:0,他引:5
L Fryklund K Uthne H Sievertsson 《Biochemical and biophysical research communications》1974,61(3):957-962
The first chemical characterization of two polypeptides from human serum which stimulate the incorporation of 35S sulfate into chick cartilage is described. These two polypeptides, designated Somatomedin A1 and A2 have a molecular weight of approximately 7000. Although each peptide contains 1 cysteine residue and has asparagine as amino terminal residue, there are apparent differences in the amino acid composition. Administration of a Somatomedin A concentrate to hypophysectomized rats gave an increase in tibial width similar to that obtained with 20 μg human growth hormone. 相似文献
9.
About 15% of the total (3H)leucine incorporated into protein by isolated rat liver mitochondria could be extracted by chloroform:methanol. This incorporation was inhibited by chloramphenicol and carbomycin, both specific inhibitors of mitochondrial protein synthesis. SDS-gel electrophoresis of the mitochondrial membrane revealed 6–7 labeled bands. Label in the proteolipid fraction was present mainly in a band of 40,000 molecular weight. Several labeled bands observed in gels of the mitochondrial membrane were not removed or changed by extraction with chloroform:methanol suggesting that some, but not all, of the proteins synthesized by rat liver mitochondria are proteolipids. 相似文献
10.
Rosario R. Trifiletti Adele M. Snowman Solomon H. Snyder 《Biochemical and biophysical research communications》1984,120(2):692-699
Binding activity for the cage convulsant [35S]--butylbicyclophosphorothionate, which appears to label a site closely associated with the chloride ionophore of the GABAA/benzodiazepine receptor complex has been solubilized from rat cerebral cortex using the zwitterionic detergent CHAPS. Of several detergents screened, only CHAPS and CHAPSO were capable of solubilizing the binding activity with good recovery. The pharmacologic specificity of soluble [35S]--butylbicyclophosphorothionate binding is very similar to the membrane state. In both the membrane and soluble state, [35S]--butylbicyclophosphorothionate binding is enhanced by anions which support inhibitory post-synaptic potentials (“Eccles anions”), suggesting that [35S]--butylbicyclophosphorothionate may label chloride channels thought to be involved in these potentials. Since this solubilization procedure also preserves GABA and benzodiazepine binding and their regulation by drugs such as barbiturates, purification and isolation of the macromolecular complex including chloride channel and GABA-benzodiazepine sites may be feasible. 相似文献
11.
James F. Hare Randall Hodges 《Biochemical and biophysical research communications》1982,105(4):1250-1256
Antibody prepared against beef heart mitochondrial NADH dehydrogenase immunoprecipitated 26 polypeptides from detergent solubilized beef heart mitochondria. All 26 polypeptides co-migrated with those present in the dehydrogenase antigen when resolved side by side on sodium dodecyl sulfateurea polyacrylamide gels. From mixed rat liver-[35S]methionine pulsed hepatoma mitochondria the antibody immunoprecipitated 24 stained liver polypeptides and 19 radio-labelled hepatoma polypeptides. The translation of three of the labelled polypeptides was resistent to inhibition by cycloheximide, indicating these are translated on mitochondrial ribosomes. These same polypeptides, however, wre previously identified as cytochrome c oxidase subunits; and, apparently, non-specifically co-precipitate with dehydrogenase associated polypeptides. We conclude that there are no mitochondrially translated polypeptides specifically associated with NADH dehydrogenase. 相似文献
12.
G. Sheir-Neiss R.V. Nardi M.A. Gealt N.R. Morris 《Biochemical and biophysical research communications》1976,69(2):285-290
[35S] labeled extracts of the fungus were copolymerized with purified porcine brain tubulin. The [35S] protein which copurified with porcine microtubules was found to be similar to [3H] chick tubulin when the two were coelectrophoresed on several polyacrylamide gel electrophoresis systems. These results strongly suggest the presence in of a tubulin-like protein. 相似文献
13.
M Yoshida K Ohno Y Takeuchi Y Kagawa 《Biochemical and biophysical research communications》1977,75(4):1111-1116
Prostatic binding protein (PBP) is a quantitatively important steroid-binding protein present in rat ventral prostate. Electrophoresis on SDS-containing polyacrylamide gels shows that PBP is composed of two subunits, F and S having molecular weights of 16,000 and 18,000. Upon reduction these subunits dissociate further into smaller components. Translation of mRNA from rat ventral prostate in a wheat germ cell-free system or in oocytes results in the formation of polypeptides immunoprecipitable with an anti-PBP antiserum. However, as opposed to the wheat germ system, only the oocytes synthesize polypeptides, that are electrophoretically identical to those of native cytosolic PBP. 相似文献
14.
R A Capaldi R L Bell T Branchek 《Biochemical and biophysical research communications》1977,74(2):425-433
The order of migration of polypeptides in both cytochrome oxidase and ubiquinone cytochrome reductase has been found to differ depending on the gel conditions used. Thus the nomenclatures or numbering systems being used for the subunits of these membrane complexes by workers using Weber-Osborn gels is not the same as that being used in laboratories which use the Swank-Munkres or Fairbanks gel procedure. 相似文献
15.
Gary K. McMaster Luciano Zardi 《Biochemical and biophysical research communications》1982,107(2):609-617
Here we report the use of the technique of transferring proteins from polyacrylamide gels to nitrocellulose sheets to identify the DNA-binding domains of human plasma fibronectin. After separation of fibronectin and its tryptic digest on a sodium dodecylsulfate-polyacrylamide gel, the polypeptides were transferred to nitrocellulose sheets and the DNA-binding polypeptides were identified by incubation of the nitrocellul se sheets with 32P-labeled human DNA followed by autoradiography.The following results were obtained: a) only two fibronectin trypsin resistant peptides (50 and 60 kd) showed DNA-binding capacity; b) in competition experiments a 300 fold excess of E. coli DNA did not inhibit the human DNA-human fibronectin binding, indicating the specificity of the interaction. 相似文献
16.
The cellular distribution of 35S from 35S- thioacetamide was determined in rabbit liver subcellular fractions following its administration. Of the various fractions isolated, only the nucleolar fraction contained 35S counts that were insoluble in 10% trichloroacetic acid but soluble in trichloroacetic acid if the fraction was treated with trypsin but not RNase or DNase. These results demonstrate that a protein bound form of thioacetamide is present in the nucleolus following administration of this drug. 相似文献
17.
R A Katz G M Maniatis R V Guntaka 《Biochemical and biophysical research communications》1979,86(2):447-453
Evidence for the synthesis and processing of Pr76 (precursor to group-specific antigens p27, p19, and , upon injection of avian sarcoma virus 70S or 35S RNA into oocytes has been presented. Further, we show that tRNAtrp primer, bound to 35S RNA, does not block translation of virion RNA under these conditions. 相似文献
18.
Electrophoretic analyses were performed to establish the polypeptide profiles of the following tick-borne typhus rickettsiae of the spotted fever group: (Sheila Smith, Bitter Root, Iowa and R strains), , , , and . Organisms were propagated in chick embryo fibroblast cells in roller bottles and purified by density centrifugation. After polyacrylamide gel electrophoresis, comparative electrophoretic profiles were examined by densitometric scanning. Of the more than 20 separate polypeptides detected, six of the most prominent were found in all rickettsiae examined except . No carbohydrate-containing moieties were detected in electrophoretic gels. 相似文献
19.
Identification of the structural gene for yeast cytochrome c oxidase subunit I on mitochondrial DNA.
E Keyhani 《Biochemical and biophysical research communications》1979,89(4):1212-1216
Mitochondrial protein synthesis was analyzed in the yeast mit? mutants of which specifically lack cytochrome oxidase. [3H]leucine labeled polypeptides synthesized in yeast OXI 3 mutant were analyzed by means of immunoprecipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When compared to control, subunit I was not detectable. This result was substantiated by growing OXI 3 mutant in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Under such conditions SDS-PAGE analysis of [3H]leucine labeled immunoprecipitate shows the absence of subunit I. These data show that the OXI 3 locus contains the structural gene for cytochrome oxidase subunit I. 相似文献
20.
Precursor of a mitochondrial enzyme accumulates in the cytoplasm of preen glands for a specialized function 总被引:1,自引:0,他引:1
W Flurkey Y S Kim P E Kolattukudy 《Biochemical and biophysical research communications》1982,106(4):1346-1352
Malonyl-CoA decarboxylase in the mitochondria of the liver of goose is immunologically identical with the decarboxylase in the cytoplasm of the uropygial gland (Buckner et al. (1978) 186, 152–163). Messenger RNA was isolated from the liver and the uropygial gland and translated in a rabbit reticulocyte system. Specific immunoprecipitation of the translation products with anti malonyl-CoA decarboxylase showed that in both cases the primary translation product was a 50 K dalton peptide identical in size to the cytoplasmic enzyme in the gland. Specific immunoprecipitation of malonyl-CoA decarboxylase from liver slices which had been incubated with [35S]methionine showed that the mature mitochondrial enzyme was a 47 K dalton peptide, 3 K daltons smaller than the primary translation product and the isolated cytoplasmic enzyme. These results suggest that the decarboxylase is proteolytically processed during transport into the mitochondria and that the large amount of the cytoplasmic decarboxylase found in the gland represents accumulation of the unprocessed precursor form of the normally mitochondrial enzyme. 相似文献