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Halophilic Nuclease of a Moderately Halophilic Bacillus sp.: Production, Purification, and Characterization

Onishi, Hiroshi; Mori, Tatsuro; Takeuchi, Setsuo; Tani, Keiko; Kobayashi, Takekazu; Kamekura, Masahiro
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1983 EN
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A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular weight was estimated to be 138,000 by Sephadex G-200 gel filtration. The enzyme had marked halophilic properties, showing maximal activities in the presence of 1.4 to 3.2 M NaCl or 2.3 to 3.2 M KCl. The enzyme hydrolyzed thymidine-5′-monophosphate-p-nitrophenyl ester at a rate that increased with NaCl concentration up to 4.8 M. In the presence of both Mg2+ and Ca2+, activity was greatly enhanced. The activity was lost by dialysis against water and low-salt buffer, but it was protected when 10 mM Ca2+ was added to the dialysis buffer. When the inactivated enzyme was dialyzed against 3.5 M NaCl buffer as much as 68% of the initial activity could be restored. The enzyme exhibited maximal activity at pH 8.5 and at 50°C on DNA and at 60°C on RNA and attacked RNA and DNA exonucleolytically and successively...

Purification and Reversible Inactivation of the Isocitrate Dehydrogenase from an Obligate Halophile

Hubbard, Jerry S.; Miller, Alan B.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1969 EN
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The nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase of Halobacterium cutirubrum is rapidly inactivated at low NaCl levels. As much as 75% of the initial activity can be restored by dialyzing the inactive enzyme against 4 m NaCl. A mixture of 4 mm isocitrate and 10 mm MnCl2 gives the same protection as 4 m NaCl but does not replace the NaCl requirement for reactivation. The reactivated and native enzymes have identical sedimentation rates on sucrose gradients, electrophoretic mobilities on polyacrylamide gels, and elution rates from Sephadex G-200. However, there are distinct differences between the active and inactive forms of the enzyme. Compared with the active enzyme, the inactive protein has a lower sedimentation rate, a lower electrophoretic mobility, and a faster elution rate from Sephadex. These differences indicate that inactivation causes a major conformational change in the protein. Presumably, the removal of NaCl permits the enzyme to expand into a less dense, inactive form. The isocitrate dehydrogenase was purified 69-fold by a procedure involving the following steps. When the enzyme is selectively protected with isocitrate and MnCl2 at low ionic strength, most of the contaminating proteins are precipitated with (NH4)2SO4 at 0.9 saturation. The enzyme in the supernatant fluid is then inactivated at low NaCl levels...

PARTIAL PURIFICATION OF THE EXTRACELLULAR HEMOLYSIN OF PSEUDOMONAS AERUGINOSA

Berk, Richard S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1964 EN
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Berk, Richard S. (Wayne State University, College of Medicine, Detroit, Mich.). Partial purification of the extracellular hemolysin of Pseudomonas aeruginosa. J. Bacteriol. 88:559–565. 1964.—Through a series of chemical fractionation steps, the extracellular hemolysin of Pseudomonas aeruginosa was purified 126-fold with a recovery of 49%. Hemolytic activity of crude preparations was irreversibly lost upon contact with anionic exchange materials such as diethylaminoethyl Sephadex or ECTEOLA-Cellulose, but traveled with the solvent front during passage through Sephadex G-200 and carboxymethyl Sephadex. The hemolysin was soluble in water and ethanol, and was partially extractable with ether, but not with trichlorotrifluoroethane (Freon). Although normal serum and serum albumin blocked hemolytic activity, it was unaffected by trypsin, deoxyribonuclease, or ribonuclease. Partially purified hemolysin was studied in vivo, but did not exert dermonecrotic activity in mice or rabbits in the concentrations tested. Although preparations were toxic to mice, lethality appeared to be more a reflection of the nonhemolytic protein content of the preparations rather than of hemolytic activity.

Identification of a nicotinamide adenine dinucleotide glycohydrolase and an associated inhibitor in isoniazid-susceptible and -resistant Mycobacterium phlei.

Davis, W B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1980 EN
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Nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was demonstrated in the catalases fraction of Sephadex G-200-chromatographed sonic extracts of isoniazid (INH)-susceptible (Inhs) and -resistant (Inhr) Mycobacterium phlei. Since crude extracts had no demonstrable activity even after heating, active fractions of the NADase were purified chromatographically by removing the inhibitor with Sephadex G-200. Assays for oxidized nicotinamide adenine dinucleotide (NAD+) hydrolytic activity were done by following the disappearance of NAD+ by the methods of alcohol dehydrogenase or cyanide addition. The NADase activity was linear with respect to time as well as concentration of enzyme and was inhibited in the presence of 0.04 M NADP, benzoic acid hydrazide, or nicotinamide. Crude extracts or pooled concentrated Sephadex G-200 fractions eluting after the catalase inhibited NADase activity by at least 70%. Inhibitor activity was present in both the Inhs and Inhr strains of M. phlei. The activity of the partially purified inhibitors was reversible by INH or nicotinic acid hydrazide at levels between 10 and 100 mM. These findings indicate that an NADase inhibitor system which is sensitive to reversal by INH functions in both the Inhs and Inhr strains; however...

Purification of Bacteroides amylophilus Protease

Lesk, E. M.; Blackburn, T. H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1971 EN
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A protease was released by Bacteroides amylophilus cells in late stationary phase, approximately 12 hr after maximum cell density was reached. The protease was concentrated by adsorption on diethylaminoethyl (DEAE)-Sephadex and was purified 532-fold by DEAE-Sephadex chromatography, by G-200-Sephadex gel filtration, and by isoelectric focusing. The purified protease was active between pH 4.5 and 12.0 with optima at pH 6.0 and 11.5. Evidence against there being a single protease was given by the differential inhibition of esterase and protease activities by some inhibitors. There was some evidence that only a single protease was present as the ratio of protease activity at various pH values did not alter significantly during purification or when the purified protease was partially heat-inactivated or treated with two specific trypsin-type protease inhibitors: N-α-tosyl-l-lysylchloromethyl ketone or phenylmethane-sulfonyl fluoride. Two forms of the same protease were found by acrylamide gel electrophoresis. Gel filtration confirmed the presence of protease in 30,000 and 60,000 molecular-weight forms. Treatment with 1 mm ethylenediaminetetraacetic acid or with 4 m urea failed to convert the 60,000-molecular-weight to the 30,000-molecular-weight species.

Further observations on the folate-binding factor in some leukemic cells

Rothenberg, Sheldon P.; daCosta, Maria
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1971 EN
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The lysates of peripheral cells as well as the serum from some patients with chronic myelogenous leukemia, contained a macromolecular factor which bound tritiated folic acid. Bound tracer folate filtered through Sephadex G-75 and G-100 columns with the early effluent and appeared with the inner volume through a Sephadex G-200 column. Bound tracer could not be extracted from solution by coated charcoal or the anion exchange resin Dowex 2-X8 and could not be reduced to tetrahydrofolate by folate reductase. The velocity of the binding reaction was very rapid and dissociation of bound tracer extremely slow. Binding decreased sharply below pH 5.0 and the binding factor as well as the folate-binder complex, resisted 56°C for 30 min. The binding factor in the leukemic lysate could be separated from endogenous folate reductase by filtration through a G-75 Sephadex column.

Type II insulin-like growth factor receptor is present in rat serum.

Kiess, W; Greenstein, L A; White, R M; Lee, L; Rechler, M M; Nissley, S P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1987 EN
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We previously identified in fetal rat serum a component capable of specifically binding radiolabeled insulin-like growth factor type II (IGF-II) that is considerably larger than both the fetal (40 kDa) and the adult (150 kDa) carrier proteins. We now present immunologic and affinity crosslinking data to show that this binding species is the type II IGF receptor. Rat serum was gel-filtered on a Sephadex G-200 column (0.05 M NH4HCO3, pH 8), and 125I-labeled IGF-II (125I-IGF-II) binding was measured in individual column fractions. 125I-IGF-II binding activity was found in the void volume of the column in addition to the carrier protein regions. Competitive binding studies using 125I-IGF-II and binding activity from the Sephadex G-200 void volume showed the characteristics of the type II receptor: IGF-II was more potent than IGF-I, and insulin did not compete. Moreover, a specific anti-type II IGF receptor antibody (no. 3637) completely blocked 125I-IGF-II binding. 125I-IGF-I did not bind to the void volume pool, demonstrating the absence of the type I IGF receptor in rat serum. Affinity crosslinking of 125I-IGF-II to the Sephadex G-200 void volume material demonstrated a specific band at 210 kDa without reduction and at 240 kDa after reduction of disulfide bonds. The serum type II IGF receptor size was confirmed by immunoblotting the void volume material with antiserum 3637...

Partial Purification of Plasma Thromboplastin Antecedent (Factor XI) and its Activation by Trypsin

Saito, Hidehiko; Ratnoff, Oscar D.; Marshall, James S.; Pensky, Jack
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1973 EN
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A persistent puzzle in our understanding of hemostasis has been the absence of hemorrhagic symptoms in the majority of patients with Hageman trait, the hereditary deficiency of Hageman factor (factor XII). One proposed hypothesis is that alternative mechanisms exist in blood through which plasma thromboplastin antecedent (PTA, factor XI) can become active in the absence of Hageman factor. In order to test this hypothesis, the effect of several proteolytic enzymes, among them thrombin, plasma kallikrein, and trypsin, was tested upon unactivated PTA. PTA was prepared from normal human plasma by Ca3(PO4)2 adsorption, ammonium sulfate fractionation, and successive chromatography on QAE-Sephadex (twice). Sephadex-G150, and SP-Sephadex. The partially purified PTA was almost all in its native form, with a specific activity of 45-70 U/mg protein; the yield was about 10%. It contained no measurable amounts of other known clotting factors, plasmin, plasminogen, nor IgG. Incubation of PTA with trypsin generated potent clot-promoting activity that corrected the abnormally long clotting time of plasma deficient in Hageman factor or PTA but not in Christmas factor. This clot-promoting agent behaved like activated PTA on gel filtration (apparent molecular weight: 185...

Intracellular receptor for somatostatin in gastric mucosal cells: decomposition and reconstitution of somatostatin-stimulated phosphoprotein phosphatases.

Reyl, F J; Lewin, M J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1982 EN
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Using 32P-labeled histone as exogenous substrate, we showed a potent stimulatory effect of somatostatin on cytosolic phosphoprotein phosphatases (PPPases; phosphoprotein phosphohydrolase, EC 3.1.3.16) in rat gastric mucosal cells. Partial purification of cytosolic fraction in DEAE-Sephadex ion-exchange chromatography and further gel filtration on Sephadex C-75 and Sephadex G-100 separated somatostatin-dependent PPPases into three distinct molecular species. One corresponding to Mr 130,000 was devoid of any PPPase activity but specifically bound [Tyr1]somatostatin 125I-labeled on the Tyr ([125I-Tyr1]somatostatin) with an apparent equilibrium dissociation constant of 3 x 10(-10) M. The two other molecular species corresponded to Mrs 64,000 and 13,000. They produced catalytic dephosphorylation of 32P-labeled histone, but they were not sensitive to somatostatin and did not show any specific binding to radiolabeled hormone. Mixing of the larger with either of the two smaller molecular species resulted in concentration -dependent inhibition of PPPase activity. However this inhibition was reversed by increased concentrations of somatostatin, with the concentration for half-maximal reactivation on being close to 0.1 nM. Furthermore somatostatin stimulation in reconstituted materials developed according to a rapid time course (t1/2...

Cholecystokinin-converting enzymes in brain.

Malesci, A; Straus, E; Yalow, R S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1980 EN
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Crude extracts of porcine cerebral cortical tissue convert cholecystokinin (CCK) to its COOH-terminal fragments, the dodecapeptide (CCK-12) and the octapeptide (CCK-8). The Sephadex G-75 void volume eluate of the crude extract cleaves the arginine-isoleucine bond and effects conversion only to CCK-12; the Sephadex G-50 void volume eluate of the same extract cleaves the arginine-aspartate bond as well, so that both CCK-12 and CCK-8 are end products. Thus, there are at least two enzymes; the one involved in the conversion to CCK-12 is of larger molecular radius than the other. The Km for the cleavage of CCK at the arginine-isoleucine bond by the Sephadex G-75 void volume eluate enzyme is 1.1 X 10(-6) M; the Km for trypsin cleavage of the same bond is 4.7 x 10(-6) M. The lower Vmax for the brain enzyme (1.5 x 10(-11) mol/min per g of extract) compared with trypsin (66 x 10(-11) mol/min per g of trypsin) simply reflects the lesser degree of purify of the brain extract than of the highly purified trypsin.

Immunological identification of high molecular weight forms common to bovine neurophysin and vasopressin

Nicolas, Pierre; Camier, Maryse; Lauber, Marc; Masse, Marie-J. O.; Möhring, Jan; Cohen, Paul
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1980 EN
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Extracts of bovine neurohypophysis made in acid/ethanol solution containing protease inhibitors were fractionated by two successive filtrations on Sephadex G-75 columns equilibrated in the presence and then in the absence of 4 M urea. Analysis of the pattern of neurophysin-like immunoreactivity in the eluate, with two different antibodies, indicated the presence of high Mr forms of neurophysin (apparent sizes, [unk]70,000 and 20,000-25,000, respectively) besides the Mr 10,000 neurophysin. [8-Arginine]vasopressin-like immunoreactivity was also detected, coeluting with the neurophysin-like species, in the material recovered in the exclusion and Mr 20,000-25,000 elution volumes of the same molecular sieve fractionation of neurohypophyseal extracts. Upon subsequent Sephadex G-150 filtration, the immunoreactive material recovered in the exclusion volume of the Sephadex G-75 filtration showed an apparent Mr of approximately 140,000. Both neurophysin-like and vasopressin-like immunoreactivities coeluted in the same volume. The elution profile of this Mr 140,000 material was unmodified when reanalyzed by the same molecular sieve filtration after exposure to 8 M urea. When these Mr 140,000 immunoreactive forms of vasopressin and neurophysin were submitted to affinity chromatography on anti-neurophysin antibodies immobilized on Sepharose...

Active site amino acid sequence of human factor D.

Davis, A E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1980 EN
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Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However...

Antigenic stimulation of T lymphocytes in chronic nononcogenic retrovirus infection: equine infectious anemia.

Shively, M A; Banks, K L; Greenlee, A; Klevjer-Anderson, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1982 EN
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Equine infectious anemia is a chronic disease of horses caused by a nononcogenic retrovirus. Studies were undertaken to determine the types of cells involved in the in vitro lymphoproliferative response to viral antigens and the dynamics of this reaction. It was observed that reactive lymphocytes were present at unpredictable times in the peripheral blood of infected horses. This reaction was shown to be specific for the interaction of equine infectious anemia virus and T lymphocytes. Enriched B-lymphocyte populations did not divide when exposed to equine infectious anemia virus. Macrophages were depleted from the reaction by two methods: adherence to Sephadex and a combination of binding to Sephadex and adherence to complement-coated erythrocytes. Both methods reduced the number of monocytes, but only the combination of Sephadex and complement-coated cells removed the accessory cells needed for lymphocyte proliferation. We conclude that during the chronic stages of equine infectious anemia the number of antigen-reactive T lymphocytes fluctuates within the peripheral blood and that these cells require a complement-binding cell for reaction. The relationship of these cells to the lymphoproliferative stages of this disease is discussed.

Purification, Properties, and Cytotoxic Effect of a Bacteriocin from Mycobacterium smegmatis

Saito, Hajime; Watanabe, Takashi; Tomioka, Haruaki
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1979 EN
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The bacteriocin produced by Mycobacterium smegmatis ATCC 14468 was isolated, and a study was made of its chemical, physical, and biological properties. No appreciable bacteriocin activity was found in the culture supernatant fluids, but it was released in appreciable quantities after disruption of the cells. The material was purified 49-fold by means of chromatography on diethylaminoethyl-cellulose, ammonium sulfate fractionation, gel filtration on Sephadex G-200, and chromatography on diethylaminoethyl-Sephadex A-50. Its molecular weight was determined to be approximately 75,000 from the elution profile on Sephadex G-200 chromatography. The bacteriocin was resistant to deoxyribonuclease, ribonuclease, lipase, ultraviolet irradiation, and freeze-thawing, whereas it was relatively less thermostable and was sensitive to proteolytic enzymes. The lethal effect of the bacteriocin was demonstrated by the decrease in viable counts of the bacteriocin-sensitive indicator strain, M. diernhoferi ATCC 19340. The bacteriocin preparation inhibited the growth of HeLa-S3 cells.

Purification and molecular characterization of adenovirus type 2 DNA-binding protein.

Sugawara, K; Gilead, Z; Green, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1977 EN
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An adenovirus type 2 (Ad2) DNA-binding protein was purified by sequential DNA-cellulose, Sephadex G-200, and DEAE-Sephadex chromatography, with a yield of 120 mug of binding protein (95 to 99% homogeneity) starting with 2 X 10(9) infected cells. By omitting the Sephadex G-200 step, 400 to 600 mug of 95% pure binding protein was obtained. To obtain high yields of highly purified binding protein, it was necessary to include deoxycholate and Nonidet P-40 at selected stages during the preparation. The highly purified binding protein appeared to have retained its native stage as indicated by: (i) binding to single-stranded but not native Ad2 DNA, (ii) almost complete precipitation by immunoglobulin G from hamsters immunized by extracts of tumors induced by Ad2-simian virus 40 hybrid viruses, and (iii) identical sedimentation coefficient with binding protein obtained from DNA-cellulose chromatography only. Zonal centrifugation in sucrose gradients and gel filtration revealed that purified binding protein has a sedimentation coefficient of 3.4S and a Stokes radius of 5.2 nm. Based on these two values, a molecular weight of 73,000 was calculated, in agreement with the estimate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A frictional ratio of 1.88 was calculated...

Developmental Pattern of a Serum Binding Protein for Multiplication Stimulating Activity in the Rat

White, Robert M.; Nissley, S. Peter; Short, Patricia A.; Rechler, Matthew M.; Fennoy, Ilene
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1982 EN
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The concentration of multiplication stimulating activity (MSA), an insulinlike growth factor (IGF), is high in fetal rat serum. We now report that MSA is exclusively associated wth an albumin-size binding protein in fetal rat serum; the growth hormone-dependent, gamma globulin-size binding protein, which predominates in the older animal, is absent from fetal rat serum. When 125I-MSA was incubated with fetal rat serum and then gel filtered on Sephadex G-200, specific radioactivity eluted in the void volume (peak I) and the albumin region (peak III); by contrast, specific radioactivity eluted mainly in the gamma globulin region (peak II) in adult rat serum. Pools of the Sephadex G-200 fractions were chromatographed on Sephadex G-50, in 1 M acetic acid, to separate the binding protein from IGF activity. Analysis of IGF activity by chick embryo fibroblast bioassay, competitive protein binding assay, and MSA by radioimmunoassay revealed that all the IGF activity and MSA in fetal rat serum resided in peak III. Measurement of MSA binding capacity of the stripped binding protein by Scatchard analysis demonstrated that the majority of binding capacity also was found in peak III in fetal rat serum; most of MSA binding capacity was in peak II in adult rat serum. In fetal rat sera...

Physicochemical and immunohistological studies of a sulfobromophthalein- and bilirubin-binding protein from rat liver plasma membranes.

Stremmel, W; Gerber, M A; Glezerov, V; Thung, S N; Kochwa, S; Berk, P D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1983 EN
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Affinity chromatography over bilirubinagarose and sulfobromophthalein (BSP)-agarose was used to isolate two proteins, with high affinities for bilirubin and BSP, respectively, from Triton X-100-solubilized rat liver plasma membranes. The protein eluted from either affinity column migrated as a single band of approximately 55,000 D on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and either protein cochromatographed with both [14C]bilirubin and [35S]BSP on Sephadex G-75. On gradient gels without reduction or SDS, or on Sephadex G-150, the native BSP-binding protein had an estimated molecular mass of approximately 100,000 D. After incubation with SDS, an additional Sephadex G-150 peak of molecular mass of 56,000 D was observed. Both, the 100,000- and 56,000-D G-150 peaks cochromatographed with [35S]BSP. The native protein had an isoelectric point of 3.5, stained with periodic acid-Schiff but not Sudan black, and contained 4 mol of sialic acid per mol of protein. A rabbit antibody to the BSP-binding protein gave a line of identity with both the BSP- and bilirubin-binding antigens, and inhibited the binding of [14C]bilirubin and [35S]BSP, but not [14C]oleate or [14C]taurocholate, to rat liver plasma membranes. Immunohistochemical studies revealed the presence of the antigen on all surface domains of rat hepatocytes...

Reconstitution of RNase P activity from inactive RNA and protein.

Kole, R; Altman, S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1979 EN
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RNase P preparations from Escherichia coli can be separated into RNA and protein by chromatography, in buffers containing 7 M urea, on Sephadex G-200, DEAE-Sephadex, or CM-Sephadex columns. Neither RNA nor protein components alone exhibits any RNase activity. RNase P activity can be reconstituted by mixing separated RNA and protein components in buffer containing 7M urea followed by dialysis of this mixture to remove the urea. Of several purified RNAs tried, only M2 RNA, the RNA species found in purified RNase P, is active in the reconstitution experiments.

Evidence for the Presence of Bacteria-specific Proteins in Sterile Crown Gall Tumor Tissue 1

Chadha, Kailash C.; Srivastava, B. I. Sahai
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1971 EN
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Cross-reacting antigens were found in bacteria-free crown gall tumor tissue tested with serum prepared against Agrobacterium tumefaciens (Smith and Towns.) Conn., but no such antigens were detected in callus tissue. Soluble proteins from tumor tissue, callus tissue, and the crown gall bacteria were fractionated on a DEAE-Sephadex (A-50) column. The diethylaminoethyl-Sephadex elution profile for tumor tissue showed three protein fractions that were not detected in the callus tissue. Two of these protein fractions were shown to be exclusively bacteria specific. Besides these qualitative differences between the two tissues, significant quantitative differences in the amount of protein fractions were also observed. The diethylaminoethyl-Sephadex column fractions from tumorigenic strain of A. tumefaciens corresponding in position to the three additional peaks in the tumor tissue also showed cross-reacting antigens when tested with serum prepared against sterile tumor tissue. It is suggested that tumor formation by A. tumefaciens involves integration of the bacterial genome into the host-cell genome.

Production of choriogonadotropin-like factor by a microorganism.

Maruo, T; Cohen, H; Segal, S J; Koide, S S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1979 EN
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Extracts from an acetone powder preparation of a culture of a microorganism tentatively named Progenitor cryptocides contain choriogonadotropin (CG)-like factor as determined by radioimmunoassay with antiserum to human (h)CG beta subunit COOH-terminal peptide and radioreceptor assay with bovine corpus luteum membranes. Possible interference by proteases in the extracts was excluded. Immunoreactive materials reacting with antisera to hCG beta subunit and hCG beta subunit COOH-terminal peptide were also found in the extracts. No free alpha subunit was detected. The CG-like factor was purified by chromatography on Sephadex G-100, concanavalin A-Sepharose, and DEAE-Sephadex A-50. The factor was adsorbed by concanavalin A-Sepharose, suggesting that it contains mannose and glucose moieties. The factor was eluted at the same position as standard hCG on Sephadex G-100. It dissociated into two bands when subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis; the bands corresponded to the respective standard hCG subunits. The biological activity of the purified factor as determined by the rat uterine weight assay and the ovarian weight assay was equivalent to 380 (95% confidence limits: 320-490) and 880 (780-1020) international units/mg...