Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cytosolic factor that stimulates transcription in isolated nuclei was purified approximately 4000-fold to near homogeneity from rat liver. The molecular weight of the factor was determined as 47 000 by SDS-polyacrylamide gel electrophoresis. The factor had no detectable deoxyribonuclease and protease activity but showed ribonuclease inhibitor activity. The factor could stimulate transcription in isolated nuclei by 50% at about 3.0 ng and the maximal stimulation was about 100%. When [gamma-S]ATP and [gamma-S]GTP were included in the reaction, the factor stimulated the synthesis of RNA which was able to bind to a mercury-Sepharose column and about 80% of the bound RNA was sensitive to a low concentration of alpha-amanitin. When heparin was added before initiation to preincubation mixture containing RNA polymerases II and DNA, a small but definite incorporation of [14C]UTP was observed. The factor alone had no stimulatory effect on the heparin-resistant incorporation of [14C]UTP but, in the presence of two rat liver nuclear fractions, phosphocellulose 0.5 and 1 M KCl step fractions, could stimulate the incorporation above the level with the combination of the two nuclear fractions. Antibody raised against the factor inhibited accurate transcription from the adenovirus 2 major late promoter in a nuclear lysate from Ehrlich ascites tumor cells, and the inhibition was neutralized by the factor.
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PMID:Purification of a cytosolic factor from rat liver that stimulates transcription in isolated nuclei and its action on purified RNA polymerase II-DNA system. 407 43

Proteins were extracted from rat liver ribosomes and ribosomal subunits: with 67% acetic acid (in the presence of 3.3 mM, 33 mM, or 67 mM Mg) with 2 M LiCL in 4 M urea; with 0.25 N HCI; with 1% SDS; and after RNase digestion. The most efficient extraction and the best recovery were either with acetic acid in the presence of 33 mM or 67 mM Mg, or with LiCI-urea. Protein extracted with acetic acid, LiCi-urea, or with HCI had little or no contamination with RNA. The ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis: the proteins extracted with acetic acid were the most soluble in the sample gel solution; their electrophoretograms displayed the maximum number of spots and the smallest number of derivatives or altered proteins. Preparations of protein extracted with SDS or RNase were relatively insoluble in the sample gel solution, and proteins extracted with HCI showed a large number of derivatives. All things considered, the most satisfactory method for the extraction of protein from eukaryotic ribosomes is with 67% acetic acid in the presence of 33 mM MgCl2.
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PMID:The extraction of proteins from eukaryotic ribosomes and ribosomal subunits. 445 58

Two ribonuclease activities have been isolated from macrophage culture medium. SDS-electrophoresis gave a molecular weight of 26,000 for both RNAases. The two RNAases differ only slightly in their enzymic properties. They are optimally active at neutral and slightly alkaline pH, and are not activated by monovalent or divalent cations or by spermine. Cu(II), Zn(II), Mn(II) and heparine inactivate them but they are not affected by the RNAase inhibitor from rat liver. They both degrade RNA endonucleolytically to mono- and oligonucleotides. They react to synthetic polynucleotides, especially poly(C), but do not degrade the synthetic double-stranded RNA poly(I) . poly(C), or double or single-stranded DNA. RNAase 1 inhibits DNA synthesis and increases degradation of RNA in granulation-tissue fibroblasts but RNAase 2 at the same concentration does not have these effects.
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PMID:Purification of two ribonucleases from macrophage culture medium and studies on their effect on granulation-tissue fibroblasts. 616 35

At present, nonspecific immunotherapy using BCG or Picibanil is popular as an immunotherapy for malignant brain tumor. On the other hand, specific immunotherapy using lymphocytes which play an important role in the passive transmission of tumor immunity is also performed. But this therapy has some disadvantage such as the problem of blocking factor or histocompatibility. To compensate for this disadvantage, specific immunotherapy using the chemical mediater, immune RNA, extracted from immunized lymphocytes has started. We extracted immune RNA from the spleen of immunized rat using phenol and SDS, and observed the effect of normal lymphocytes preincubated with immune RNA in vitro and in vivo experiments. In in vitro experiment, following results were obtained. (1) Normal lymphocytes incubated with immune RNA revealed a significant cytotoxicity and this effect was disappeared by the pretreatment of immune RNA with RNase. (2) Cytotoxic effect of immune RNA seemed to be a tumor specific response. (3) Not only syngeneic RNA, but also xenogeneic RNA were effective. In addition to these results, we decided the optimal condition in which immune RNA transferred the cell mediated cytotoxicity to normal lymphocytes. Next, we tried the purification of immune RNA by way of sucrose density gradient centrifugation method and oligo (dT) cellulose affinity chromatography method. Active fraction of immune RNA was contained in 8-16 S fraction and in poly (A) containing fraction. As in vivo experiment, we injected immune RNA incubated lymphocytes intravenously to brain tumor-bearing rats and observed for death daily. In this experiments, significant prolongation of mean survival time was recognized in the group treated with immune RNA. We now consider the clinical application of immune RNA for malignant brain tumor by the following method: immune RNA is obtained from the lymphocytes of sheep which has immunized with patient's tumor cells. And then patient's own lymphocytes strengthened by incubation with immune RNA is injected intratumorally by way of Ommaya's reservoir. To do this therapy safely and effectively, further purification of immune RNA and solution of its physiochemical characteristics will be necessary.
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PMID:[The role of immune RNA in the immunotherapy of malignant brain tumor (author's transl)]. 617 20

We have examined the secretogogue responsiveness and the pattern of secretory proteins produced by a transplantable rat pancreatic acinar cell tumor. Dispersed tumor cells were found to discharge secretory proteins in vitro when incubated with hormones that act on four different classes of receptors: carbamylcholine, caerulein, secretin-vasoactive intestinal peptide, and bombesin. With all hormones tested, maximal discharge from tumor cells was only about one-half that of control pancreatic lobules, but occurred at the same dose optima except for secretin, whose dose optimum was 10-fold higher. Biochemical analysis of secretory proteins discharged by the tumor cells was carried out by crossed immunoelectrophoresis and by two-dimensional isoelectric focusing-SDS polyacrylamide gel electrophoresis. To establish a baseline for comparison, secretory proteins from normal rat pancreas were identified according to enzymatic activity and correlated with migration position on two-dimensional gels. Our results indicate that a group of basic polypeptides including proelastase, basic trypsinogen, basic chymotrypsinogen, and ribonuclease, two out of three forms of procarboxypeptidase B, and the major lipase species were greatly reduced or absent in tumor cell secretion. In contrast, the amount of acidic chymotrypsinogen was notably increased compared with normal acinar cells. Although the acinar tumor cells are highly differentiated cytologically and express functional receptors for several classes of pancreatic secretagogues, they show quantitative and qualitative differences when compared with normal pancreas with regard to their production of secretory proteins.
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PMID:Biochemical analysis of secretory proteins synthesized by normal rat pancreas and by pancreatic acinar tumor cells. 618 2

We have previously established that secretory proteins from a rat acinar cell tumor lack two forms of procarboxypeptidase B, are deficient in a major lipase species, and possess markedly reduced amounts of the basic proteins proelastase, basic chymotrypsinogen, basic trypsinogen and ribonuclease (Iwanij, V., and J.D. Jamieson, J. Cell Biol., 95:734-741). Because secretory proteins are markers for acinar cell differentiation, we sought to establish whether the secretory protein profile of the acinar cell tumor is unique to the transformed cell or whether it resembles that of a stage of normal pancreatic development. To this end, we compared the secretory protein pattern from acinar tumor cells with that of rat pancreatic rudiments at days 19-22 of gestation and through day 21 of the postnatal period. Two-dimensional IEF-SDS gel electrophoresis coupled with biosynthetic labeling and fluorography indicates a time-dependent appearance of individual secretory proteins with basic polypeptides, except for amylase, appearing in the terminal stages of differentiation. In comparison, the secretory protein pattern of the acinar tumor cells most closely resembles that of day-19 embryonic pancreatic rudiments. We propose that the cells of the acinar cell tumor may, in part, mirror a stage of normal pancreatic development.
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PMID:Comparison of secretory protein profiles in developing rat pancreatic rudiments and rat acinar tumor cells. 618 3

24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high-resolution separation of proteins by two-dimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides. Beginning in the third hour of optimal hormone infusion at 0.25 microgram kg-1 h-1, changes were observed in the synthetic rates of 12 proteins, which progressed over the course of the 24-h study. Based on coordinate response patterns, exocrine proteins could be classified into four distinct groups. Group I (trypsinogen forms 1 and 2) showed progressive increases in synthetic rates reaching a combined 4.3-fold increase over control levels. Group II (amylase forms 1 and 2) showed progressive decreases in synthesis to levels 7.1- and 14.3-fold lower than control levels, respectively. Group III proteins (ribonuclease, chymotrypsinogen forms 1 and 2, procarboxypeptidase forms A and B, and proelastase 1) showed moderate increases in synthesis, 1.4-2.8-fold, and group IV proteins (trypsinogen 3, lipase, proelastase 2, and unidentified proteins 1-4) did not show changes in synthesis with hormone stimulation. Regulation of protein synthesis in response to caerulein infusion was specific for individual isoenzymic forms in the case of both trypsinogen and proelastase. The ratio of biosynthetic rates of trypsinogen forms 1 + 2 to amylase forms 1 + 2 increased from a control value of 0.56 to 24.4 after 24 h of hormonal stimulation (43.5-fold increase). Biosynthetic rates for an unidentified protein (P23) with an Mr = 23,000 and isoelectric point of 6.2 increased 14.2-fold, and the ratio of synthesis of P23 to amylase 2 increased 200-fold during caerulein infusion. During hormone stimulation the anticoordinate response in the synthesis of pancreatic glycosidases (decreased synthesis) and serine protease zymogens (increased synthesis) explain previous observations that showed little change in rates of total protein synthesis under similar conditions.
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PMID:Hormonal stimulation in the exocrine pancreas results in coordinate and anticoordinate regulation of protein synthesis. 620 98

An inhibitory factor, which has been shown to suppress the uptake of 125I-iododeoxyuridine by both lymphoid and nonlymphoid cells, was isolated from the supernatant of an Epstein-Barr virus- (EBV) transformed B cell line (1605L) established from a cotton-topped marmoset. Purification of the inhibitor, which was produced in serum-free medium by crowded cultures of the 1605L cells, was achieved by DEAE-cellulose chromatography followed by preparative polyacrylamide gel electrophoresis. The apparent m.w. of the 1605L factor was determined to be 65,000 to 70,000 by SDS-polyacrylamide gel electrophoresis. The inhibitor was sensitive to digestion by trypsin and chymotrypsin but not RNase or DNase, indicating that it was protein in nature. Exposure of the 1605L factor to 56 degrees C for 1/2 hr or pH 2 for 48 hr at 4 degrees C destroyed its inhibitory activity. The biochemical characteristics and activity of the 1605L inhibitor distinguish it from Type I interferon and several other soluble immunologic mediators known to be produced by lymphoid cell lines.
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PMID:Purification and biochemical characterization of an inhibitor of DNA synthesis produced by an Epstein-Barr virus-transformed B cell line. 624 72

The mechanism of poly ADPR synthesis and the transfer of poly ADPR to histone H1 molecule by electrophoretically homogenous calf thymus poly ADPR polymerase containing DNA was examined. 1) An acid insoluble radioactive complex (I) was obtained after incubation of purified enzyme with [3H] NAD. The stability of (I) was examined by SDS-polyacrylamide gel electrophoresis. The complex (I) was stable against acid, SDS, urea, DNase and RNase, but labile against pronase, trypsin, alkali and snake venom phosphodiesterase treatment. The molecular weight of (I) was about 130 000 daltons estimated by SDS-gel electrophoresis. The radioactive products of successive alkali, venom phosphodiesterase and Pronase hydrolysis of (I) were PR-AMP and AMP. The mean chain length of poly ADPR of (I) was 20--30. These results suggest that the complex (I) is poly ADP-ribosylated poly ADPR polymerase. 2) Besides (I), a second radioactive peak (II) was observed when acid insoluble products obtained from an incubation mixture containing purified poly ADPR polymerase, [3H] NAD and purified histone H1 were analyzed on SDS-polyacrylamide gel electrophoresis. The molecular weight of (II) was estimated to be about 23 000 daltons. The complex (II) is eluted like histone H1 on CM-cellulose columns and hydrolyzed by alkali, trypsin and snake venom phosphodiesterase but not by DNase, or RNase. The comples (II) was extracted selectively by 5 per cent perchloric acid or 5 per cent trichloroacetic acid from mixture of (I) and (II). The mean chain length of poly ADPR of complex (II) and 5--20; these results suggest that the complex (II) is poly ADP-ribosylated histone H1. 3) Results 1) and 2) indicate that purified DNA containing, thus DNA independent, poly ADPR polymerase catalyzes two different reactions, the ADPR transfer onto the enzyme itself and onto histone H1 and the elongation of ADPR chains. Dimeric forms of ADP-ribosylated histone H1 was not observed. Free poly ADPR was observed only when very small quantities of enzyme were used for incubation.
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PMID:Adenosine diphosphate ribosylation of histone H1 by purified calf thymus polyadenosine diphosphate ribose polymerase. 624 65

1. DNase I from porcine pancreas, if Mg2+ was present, hydrolyzed both sDNA and dDNA, whether free or bound to Sepharose. The hydrolysis rates were maximum at pH 7.5 with the bound DNAs and at pH 7.0 with the free DNAs negligible at pH 4.0 and pH 10.5 with the free and bound DNAs. The hydrolysis was completely inhibited by 50 mM sodium citrate. 2. With 50 mM citrate buffer (Ph 4.0), DNase I was effectively adsorbed on the DNA-Sepharoses in the absence of 5 mM Mg2+. The adsorbed enzyme was effectively eluated by the buffer containing 1 M KCl (eluate). The amounts of the eluated enzyme were approximately 1.5 X 10(5) units/mg DNA with sDNA-Sepharose and approximately 3.0 X 10(5) units/mg DNA with dDNA-Sepharose. This simple adsorption-elution of the pancreas extract resulted in approximately 300-fold purification of DNase I with a yield of 95%. In the elute, the ratios in activity of trypsin, chymotrypsin and RNase to DNase I were 1/(4.0 X 10(5)), 1/(5.3 X 10(3)), and 1/(4.1 X 10(2)) as low as in the extract, respectively. In addition, the eluate was not contaminated by kallikrein or carboxypeptidases A and B. 3. Upon repeating the adsorption-elution described above, the adsorbing capacities of DNA-Sepharoses gradually deteriorated with the whole pancreas extract, but not with the precipitate of the extract formed on 60% ammonium sulfate saturation, which contained 90% of the DNase I. With the precipitate, one dDNA-Sepharose solumn was repeatedly usable at least 20-times without deterioration. The DNase I preparation thus obtained was homogeneous on SDS-polyacrylamide gel electrophoresis. 4. Conceivably, the above-mentioned adsorption of DNase I on DNA-Sepharoses was mainly due to the steric and electrostatic affinity of a relatively large moiety of the DNA molecule to the substrate-binding site, but not to the catalytic site, of the enzyme.
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PMID:Affinity chromatography of porcine pancreas deoxyribonuclease I on DNA-binding sepharose under non-digestive conditions, using its substrate-binding site. 625 6


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