Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The presence of a nuclear DNA polymerase in mouse sperm from adult testes has been confirmed and the properties of this enzyme further investigated. This activity was shown to be greatly enhanced by treating the spermatozoa with methanol or ethanol before incubation in the reaction medium or by their addition in small amounts to this medium. It was protected against degradation by nuclear proteases by adding soybean trypsin inhibitor and was stimulated by ATP. It was found to be Mg2+ dependent (optimum concentration: 7.5 mM), DNA dependent, and all four deoxynucleoside triphosphates were needed for optimal reaction. The radioactive acid-precipitable product of polymerization was not eliminated by organic solvents, nor by pronase, ribonuclease or by nuclease S1; however, it was converted to a large extent to acid-soluble products by pancreatic deoxyribonuclease. Since it was only partially solubilized by Triton X-100, it therefore did not appear to be preferentially associated with the nuclear membranes. The activity recovered after incubation depended also on the pH (optimum at pH 8.3) and did not work well in a medium for DNA polymerase alpha. The temperature for maximum incorporation of nucleotides was found to be 32 degrees C and, under our conditions, the reaction was linear for 30 min. The DNA polymerase activity was inhibited by low and high concentrations of KCl. It was not lowered by N-ethylmaleimide or p-hydroxymercuribenzoate; urea slightly stimulated the reaction and this stimulation was reversed by subsequent treatment with N-ethylmaleimide. Actinomycin D (40 mug/ml), ethidium bromide (25--50 muM), netropsin (5--50 mug/ml), and spermidine (0.5--2.5 mM) lowered the polymerization of DNA precursors. The nuclear enzyme could shift from the endogenous template to activated exogenous calf thymus DNA, the resulting nuclear radioactivity being reduced. The endogenous DNP template ability was not increased by deoxyribonuclease activation according to the method of Aposhian and Kornberg (J. Biol. Chem. (1962) 237, 519--525) suggesting that the amount of DNA polymerase associated with chromatin was probably limiting the reaction. The DNA polymerase activity detected in mouse sperm nuclei has numerous properties of low molecular weight DNA polymerases (DNA polymerase beta) reported in several eukaryotic organisms.
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PMID:Further characterization of a DNA polymerase activity in mouse sperm nuclei. 1 3

A macromolecular binder of folic acid and folic acid derivatives has been identified in the particulate fraction of homogenates of rabbit choroid plexus. Within the choroid plexus, there are 2.3 nmol of folate-binding activity (binder) per g of tissue. The molecular weight of the folate binder complex, separated from the particulate fraction after solubilization with Triton X-100, was 340,000 to 400,000 by Sephadex gel filtration. The partially purified binder, when freed of endogenous folates, bound equivalent amounts of both [3H]folic acid and [methyl-14C]methyltetrahydrofolic acid per mg of protein. Folic acid, homofolic acid, 5-methyltetrahydrofolic acid, and to a lesser degree, methotrexate, inhibited the binding of both [3H]folic acid and [14C]methyltetrahydrofolic acid. Binding activity, which decreased below pH = 7.0, was unaffected by pretreatment with ribonuclease but was eliminated completely by papain and a protease (Streptomyces griseus). Although dihydrofolate reductase was present in choroid plexus, the binder was distinct from dihydrofolate reductase as judged by gel filtration and methotrexate sensitivity. This high affinity binder of folates may be responsible, in part, for the rapid, saturable uptake of folic acid and methyltetrahydrofolic acid by rabbit choroid plexus in vitro.
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PMID:Identification of folate binding macromolecule in rabbit choroid plexus. 1 98

Western equine encephalitis virus was disrupted with Triton X-100 and subjected to isoelectric focusing in a sucrose or urea gradient. The two envelope proteins, E1 and E2 were not well separated in a sucrose gradient, while the E1 and E2 proteins were distinguished as two major peaks which focused in a urea gradient at about pH 7.5 and 10, respectively. Isolated E1 protein refocused at pH 6.5 in a sucrose gradient isoelectric focusing column. When Western equine encephalitis virus was treated with Triton X-100 in 0.01 M phosphate buffer (pH6), hemagglutinating E1 protein was solubilized, which isoelectrofocused at pH 6.5. Purified nucleocapsids focused at pH 4 in a sucrose gradient on an isoelectric focusing column. After ribonuclease treatment of the purified nucleocapsid more than 95 per cent of the viral RNA was acid-soluble, and hte nucleocapsid protein isoelectrofocused at about pH 4.
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PMID:Isolation of the structural proteins of western equine encephalitis virus by isoelectric focusing. 4 5

This study describes the isolation and partial characterization of a Chlamydia trachomatis specific antigen. A species-specific antigen of C. trachomatis (antigen-0.65) was identified by two-dimensional immunoelectrophoresis. Antiserum specific for antigen-0.65 was prepared in rabbits by immunizing with agarose-gel precipitates excised from two-dimensional immunoelectrophorograms. Purified gamma-globulins from antigen-0.65 specific serum were coupled to the N-hydroxysuccinimide ester derivative of agarose which was then used for the immunoadsorbent purification of antigen-0.65 from Triton X-100 solubilized lymphogranuloma venereum (L2/434/Bu) organisms. The isolated antigen was immunochemically pure when tested against rabbit antiserum prepared to LGV-434 organisms by using rocket and two-dimensional immunoelectrophoresis. Antigenicity was destroyed by protease treatment and heating at 56 degrees C for 30 min, but the antigen was stable to ribonuclease, deoxyribonuclease, periodate oxidation and pH extremes of 2.2 and 10.6. Polyacrylamide gel electrophoresis of purified antigen showed a major protein band with an apparent m.w. of 155,000.
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PMID:Purification of a Chlamydia trachomatis-specific antigen by immunoadsorption with monospecific antibody. 6 24

After homogenization of intestinal mucosa from vitamin D-replete chicks and high speed centrifugation, the major proportion of the vitamin D-induced calcium-binding protein is present in the supernatant fraction. However, the centrifugate, after repeated washing, contains significant amounts of bound calcium-binding protein that can be solubilized by Triton X-100. The bound calcium-binding protein is identical to soluble calcium-binding protein by the criteria of immunological identity, electrophoretic mobility, and molecular size, as determined by gel filtration chromatography. The bound calcium-binding protein is only partially released by sonication, osmotic shock or by ribonuclease treatment. Bound and soluble calcium-binding protein are not present in rachitic chick intestine. The addition of calcium-binding protein to rachitic mucosa prior to homogenization does not yield a Triton X-100 solubilizable form, indicating that bound calcium-binding protein in vitamin D-replete intestine is not due to adsorption or vesicular entrapment of soluble calcium-binding protein. The overall evidence suggests that part of the intestinal calcium-binding protein is membrane-bound.
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PMID:Evidence for a membrane-bound fraction of chick intestinal calcium-binding protein. 63 6

A procedure allowing the isolation of intact polysomes from rooster liver is described. Good recovery of polysomes is achieved by the presence of Triton X-100 in the homogenization and centrifugation steps since the detergent prevents the sedimentation of microsomes with the nuclear fraction. This sedimentation of microsomes leads to considerable losses of polysomes, especially the larger ones. In the detergent-treated homogenate the integrity of the polysomes is threatened by various ribonucleases, some of which can be effectively inhibited by the addition of both heparin and yeast RNA. The remaining nuclease activity is counteracted by the endogenous ribonuclease inhibitor of the liver. In estradiol-treated roosters, sufficient endogenous inhibitor is present to inhibit its specific ribonuclease, but in control roosters there is not. This difference is due to a hormone-mediated increase in inhibitor level and decrease in nuclease level. Consequently, for an estrogenized rooster, the addition of both heparin and yeast RNA to the homogenate suffices to stabilize the polysomes, whereas control rooster liver homogenate needs supplementation with endogenous ribonuclease inhibitor. The cytosol of estrogenized rooster liver can be used as a crude inhibitor preparation. Rat liver cytosol is only partially effective; this may indicate a certain degree of species specificity of the inhibitor. The isolation procedure described also yields large polysomes from the livers of duck and Xenopus.
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PMID:Estradiol-induced synthesis of vitellogenin. IV. The isolation of non-degraded polysomes from avian liver using an endogenous ribonuclease inhibitor. 71 34

An endogenous DNA-synthesizing complex sensitive to ribonuclease has been found in purified preparations of swollen human sperm heads. Incorporation of [3H]dTTP into acid-precipitable material occurred in the presence of actinomycin D and required addition of dGTP, dCTP, dATP, plus Mg++. Polymerization was sensitive to pretreatment of the complex with pancreatic RNase A or Triton X-100. Exogenous activity was elicited by the synthetic template (dT)12--18-(rA)n but not by (dT)12--18-(dA)n or (dT)10. The complex sedimented from a 10,000 X g supernatant by centrifugation at 165,000 X g for 60 min and banded in sucrose at a density of 1.21--1.25 g/cm3. Endogenous RNase-sensitive DNA polymerase activity from cell-free seminal fluid was also detected in a fraction in sucrose at a density of 1.22 g/cm3. This activity was labile to freezing and stimulated by 0.04% Triton X-100, and thus differed from that of sperm heads.
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PMID:Ribonuclease-sensitive DNA-synthesizing complex in human sperm heads and seminal fluid. 105 11

Polysomes consisting of two to eight monosomes were isolated from yeast mitochondria by lysing the mitochondria with Triton X-100 and centrifugation in a 20 to 40% linear sucrose gradient. When yeast spheroplasts were pulse-labeled with [3H]-Leucine in the presence of cycloheximide to block cytoplasmic protein synthesis, radioactivity which was trichloroacetic acid-precipitable was present mainly in the polysome region. Incorporation of leucine was blocked by erythromycin, a specific inhibitor of mitochondrial protein synthesis. Release of radioactivity to the top of the gradient resulted from treating labeled polysomes with either puromycin or ribonuclease (in the latter case with the breakdown of polysomes), indicating that the radioactivity was present in nascent polypeptide chains. Yeast cells were grown in chloramphenicol for 3 hours and in fresh medium for 1 hour and then pulse-labeled with either [3H]leucine or [14C]formate. Three parameters showed a 2-fold increase in cells grown in chloramphenicol prior to pulse labeling: the polysome to monosome ratio, the amount of labeled precursor incorporated into proteins, and the rate of polypeptide chain initiation as judged by the formation of fMet-puromycin. Conversely, these parameters were all decreased approximately 50% in cells treated with cycloheximide prior to pulse labeling. Mitochondria were also isolated from cells previously grown in chloramphenicol or cycloheximide and incubated in vitro with [3H]leucine under optimal conditions. Acid-precipitable radioactivity in the polysome region was increased 3-fold in mitochondria from cells grown previously in chloramphenicol and decreased 75% in those grown in cycloheximide. Furthermore, chain initiation was deomonstrated in the isolated mitochondria by formation of fMet-puromycin. The rate of chain initiation in vitro was increased 2-fold in mitochondria isolated from chloramphenicol-treated cells.
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PMID:Regulation of mitochondrial protein synthesis at the polyribosomal level. 110 23

The administration of ethionine to female rats causes breakdown of hepatic polysomes. The state of mRNA and monomeric ribosomes after the polysome dissociation was studied. The mRNA was selectively labeled with [14C] orotate after a low dose of actinomycin D. Sucrose density gradient centrifugation of Triton X-100-treated cytoplasm revealed an accumulation of heterodisperse radioactive material with very large S values. This material was converted to smaller S values with deoxycholate treatment and was extremely sensitive to mild ribonuclease treatment. Since this material was banded at around 1.43 g/cm3 in CsCl gradient centrifugation and contained RNA with a distribution of S values characteristic of polysomal mRNA, this material was identified as mRNA-containing ribonucleoprotein particles. The monomeric ribosomes were shown to be dissociated into subunits in the presence of 0.5 M KCl, indicating that these lacked nascent polypeptide chains. When the animals were recovered from the ethionine treatment by subsequent administration of adenine and methionine, the heterodisperse ribonucleoprotein particles and monomeric ribosomes appeared to be utilized for the reformation of polysomes.
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PMID:The state of messenger ribonucleic acid and ribosomes in the cytoplasm of ethionine-treated rat liver. 111 2

An in vitro system of guinea pig pancreatic lobules convenient for the study of secretory processes is described in this paper. In this system: (a) the over-all glandular architecture of the tissue is preserved: lobules remain morphologically intact through 5 hours; (b) amylase discharge from unstimulated lobules is low (similar to 4%/hour) and linear over the 5 hours tested; (c) response to carbamylcholine chloride (10-5 M) is energy-dependent, rapid, and extensive (92% discharge of amylase by 5 hours); (d) initial rates of discharge remain stable over the first 3 hours; and (e) no autoactivation of zymogens occurs in incubation medium or tissue. The activation of four zymogens, i.e. chymotrypsinogen, trypsinogen, and procarboxypeptidases A and B, was studied using the following criteria for optimal activation: (a) maximal activation attainable under experimental conditions; (b) stability at the level of maximal activation; and (c) linear relationship between amounts of protein activated and enzyme activity elicited by activation. The concentration of activators (trypsin or enterokinase) and secretory protein, the presence or agents (bovine plasma albumin or Triton X-100) which minimize adsorptive losses of secretory protein on glass or plastic surfaces, and the temperature at which activation is carried out were found to be critical and different for each of the zymogens tested. The kinetics of the appearance of three enzyme activities (amylase, lipase, and ribonuclease) and four potential proteolytic activities (chymotrypsinogen, trypsinogen, and procarboxypeptidases A and B) into the incubation medium was studied under different conditions; i.e. rest and stimulation with various secretogogues (carbamylcholine chloride, caerulein, and pancreozymin). All seven activities estimated to represent similar to 75% of the secretory protein output of the exocrine pancreas were discharged in synchrony and in constant proportions and were released from the tissue to the same extent under each experimental condition investigated.
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PMID:Studies on the guinea pig pancreas. Parallel discharge of exocrine enzyme activities. 112 25


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