Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purpose of this study was to determine how RI alpha, the R subunit of the type I cAMP-dependent protein kinase, is regulated in rabbit ovarian follicles in response to the preovulatory luteinizing hormone surge. When soluble extracts from rabbit preovulatory follicles and 7-day-old corpora lutea were photoaffinity-labeled with 8-N3-[32P]cAMP, 3-fold more RI alpha was detected in corpora lutea than in follicles. Based on DEAE-cellulose chromatography, both type I holoenzyme and free RI alpha increased during luteinization. Western blot analysis of soluble extracts obtained from follicles and corpora lutea at various time points after human chorionic gonadotropin (hCG) injection revealed a 6-10-fold increase in RI alpha protein by 5 h after hCG injection. However, based on Northern blot analysis and solution hybridization/RNase protection assays, this increase in RI alpha protein was not due to an increase in RI alpha mRNA. These results suggested that RI alpha subunit levels were post-transcriptionally regulated. Half-life determinations indicated a 2.1-fold increase in the stability of RI alpha when follicles were incubated in the presence of hCG. The effect of hCG on the stability of RI alpha could also be mimicked by forskolin, thus suggesting that a rise in cAMP levels in follicles during the luteinizing hormone surge plays a role in RI alpha subunit stability. We conclude that RI alpha protein is stabilized in follicles by hCG treatment and the consequent rise in follicular cAMP levels.
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PMID:Luteinization-associated changes in protein stability of the regulatory subunit of the type I cAMP-dependent protein kinase. 132 Nov 43

A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE-cellulose and heparin--avidgel-F, followed by FPLC chromatography on mono S, Superose 12 and a second mono S column. This enzyme digests double-stranded DNA more efficiently than heat-denatured DNA. The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel-filtration Superose 12 column. The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein. The enzyme has an absolute requirement for divalent cations, preferring Mg2+ over Mn2+. No activity could be detected when these cations were replaced by Ca2+ or Zn2+. The pH optimum for this enzyme activity is 6.5-7.4 and its isoelectric point is 4.9. Both single-strand and double-strand breaks are introduced simultaneously into a supercoiled substrate in the presence of MgCl2 or MnCl2. Endonuclease-treated DNA serves as a substrate for DNA polymerase I from Escherichia coli, suggesting that 3'-OH termini are generated during cleavage. The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity. Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharomyces cerevisiae have revealed a potential structural homology between these nucleases.
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PMID:Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos. 133 52

A simple procedure, consisting of water extraction, heat treatment at pH 2.0, negative adsorption on DEAE-cellulose at pH 4.9, and concanavalin A-Sepharose chromatography, was developed for the partial purification of ribonuclease (RNase) T2 from taka-diastase powder with an overall yield of 5.5%. The partially purified enzyme when coupled to aminoethyl Bio-Gel P-60, retained 12-16% of the activity of the soluble enzyme. Temperature stability studies on RNase T2 bound to matrices, activated with increasing concentrations of glutaraldehyde, and the influence of lysine modification on the activity of the soluble enzyme revealed that the low activity observed for the gel-bound enzyme is probably due to the masking of the active site of the enzyme as a result of the involvement of lysine residues, situated near the active site, during coupling. Immobilization did not affect the pH and temperature optima of RNase T2. On repeated use, the bound enzyme retained approximately 55% of its initial activity after six cycles. These results are discussed, taking into consideration the factors affecting immobilized enzymes.
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PMID:Partial purification and immobilization of ribonuclease T2. 141 89

The steroid-binding capacity of the adrenocortical pregnenolone-binding protein (PBP) is effectively destroyed by extreme temperature (boiling water for 2-5 min); however, the boiled preparation contains a factor that potentiates ligand binding when readded to native PBP. Treatment of the boiled fraction with calf intestinal alkaline phosphatase at pH 9 reverses the stimulatory effect on PBP activity. Additionally, if native PBP is first incubated with alkaline phosphatase, which converts it to a nonbinding form, activity can be fully restored in a dose-dependent manner by the addition of the boiled preparation. The factor (itself devoid of binding capacity) can also be generated by exposing native PBP to acidic conditions (pH 4). The molecule is small (mol wt, less than 2000), as judged by Sephadex G-25 gel filtration and equilibrium dialysis. It is not retained on Concanavalin-A-Sepharose and is not extractable with a variety of organic solvents. The factor remains active after lyophilization and has a net negative charge at pH 7.4 (determined by DEAE-cellulose chromatography). While the binding capacity of native PBP is destroyed by a variety of proteases, the heat-stable factor is unaffected by similar treatment. Additionally, factor activity is not susceptible to RNase, DNase, or lipase digestion. Thus, the protein moiety of the PBP has an absolute requirement for a distinct phosphorylated heat-stable factor for expression of ligand-binding activity, and it may be through this factor that binding activity is regulated. It is not yet known whether the factor is acting allosterically or actually functions as part of the steroid-binding site.
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PMID:Adrenocortical pregnenolone-binding protein activity requires a small heat-stable factor: evidence that regulation by phosphorylation/dephosphorylation occurs at the level of the factor, not the protein. 177 Sep 49

Antibodies to uridylic acid rich small nuclear ribonucleoprotein particles (UsnRNP) are mainly detected in patients with systemic lupus erythematosus (SLE) or mixed connective tissue disease (MCTD). Particularly those directed against epitopes of the 70K protein of U1snRNP serve as important markers for the diagnosis of MCTD. To establish an ELISA for determination of anti-70K protein antibodies in patients' sera a 1239 bp long cDNA insert coding for the epitopes of the 70K protein was ligated into a fusion expression vector. The bacterially expressed fusion protein was purified by chromatography on DEAE cellulose. Microtiter plates were coated with the fusion protein as well as with partially purified calf thymus extract (CTE) containing all natural UsnRNP antigens and RNase digested calf thymus extract (CTERNase) in which the natural 70K antigen was destroyed by the nuclease treatment. 10,888 sera of patients with suspected or overt rheumatic disease were analyzed for antibodies against these antigens simultaneously. Antibodies against CTE or CTERNase were not detected in 9123 sera, none of these showed reactivity with the 70K protein indicating a high degree of specificity of the assay. Positive results in each the 70K protein, CTE as well as the CTERNase ELISAs were obtained with 474 sera. 319 sera were only positive with CTE and 70K protein. Of these 793 anti-70K protein ELISA positive sera, 79% could be confirmed by immunoblot. Of 967 sera reacting with CTE and CTERNase but not with the recombinant 70K protein, 31% contained antibodies against various other UsnRNP proteins as shown by immunoblotting. 2.4% of these sera revealed also antibodies against the 70K protein. The use of the recombinant 70K protein as antigen meets the criterion for a simple and specific assay to detect anti-U1snRNP antibodies. Nevertheless, the sole use of this recombinant protein for anti-U1snRNP antibody screening may not be appropriate, because antibodies against other frequently occurring U1snRNP proteins (A, C) cannot be detected with this test. Therefore it should be used together with a natural UsnRNP antigen until further studies in patients with well established diagnoses will show whether natural antigens may be omitted.
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PMID:A recombinant 70K protein ELISA. Screening for antibodies against U1snRNP proteins in human sera. 183 68

Protein disulfide-isomerase (PDI), which reactivates inactive scrambled RNase, was purified from Saccharomyces cerevisiae. The enzyme was purified 1,850-fold to apparent homogeneity by five purification steps: 30-70% ammonium sulfate fractionation, DEAE Toyopearl-650S and Butyl Toyopearl-650S chromatographies, and differential Phenyl-5PW HPLC with or without cysteine. The native enzyme had an apparent Mr of 140,000 on gel filtration chromatography, and its NH2-terminal was blocked. The Mr of its subunits were estimated to be 70,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the enzyme is probably composed of two identical subunits. The Mr of the subunits changed to 60,000 on endoglucosaminidase H treatment, indicating that the enzyme is transported into the endoplasmic reticulum. The enzyme has a pH optimum of 8.5, and pI of 4.02. Its enzymic properties were compared with those of purified bovine liver PDI. The Km values of yeast and bovine PDIs for scrambled RNase were 1 x 10(-5) and 2 x 10(-5) M, and their Vmax values were 6 and 7 units/mg protein, respectively. The two enzymes showed no significant differences in Km or Vmax values with respect to thiol compounds. Bacitracin inhibited both PDIs in the same fashion. These results indicate that this yeast PDI corresponds to mammalian PDI.
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PMID:Purification and characterization of yeast protein disulfide isomerase. 208 37

The first ATP-dependent complex formed in pre-mRNA splicing is the prespliceosome, a 30 S complex. This reaction was investigated using partially purified fractions isolated from nuclear extracts of HeLa cells. Previous studies (Furneaux, H. M., Perkins, K. K., Freyer, G. A., Arenas, J., and Hurwitz, J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4351-4355) have shown that DEAE-cellulose chromatography of nuclear extracts yielded two fractions (fractions I and II, eluted at 0.2 and 1 M NaCl, respectively) which carried out pre-mRNA splicing only when combined. Fraction II, alone and in the presence of ATP, supported the formation of the 30 S complex. In this report, we have separated fraction II into ribonucleoprotein and protein-rich fractions by isopycnic banding in CsCl. The combination of these two fractions completely replaced fraction II in prespliceosome formation; when supplemented with fraction Ib (1 M NaCl Biorex fraction derived from fraction I), the preparations supported spliceosome formation; when supplemented with fraction I, they yielded spliced products. The CsCl fractions, like fraction II, efficiently converted pre-mRNA to the 30 S complex with high yields (30-70%). The 30 S complex was shown to contain pre-mRNA complexed to U2 small ribonucleoproteins and small amounts of U1 small ribonucleoproteins. The 30 S complex protected a 50-nucleotide region at the 3'-end of the intron from T1 RNase attack. This region included sequences spanning the branch site, the polypyrimidine stretch and the AG dinucleotide of the 3'-splice site. When the 30 S complex was first generated with partially purified fractions, followed by the addition of a large amount of poly(U) or unlabeled pre-mRNA, the 30 S complex could be chased into a 55 S spliceosome complex by the addition of fraction Ib. These results support the conclusion, initially derived from kinetic data, that the 30 S complex is a precursor of the 55 S complex.
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PMID:Assemblage of the prespliceosome complex with separated fractions isolated from HeLa cells. 213 50

Infection of mouse L cells with mengovirus resulted in the activation of a protein kinase (PK) that selectively phosphorylated the small, 38,000-molecular-weight alpha subunit of eucaryotic initiation factor 2 (eIF-2) in vitro. The mengovirus-activated kinase was detected in vitro approximately 3 h after virus adsorption. The ratio of phosphorylated to unphosphorylated eIF-2 also increased in vivo between 3 and 7 h after adsorption. The virus-activated kinase fractionated with the ribosomal pellet and had a high affinity for DEAE-cellulose and Mono Q ion-exchange columns. Gel electrophoresis of the kinase activity eluting from the Mono Q column and silver staining of the gel revealed only one protein band with a molecular mass of 70 kilodaltons. The optimal assay conditions for the mengovirus-activated kinase paralleled those of the double-stranded RNA-activated PK (dsRNA-PK). Lysates from infected cells contained elements capable of activating partially purified dsRNA-PK. These elements were identified as double-stranded RNA by their sensitivity to double-stranded RNase. The phosphorylation of the alpha subunit of eIF-2 coincided with the synthesis of dsRNA in infected cells, suggesting that the mengovirus-activated kinase is the dsRNA-PK. The phosphorylation of the alpha subunit of eIF-2 correlated with the global inhibition of protein synthesis that occurs at late times after infection.
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PMID:The alpha subunit of eucaryotic initiation factor 2 is phosphorylated in mengovirus-infected mouse L cells. 216 23

T. brucei cytoplasmic calcium-dependent alkaline ribonuclease activity from DEAE-cellulose fractionation was separated into endoribonuclease and exoribonuclease activities by hydroxyapatite chromatography. T. brucei cytoplasmic extract markedly decreased the endoribonuclease activity, but slightly potentiated the activities of the exoribonuclease and bovine ribonuclease A. While the endoribonuclease was activated by trypsin, the exoribonuclease and bovine ribonuclease A were partially inactivated by trypsin. The endoribonuclease was activated by p-chloromercuribenzoate or N-ethylmaleimide; the exoribonuclease was not affected by these sulfhydryl group reagents. Free ribonuclease was separated from the latent endoribonuclease by 1 M NaCl-Sephadex G-100 gel filtration. The results demonstrate that T. brucei cytoplasm contains a latent endoribonuclease consisting of ribonuclease and inhibitor protein.
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PMID:Trypanosoma brucei: calcium-dependent endoribonuclease is associated with inhibitor protein. 222 4

The initiation of the processing of apomucin was investigated using mucus glycoprotein synthesizing polysomes from rat gastric epithelial cells. The polysomes were isolated from cells labeled with [3H]palmitic acid and [14C]N-acetylgalactosamine, purified on Helix pomatia-Sepharose affinity column, dissociated to release peptidyl-tRNA, and chromatographed on DEAE-HPLC column to separate peptidyl-tRNA complexes from the free and ribosomal RNA and proteins. The analysis of the HPLC purified peptidyl-tRNA revealed that complexes were labeled with [3H]palmitic acid and [14C]N-acetylgalactosamine. Digestion of the peptidyl-tRNA with RNase released 3H and 14C labeled peptides, while alkaline degradation destroyed the complex and rendered the [3H]palmitic acid extractable with hexane. The treatment of the 3H and 14C labeled peptidyl-tRNA complexes with alpha-N-acetylgalactosaminidase led to the release of radiolabeled N-acetylgalactosamine, whereas alkaline borohydride reduction produced N-acetylgalactosaminitol. The fatty acid residues have been detected in peptidyl-tRNA containing 2,000Da peptides, whereas N-acetylgalactosamine was discernible on 5,000Da peptides.
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PMID:Synthesis and initial processing of gastric apomucin. 276 78


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