Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.
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PMID:Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies. 620 91

Using a rapid phenol extraction assay, an enzyme was purified from uninfected HeLa cells that can cleave the 5'-terminal protein (VPg) from poliovirus RNA. Both cytoplasmic and nuclear extracts had enzymes with similar behavior. A polypeptide of molecular weight 27,000 was the major one present in the purified preparation. Assuming that this protein is the enzyme, a very low turnover number was calculated for it. The purified enzyme would cleave the tyrosine-phosphate bond linking VPg to poliovirus RNA with minimal degradation of the RNA or of VPg. If the RNA was first treated with proteinase K to degrade VPg, leaving a small peptide on the RNA, this peptide could also be removed by the enzyme. If the RNA was degraded with T1 RNase, leaving VPg attached to a nonanucleotide, the enzyme still would cleave off VPg, although incompletely. If the RNA was degraded completely, leaving either pUp or pU attached to VPg, the enzyme would not remove the nucleotides from the protein. Thus, for the enzyme to be active requires some length of polynucleotide attached to the protein but only a short peptide need be present for the enzyme to act.
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PMID:Purification and properties of a HeLa cell enzyme able to remove the 5'-terminal protein from poliovirus RNA. 624 32

Ribonuclease St consists of 101 amino acid residues in a single polypeptide chain with one disulfide bond. It has two histidines located at positions 60 and 91 from the amino terminus. The pKa values of His-60 and His-91 were estimated by hydrogen-tritium exchange titration to be 8.0 and 6.3, respectively, and these values were confirmed by 1H NMR titration. The high pKa value of His-60 suggests that it interacts with a neighboring negative charge, presumably of a carboxylate. This is suggested by the presence of an inflection at pH 4.5 in the 1H NMR titration plot for His-60. The 1H-NMR titration plot for His-91 also suggests its interaction with a carboxylate, although the pKa of His-91 was close to that of unperturbed histidine residues. This suggests that a positively charged group is also located in the vicinity of His-91. It was concluded that His-91 is one of the active site residues of the enzyme. The pKa for His-91 was shifted to the alkaline side in the presence of 3'-GMP, a competitive inhibitor, in the titration plots observed by both hydrogen-tritium exchange and 1H NMR spectroscopy. The 31P NMR titration data suggest that in the 3'-GMP-RNase St complex the dianion form of the nucleotide participates in the interaction with the protonated form of His-91. The existence of another positively charged group with pKa of 7.0 was also suggested on the basis of the 31P NMR data.
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PMID:Hydrogen-tritium exchange and nuclear magnetic resonance titrations of the histidine residues in ribonuclease St and analysis of their microenvironment. 626 Jul 63

An endonuclease, which was originally identified for its RNA polymerase inhibitory activity, was isolated from rat liver endoplasmic reticulum. The enzyme yields on gel chromatography four active fractions of different molecular weights (Mr 5.3 X 10(4), 9 X 10(4), 1.55 X 10(5) and Sephacryl S-200 fraction at V0). Each fraction contains polypeptide chains which give a single band on sodium dodecylsulphate electrophoresis (Mr 5.4 X 10(4). This indicates that the enzyme is an oligomeric protein and each of its subunits exhibits the same or very similar molecular weights. Deoxyribonucleoside and ribonucleoside triphosphates can bind to the endoplasmic reticulum nuclease. Binding is enhanced in the presence of divalent cations particularly Mg2+. The enzyme exhibits mainly RNase activity but can also degrade denatured DNA and DNA . RNA hybrids which contain breaks in one of the two strands. Poly(A) and mainly poly(U) are most susceptible to its nucleolytic activity whereas poly(C) is completely resistant.
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PMID:Endoplasmic reticulum nuclease. Purification and specificity. 627 70

A partially purified pig heart phosphoprotein phosphatase was dissociated into three distinct components, namely alpha, beta, and gamma, by gel filtration on Sephacryl S-200 followed by chromatography on DEAE-Sephadex in the presence of 6 M urea. Although alpha itself had phosphatase activities toward P-H2B histone, P-H1 histone, phosphorylase a, and glycogen synthase b, beta and gamma had no activity toward these substrates even in the presence of 1 mM Mn2+. The beta component (Mr = 80,000) combined with alpha (Mr = 31,000) in the absence of urea to produce Form 2 (Mr = 123,000) with concomitant increase in P-H1 histone phosphatase activity and Mg2+ requirement for P-H2B histone phosphatase activity (Imazu, M., Imaoka, T., Usui, H., Kinohara, N., and Takeda, M. (1981) J. Biochem. 90, 851-862). The gamma component (Mr = 62,000) reassociated with Form 2 to produce Form 1 (Mr = 199,000) which was similar to the original phosphoprotein phosphatase in substrate specificity and Mg2+ requirement. Binding of gamma to Form 2 strongly suppressed the phosphatase activities toward phosphorylase a and glycogen synthase b with marginal effects on the other phosphatase activities and Mg2+ requirement. However, gamma alone could not associate with alpha. The gamma component was sensitive to treatment with heat (60 degrees C for 2 min) or trypsin and was resistant to treatment with DNase or RNase. The pig heart phosphoprotein phosphatase was further purified to near homogeneity, as judged by polyacrylamide gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis revealed that the purified enzyme (Mr = 171,000) was composed of three polypeptide components, namely alpha', beta', and gamma' with molecular weights of 34,000, 69,000, and 56,000, respectively. The component stoichiometry was determined to be alpha' 1 beta' 1 gamma' 1 by densitometric tracing of the Coomassie blue-stained bands on the acrylamide gel. After dissociation of alpha ' and other components by gel filtration of the purified enzyme on Sephacryl S-200 in the presence of 6 M urea, one alpha ' combined with one beta' to produce Form 2' of Mr = 106,000. Since Form 1 and the purified enzyme as well as Form 2 and Form 2' had similar catalytic properties and s20,w values, respectively, component compositions are suggested to be alpha 1 beta 1 gamma 1 for Form 1 and alpha 1 beta 1 for Form Form 2.
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PMID:Resolution and reassociation of three distinct components from pig heart phosphoprotein phosphatase. 629 3

Employing the recombinant runaway replication plasmid pDPK13 [sbcB+], an exonuclease I-overproducing derivative of Escherichia coli K12 has been constructed. The strain SK4258 has exonuclease I activity 140-400-fold higher than wild type control levels. A new purification procedure has been developed such that the protein can be purified to near homogeneity and is free of endonuclease and RNase activities. The specific activity of the purified enzyme is 10-fold higher than reported previously (Ray, R.K., Reuben, R., Molineux, I., and Gefter, M. (1974) J. Biol. Chem. 249, 5379-5381). Native exonuclease I is a single polypeptide having Mr = 55,000 with a Stokes radius of 3.12 nm.
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PMID:Amplification and purification of exonuclease I from Escherichia coli K12. 634 75

The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme Ribonuclease A are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30 degrees for periods up to 20 h have been analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components were found to elute earlier to RNase A; these have been designated RNase Aa2, Aa1c, Aa1b, and Aa1a in order of their elution. With the exception of RNase Aa2, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa1a, Aa1b, and Aa1c are monodeamidated derivatives of RNase A; RNase Aa1c contains, in addition, a small amount of a dideamidated component. RNase Aa2, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives were found to have very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives have shown that the deamidations are restricted to an amide cluster in the region 67-74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65-72 disulfide loop giving rise to at least three distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the ribonuclease molecule. Significance of this specific deamidation occurring in highly acidic conditions, and the biological implications of the physiological deamidation reactions of proteins are discussed.
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PMID:Isolation and characterization of monodeamidated derivatives of bovine pancreatic ribonuclease A. 642 73

A monoclonal murine antibody (No-114) is described which reacts specifically with a polypeptide of molecular weight (Mr) 180 000 present in low-speed nuclear pellets from oocytes and somatic cells of Xenopus laevis and X. borealis and in isolated amplified nucleoli. Two-dimensional gel electrophoresis has revealed the acidic nature of this polypeptide (isoelectric at pH of ca 4.2 in the presence of 9.5 M urea). A relatively large proportion of the protein is extracted at elevated ionic strength (i.e., at 0.4-0.5 M alkali salt) in a form sedimenting at approx. 7-8S, compatible with a monomeric state. It is also extracted by digestion with RNase but not with DNase. In immunofluorescence microscopy, antibody No-114 stains intensely nucleoli of oocytes and all somatic cells examined, including the residual nucleolar structure of Xenopus erythrocytes which are transcriptionally inactive. During mitosis the antigen does not remain associated with the nucleolar organizer regions (NOR) of chromosomes but is released and dispersed over the cytoplasm until telophase when it re-associates with the reforming interphase nucleoli. At higher resolution the immunofluorescent region is often resolved into a number of distinct subnucleolar components of varied size and shape. Immunoelectron microscopy using colloidal gold-coupled secondary antibodies reveals that the Mr 180 000 protein is confined to the dense fibrillar component of the nucleolus. This conclusion is also supported by its localization in the fibrillar part of segregated nucleoli of cells treated with actinomycin D. We conclude that nucleoli contain a prominent protein of Mr 180 000 which contributes to the general structure of the dense fibrillar component of the interphase nucleolus, independent of its specific transcriptional activity.
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PMID:Identification and localization of a novel nucleolar protein of high molecular weight by a monoclonal antibody. 653 10

A factor preventing spontaneous dissolution of germinal vesicle (GVBD) of mouse oocytes in vitro was isolated from bovine granulosa cells and designated as granulosa cell factor (GCF). Granulosa cells from medium sized (2-5 mm) follicles were suspended in 10 mM Tris-HCl buffer, pH 8.3, containing 1 M urea and 5 mM EDTA. GCF was separated from the extract by gel filtration on Sephadex G-25 column. The molecular weight (Mr) of GCF was estimated to be less than 6,000 daltons. Oocytes treated with GCF and resuspended in control medium resumed GVBD. The partially purified GCF lost GVBD-preventing activity when digested with pronase but retained its activity when treated with DNase, RNase, or glycosidase. GCF was stable to heating at 100 degrees C for 15 min, not absorbed by charcoal, and did not bind to Concanavalin A-Sepharose 4B. The present findings suggest that GCF is a heat-stable polypeptide and its action is reversible.
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PMID:A factor from bovine granulosa cells preventing oocyte maturation. 671 45

The 38,000-Mr poly(A)-binding protein has been purified to near homogeneity from non-polysomal messenger ribonucleoprotein of Artemia salina [Slegers, H., De Herdt, E., and Kondo, M. (1981) Eur. J. Biochem. 117, 111-120]. The protein consists of approximately 357 amino acids and is characterized by a high glycine content of 22.5% and the presence of dimethylarginine. From polynucleotide-protein binding experiments a stoichiometry of 9-11 adenylate and 10-12 uridylate residues per protein molecule is calculated. The polypeptide is devoid of poly(A) polymerase and RNase activities. The poly(A)-binding protein and the helix-destabilizing protein HD40 [Marvil, D. K., Nowak, L., and Szer, W. (1980) J. Biol. Chem. 255, 6466-6472] have the same mobility in polyacrylamide/dodecylsulphate gel electrophoresis and exhibit a comparable amino acid composition and protein-polynucleotide stoichiometry. Based on the length of poly(A) sequences of mRNA and from protein-poly(A) binding experiments, a repetitive binding of the 38,000-Mr protein on the poly(A) sequence is demonstrated. The 38,000-Mr protein of cytoplasmic and membrane-bound non-polysomal messenger ribonucleoproteins is also compared.
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PMID:The 38,000-Mr poly(A)-binding protein of non-polysomal messenger ribonucleoproteins of cryptobiotic gastrulae of Artemia salina. 706 May 86


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