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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pathways of ubiquitin-dependent protein degradation have in common two requirements for ATP. Ubiquitin activation by the enzyme E1 is accompanied by ATP hydrolysis to yield AMP and PPi, and during conjugate breakdown, the ubiquitin-dependent protease hydrolyzes ATP to ADP and Pi. We show here that either of two beta, gamma-nonhydrolyzable ATP analogues, 5'-adenylyl imidodiphosphate or 5'-adenylyl methylenediphosphate, can support ubiquitin-protein conjugation. With the ubiquitin-dependent protease, however, neither analogue could substitute for ATP. Thus, the substitution of a beta, gamma-nonhydrolyzable analogue for ATP offers a simple method to uncouple ubiquitin conjugation from proteolysis in crude systems. On the basis of pyrophosphate exchange kinetics, E1 has apparent Km and Vmax values that are similar for ATP and the analogues, but substrate inhibition by 5'-adenylyl methylenediphosphate made use of the beta, gamma-imido analogue preferable. In one application, beta, gamma-imido-ATP was used in combination with ubiquitin aldehyde (an inhibitor of ubiquitin-protein isopeptidases) to establish that several unfolded RNase A derivatives are recognized equally as ubiquitination substrates. This result extends an earlier study [Dunten, R. L., & Cohen, R. E. (1989) J. Biol. Chem. 264, 16739-16747] to show that conjugate yields, upon which relative ubiquitination rates were based, were not influenced by differential ubiquitin-dependent proteolysis. In a second application, ATP and beta, gamma-imido-ATP were compared in a pulse-chase experiment to investigate the contributions of ATP-dependent proteolysis and isopeptidase activities to conjugate stability.
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PMID:Uncoupling ubiquitin-protein conjugation from ubiquitin-dependent proteolysis by use of beta, gamma-nonhydrolyzable ATP analogues. 164 32

Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media. The extracellular ribonuclease from strain W has been purified and partially characterized. The molecular weight was determined by gel filtration to be 26,500. The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated. The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity. The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid. The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP. No inhibition of the hydrolysis of Torula yeast RNA was detected. The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes.
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PMID:Characterization of the extracellular ribonuclease of Tetrahymena pyriformis W. 192 Jan 45

To assess whether myoglobin adversely affects renal adenylate pools, rats were infused with purified myoglobin (50 mg/100 g body wt) for two hours and renal ATP, ADP, and AMP levels were measured in the absence of shock, after 25 minutes of hemorrhagic shock (55 to 60 mm Hg) or 30 minutes post-recovery. In the absence of shock, myoglobin lowered ATP by 24% (assessed 65 min post-infusion) without affecting renal blood flow (RBF). This effect was completely blocked by deferoxamine (DFO) treatment and it could not be reproduced by ribonuclease infusion (a non-Fe containing, but filtered, protein). Myoglobin + shock caused a three- to fourfold greater decline in ATP than did shock alone despite comparable RBFs. Shock plus myoglobin, but neither one alone, induced substantial S1/S2 proximal tubular morphologic damage and a severe reduction in creatinine clearance, confirming synergistic injury. Ribonuclease completely reproduced myoglobin's effect on shock-induced adenylate profiles. DFO +/- hydroxyl radical scavenger therapy (Na benzoate) did not block the myoglobin shock effect on adenylate pools. Post-shock adenylate recovery was not compromised by myoglobin pre-treatment. If renal artery occlusion (RAO), rather than shock, was used as the ischemic challenge, myoglobin had no discernible impact on adenine nucleotide content. This study concludes that: 1) myoglobin modestly lowers baseline adenylate pools due to an Fe dependent mechanism; 2) myoglobin drastically accentuates shock-induced adenylate depletion by a non-hemodynamic/non-Fe dependent mechanism; 3) myoglobin nephrotoxicity cannot be attributed solely to tissue iron loading; and 4) the RAO model can completely mask important influences on ischemic cellular energetics.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Myoglobin depletes renal adenine nucleotide pools in the presence and absence of shock. 200 25

Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.
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PMID:Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells. 201 59

We present a calculation of the relative changes in binding free energy between the complex of ribonuclease T1 (RNase Tr) with its inhibitor 2'-guanosine monophosphate (2'GMP) and that of RNase T1-2'-adenosine monophosphate (2'AMP) by means of a thermodynamic perturbation method implemented with molecular dynamics. Using the available crystal structure of the RNase T1-2'GMP complex, the structure of the RNase T1-2'AMP complex was obtained as a final structure of the perturbation calculation. The calculated difference in the free energy of binding (delta delta Gbind) was 2.76 kcal/mol. This compares well with the experimental value of 3.07 kcal/mol. The encouraging agreement in delta delta Gbind suggests that the interactions of inhibitors with the enzyme are reasonably represented. Energy component analyses of the two complexes reveal that the active site of RNase T1 electrostatically stabilizes the binding of 2'GMP more than that of 2'AMP by 44 kcal/mol, while the van der Waals' interactions are similar in the two complexes. The analyses suggest that the mutation from Glu46 to Gln may lead to a preference of RNase T1 for adenine in contrast to the guanine preference of the wild-type enzyme. Although the molecular dynamics equilibration moves the atoms of the RNase T1-2'GMP system about 0.9 A from their X-ray positions and the mutation of the G to A in the active site increases the deviation from the X-ray structure, the mutation of the A back to G reduces the deviation. This and the agreement found for delta delta Gbind suggest that the molecular dynamics/free energy perturbation method will be useful for both energetic and structural analysis of protein-ligand interactions.
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PMID:Calculation of the relative binding free energy of 2'GMP and 2'AMP to ribonuclease T1 using molecular dynamics/free energy perturbation approaches. 215 20

Poly(A)-specific ribonuclease was co-purified with poly(A) polymerase from Vigna unguiculata seedlings. Both activities were separated into two forms (enzymes I and II) by a final hydrophobic column chromatography. The enzyme I preparation, which was homogeneous as examined by SDS/PAGE, had both poly(A) polymerase and poly(A)-specific ribonuclease activities. The antibody raised to the enzyme I preparation precipitated both enzyme activities. These indicate that a single polypeptide (Mr 63,000) is responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. The poly(A)-specific ribonuclease was a 3'-exonuclease specific to single-stranded poly(A), forming 5'AMP as the sole reaction product. The hydrolytic activity required either Mn2+ or Mg2+ with different optimum concentrations, whereas the polymerizing activity required Mn2+ but not Mg2+. ATP and PPi had little or no effect on the poly(A)-specific ribonuclease activity.
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PMID:Poly(A) polymerase from Vigna unguiculata seedlings. A bifunctional enzyme responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. 255 12

The reaction of ribonuclease A with either 6-chloropurine riboside 5'-monophosphate or the corresponding nucleoside yields one derivative, with the reagent covalently bound to the alpha-amino group of Lys-1, called derivative II and derivative E, respectively. We studied by means of 1H-n.m.r. at 270 MHz the interaction of these derivatives with different purine ligands. The pK values of His-12- and -119 were obtained and compared with those resulting from the interaction with ribonuclease A. The results showed that the interaction of derivative E with 3'AMP is similar to that described for RNase A as the pK2 of His-12 is increased while that of His-119 remains unaltered. However, derivative II presents some differences as it was found an enhancement of the pK2 values of both His-12 and His-119. Interaction of derivative II and derivative E with dApdA increases the pK2 of His-119, whereas a decrease is found when it interacts with ribonuclease A. These results suggest that the phosphate group and the nucleoside of both derivatives are located in regions of the enzyme where natural substrate analogues have secondary interactions and they can be interpreted as additional binding sites.
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PMID:1H-n.m.r. studies on the existence of substrate binding sites in bovine pancreatic ribonuclease A. 279 10

Extensive 15N-NMR investigations of active-site amino acids were made possible by the solid-phase synthesis of the N-terminal pentadecapeptide of RNase A with selectively 15N-enriched amino acids. On complexation with S-protein a fully active RNase S' complex was obtained. The 15N resonances of the side chains of lysine-7 (N epsilon), glutamine-11 (N gamma), and histidine-12 (N pi, tau) were studied in the free synthetic peptide, in the RNase S' complex and in the nucleotide complexes RNase S' with 2'CMP, 3'CMP, and 5'AMP. The analysis of the 15N-1H couplings, the 15N line broadenings due to proton exchange, and the chemical shift values showed that, while the imidazole ring is directly involved in the peptide-protein interaction, the side chains of Lys-7 and Gln-11 do not contribute to this interaction. In the nucleotide complexes the resonances of His-12 and Gln-11 are shifted downfield. In the 2'CMP complex a doublet for the N tau signal of His-12 indicates a stable H bond between this nitrogen and the phosphate group of nucleotide. The other nucleotide influence the resonances of the imidazole group much less, possibly due to a slightly different orientation of the phosphate group. The downfield shift of the Gln-11 resonance indicates an interaction between the carbonyl oxygen of the amide group and the phosphate moiety of the nucleotide. The only observable effect of nucleotide complexation on the Lys-7 signal is line broadening due to reduced proton exchange. For comparison with the 15N-NMR titration curves of His-12 in RNase S' the 1H-NMR titration curves of RNase A were also recorded. Both shape and pK values were very similar for the 15N and the 1H titration curves. An extensive analysis of the protonation equilibria with several fitting models showed that a mutual interaction of the imidazole groups of the active-site histidines results in flat titration curves. The Hill plots of all resonances of the imidazole rings, including the 15N resonances, show a small inflection in the pH range 5.8-6.4. Since the existence of a diimidazole system is most likely in this pH range, the inflection could be interpreted as a disturbance of the mutual electrostatic interaction of the active-site histidines by a partial H-bond formation between the imidazole groups.
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PMID:15N- and 1H-NMR investigations of the active-site amino acids in semisynthetic RNase S' and RNase A. 283 66

To facilitate further studies of flavivirus transcription, cell extraction methods and in vitro reaction conditions which increased West Nile virus (WNV) RNA-dependent RNA polymerase activity were determined. Subcellular fractions from WNV-infected BHK-21/W12 cells were characterized with regard to their protein and RNA content and in vitro polymerase activity. In both a cytoplasmic fraction, designated S1, and a fraction enriched for outer nuclear membranes, designated S2, seven virus-specific proteins, NS5 (96 kilodaltons [kDa]), NS3 (67 kDa), E (48 kDa), NS1 (47 kDa), ns4a (26 kDa), ns2a (17 kDa), and ns2b (14.5 kDa), were detected. The fractions also contained virus-specific RNA and cellular rRNA and mRNA. Polymerase activity in S1 and S2 fractions from WNV-infected cells was concentrated by pelleting and consisted of two types of enzyme activities: the WNV RNA-dependent RNA polymerase and terminal transferases of cellular origin. Enhanced levels of WNV polymerase activity were obtained from these cell fractions by altering several of the in vitro reaction conditions. Although Mg2+ was the divalent cation preferred by WNV polymerase, virus-specific in vitro transcription was detected at reduced levels when Mn2+ (0.05 or 0.5 mM) was present as the sole divalent cation. Product analysis revealed that the viral polymerase incorporated radiolabeled ribonucleotides into three distinct RNA species. Free single-stranded genome-sized RNA which was LiCl insoluble and RNase sensitive was found by fingerprint analysis to have an oligonucleotide pattern similar to that of WNV genomic RNA. RNA molecules which comigrated as a broad band near the top of the gel were separable into LiCl-insoluble, partially RNase-sensitive replicative-intermediate RNA and LiCl-soluble, RNase-resistant replicative-form RNA. The cellular transferases added UMP or AMP residues to the 3'-termini of cellular mRNA, tRNA, and 18S and 28S rRNA. Although a cellular terminal transferase has been reported to function in initiation of poliovirus transcription, no labeling of the WNV RNA by either of these cellular enzymes was detected. Therefore, they appear to play no specific role in flavivirus RNA synthesis.
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PMID:Characterization of West Nile virus RNA-dependent RNA polymerase and cellular terminal adenylyl and uridylyl transferases in cell-free extracts. 302 63

A soluble ATP/Mg2-dependent proteolytic system from rabbit cardiac muscle has been identified (m ca. 310 kDa) and purified ca. 9-fold. This enzyme which splits the substrate [3H]globin and 125I-bovine serum albumin (125I-BSA) has many similarities to the ATP-dependent proteolytic enzyme system from reticulocytes which utilizes ubiquitin: 1) The specific activities in reticulocyte lysates and cardiac muscle extracts are of the same magnitude (0.5-1 arb. unit/mg). 2) The binding and elution behavior on DEAE-cellulose is similar. 3) In both cases the pH optimum (substrate 125I-BSA) is pH 7.6. 4) Both enzymes are inhibited by hemin, NEM and iodoacetate but not e.g. by leupeptin, or inhibitors of serine proteases. 5) Neither enzyme system can utilize ATP-analogs such as AMP-CPP, AMP-PCP, AMP-PNP or ATP-gamma-S. There are however also significant differences: 1) The enzyme system from cardiac muscle is fully active in the absence of ubiquitin and cannot be activated by this peptide. 2) The enzyme from cardiac muscle can degrade methylated BSA. 3) The cardiac muscle enzyme can be further purified on Sepharose 4B; the enzyme from reticulocytes is inactivated by this procedure. 4) The cardiac enzyme cannot be inactivated by ribonuclease as the reticulocyte counterpart. Although ubiquitin does not appear to play a role in the isolated ATP/Mg2-dependent proteolytic system from cardiac muscle, it is demonstrated for the first time that 125I-ubiquitin can be conjugated to a wide variety of cardiac muscle proteins in vitro in an ATP-dependent manner. Apparent molecular masses of major conjugates were: 185 kDa, 140 kDa, 85 kDa, 65 kDa, 46 kDa, 38 kDa and 36 kDa as estimated by discontinuous SDS gel electrophoresis. Addition of purified phosphorylase kinase to cardiac muscle extract changed the ubiquitination pattern by the appearance of two novel protein bands. It is concluded that the ATP/Mg2-dependent proteolytic system of cardiac muscle must be differentiated from the proteolytic system of reticulocytes mainly because of its ubiquitin-independence. Nevertheless the conjugation of 125I-ubiquitin to many muscle proteins is a strong indication for a crucial role of this interesting peptide in striated muscle.
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PMID:ATP-dependent proteolysis and the role of ubiquitin in rabbit cardiac muscle. 304 36


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