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)

A ribonuclease was isolated and completely purified from sporulating cells of Bacillus subtilis. This RNase has a M.W. of about 150,000 daltons. It hydrolyzes single stranded RNA and single stranded synthetic polynucleotides yielding nucleoside 5'-monophosphates. The enzyme is an exonuclease which degrades polynucleotides from the 3'-end in the direction of the 5'-terminal. The RNase activity is strikingly inhibited by cGMP and to a lesser extent by cAMP. This inhibition (Ki = 0.1 mM) is of a non competitive nature. It appeared that in addition to the inhibition site, the enzyme contains a high affinity binding site for the two cyclic mononucleotides (K (cAMP) = 8.3 x 10-8; K (cGMP) = 2.5 x 10-7). The RNase activity is also strongly inhibited by spermidine. This inhibition appeared to be due to the polyamine binding with the RNA, thus lowering the affinity of the substrate for the active site of the enzyme. This RNase may play a role in vivo in selective degradation of newly synthesized mRNA during sporulation.
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PMID:Isolation and properties of a cyclic guanosine-monophosphate sensitive intracellular ribonuclease from Bacillus subtilis. 18 87

Membranes prepared from A-431 human epidermoid carcinoma cells retained the ability to bind 125I-labeled epidermal growth factor (EGF) in a specific manner. In the presence of [gamma-32P]ATP and Mn2+ or Mg2+, this membrane preparation was capable of phosphorylating endogenous membrane components, including membrane-associated proteins; the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. The binding of EGF to these membranes in vitro resulted in a severalfold stimulation of the phosphorylation reaction; again, the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. Membrane-associated dephosphorylation reactions did not appear to be affected by EGF. The phosphorylation reaction was not stimulated by cyclic AMP or cyclic GMP in the absence or presence of EGF. The phosphorylation system of the membrane was able to utilize [gamma-32P]GTP in both the basal and EGF-stimulated reactions. The enhanced membrane phosphorylation was specific for EGF and its derivatives; a wide variety of other peptide hormones were ineffective. The A-431 membrane preparation also was capable of phosphorylating exogenous proteins, such as histone, phosvitin, and ribonuclease, by a process which was stimulated by EGF. These findings suggest that one of the biochemical consequences of the binding of EGF to membranes is a rapid activation of a cyclic AMP-independent phosphorylating system.
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PMID:Rapid enhancement of protein phosphorylation in A-431 cell membrane preparations by epidermal growth factor. 31 92

The X-ray structures of two complexes of bovine ribonuclease-A produced by soaking pre-grown crystals in solutions of the inhibitors cytidylyl-2',5'-guanosine (2',5' CpG) and deoxycytidylyl-3',5'-guanosine (3',5'dCpdG) have been determined at 1.5 A resolution and refined by restrained least squares to R = 21.0% for 17,855 reflections, and R = 19.1% for 16,347 reflections, respectively. Binding of the substrate analogs to the protein has taken place in a completely unexpected and previously unreported manner. In each case the guanine base occupies the well characterized B1 pyrimidine binding site adjacent to Thr-45 (described by Richards, F.M., Wyckoff, H.W., Carlson, W.D., Allewell, N.M., Lee, B. and Mitsui, Y. (1971) Cold Spring Harbor Symp. Quant. Biol. 36, 35-54, and others including Palmer, R.A., Moss, D.S., Haneef, I. and Borkakoti, N. (1984) Biochim. Biophys. Acta 785, 81-88) having entered through a secondary channel external to the active site itself. We designate this reversed non-productive mode as retro-binding. In this mode of binding the SO4(2-) anion bound in the active site of the native protein crystals (Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38 2210-2217) has not been displaced by the phosphate of the inhibitor molecule as originally anticipated and observed in other studies. Instead the CMP or dCMP moiety of the inhibitor molecule is held loosely in a channel running towards the surface of the protein molecule and is thus completely external to the active site. Consequently, although it has been possible to model them, no attempt has been made to refine either the disordered cytosine in the CpG complex or the deoxycytosine in the dCpdG complex. The traditional B2 purine binding site of RNase (Richards et al., 1971) is unoccupied by the soaked inhibitors. Important changes that have taken place in the protein structure include: stabilization of both Lys-41 and Gln-11 via H-bonding to SO4(2-); stabilization of His-119 in the A conformation (Borkakoti, N., Moss, D.S. and Palmer, R.A. (1982) Acta Crystallogr. B38 2210-2217); and stabilization of SO4(2-) by H-bonds formed with the retro-bound guanine base. Binding of the inhibitors and stabilization of the active site is accompanied by displacement and redistribution of solvent molecules.
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PMID:Novel non-productively bound ribonuclease inhibitor complexes--high resolution X-ray refinement studies on the binding of RNase-A to cytidylyl-2',5'-guanosine (2',5'CpG) and deoxycytidylyl-3',5'-guanosine (3',5'dCpdG). 176 78

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activities, and is a competitive inhibitor of guanylate cyclase activity. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses. In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity. The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This preparation was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons. The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse). As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system. DNA synthesis was slightly stimulated. The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h. The cytostatic component had no effect on cellular cyclic GMP metabolism.
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PMID:Purification and characterization of a cytostatic factor with anti-viral activity from the bitter melon. 614 53

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activities, and is a competitive inhibitor of guanylate cyclase activity. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses. In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity. The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This preparation was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons. The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse). As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system. DNA synthesis was slightly stimulated. The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h. The cytostatic component had no effect on cellular cyclic GMP metabolism.
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PMID:Purification and characterization of a cytostatic factor with anti-viral activity from the bitter melon. 619 72

We have measured the expression levels of a range of distinct ion channel genes in the apex/ventricle region and sino-atrial node (SAN) sub-regions of heart under conditions in which conventional Northern hybridization or ribonuclease protection methods were too insensitive or non-quantitative. The abundance of six potassium channel mRNAs was determined in relation to a single synthetic competitor RNA template which was co-reverse transcribed and PCR-amplified. By these methods we have shown that coronary artery ligation procedures which induce anoxia and ischaemic scarring in the apical region reduce amplifiable message abundance in a time-dependent, but non-specific manner. There was no evidence for any selective reduction of individual mRNA levels during this process. Despite a high reproducibility of titration endpoints, competitive RNA template amplification assays did not provide a simple marker for ischaemic damage, since it was not possible to control for tissue sample heterogeneity. We have also applied these competitive nucleic acid titration techniques to demonstrate expression of cAMP- and cGMP-gated ion channel sequences in small pieces of tissue derived from the SAN sub-region of rabbit heart. Although the ion channels encoded by these sequences are obligately coupled to intracellular signalling agonists commonly found in cardiac cells, they have not been described in functional terms within SAN or any other cardiac subregion. For rapid determination of cDNA molecular numbers, we have devised single-gene, DNA template controls to measure absolute abundance of a cAMP-gated cDNA derived from heart tissue. Competitive titration procedures therefore provide an important technique for probing gene induction and/or repression accompanying pharmacological or surgical interventions, or in progression of disease states. For rare cDNAs, they can estimate the representativeness of a given preparation prior to library construction, screening and retrieval of clones, while eliminating 'false positive' or 'false negative' signals.
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PMID:Competitive titration for probing low-abundance ion channel mRNA molecules in normal and regionally-ischaemic heart tissue. 752 71

Hypertension is commonly associated with diabetes mellitus. The aim of the present study was to explore the pathophysiological significance of the natriuretic peptide (NP) system in hypertension associated with genetically obese/hyperglycemic Wistar fatty rats. The messenger RNA (mRNA) levels of the two biologically active NP receptors, NP-A receptor [more specific for atrial natriuretic peptide (ANP)] and NP-B receptor [more specific for C-type natriuretic peptide (CNP)], and CNP mRNA levels were determined in the aorta and kidney by ribonuclease protection assay. Plasma ANP levels were determined by RIA. Both NP-A and NP-B receptor mRNA levels in the aortae of Wistar fatty rats were double those in Wistar lean rats. Plasma ANP levels and CNP mRNA levels in the aorta of Wistar fatty rats were also significantly higher than those in Wistar lean rats. In contrast, there was no significant difference in renal levels of the mRNA for both NP receptors and CNP between the two strains. Administration of a NP-A and -B receptor antagonist, HS-142-1, to Wistar fatty rats resulted in a significant increase in systolic blood pressure and a larger decrease in plasma cGMP level than that in Wistar lean rats, with no difference in the extents of decrease in urine volume and urinary sodium excretion between the two strains. These results suggest that both the ANP/NP-A system and the CNP/NP-B system in vessels are up-regulated at the level of gene expression and may, thus, play an important role in counteracting the hypertension associated with diabetes mellitus.
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PMID:Vascular action of circulating and local natriuretic peptide systems is potentiated in obese/hyperglycemic and hypertensive rats. 894 Mar 83

Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate. The extracellular ribonuclease from Bacillus intermedius (binase, RNase Bi) shares a common mechanism for RNA hydrolysis with mammalian RNases. Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage. Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GpC and guanosine 2',3'-cyclic phosphate (cGMP) substrates were studied. The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.
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PMID:RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations. 915 77

An extracellular ribonuclease from Rhizopus stolonifer (designated as RNase Rs) was purified to homogeneity by chromatography on DEAE-cellulose followed by CM-cellulose. The Mr of the purified enzyme determined by gel filtration and SDS-PAGE is 25,000 and 28,200, respectively. RNase Rs is a glycoprotein and contains 10.5% neutral sugar. It is an acidic protein with a pI of 5.0 and has a blocked N-terminus. The optimum pH and temperature are 5.5 and 45 degrees C, respectively. RNase Rs shows high stability between pH 6.0-10.0. Divalent cations like Zn2+, Hg2+ and Cu2+ inhibit the enzyme activity whereas, mononucleotides does not have any significant effect. The enzyme cleaves RNA to 3'-mononucleotides via 2',3'-cyclic nucleotides, with preferential liberation of 2',3'-cyclic GMP, suggesting that RNase Rs is a guanylic acid preferential cyclizing RNase. Moreover, cyclic nucleotides generated are highly resistant to further hydrolysis.
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PMID:Extracellular ribonuclease from Rhizopus stolonifer: characteristics of an atypical--guanylic acid preferential--enzyme from ribonuclease T2 family. 952 62

Rod photoreceptor cGMP phosphodiesterase (PDE6) is a three-subunit (a, b, g2) enzyme that functions to reduce intracellular cytoplasmic cGMP levels, an integral feature of the phototransduction cascade of vision. To allow assessment of the potential for defects in the gene encoding the alpha-subunit (PDE6A) to cause visual dysfunction, and to begin to dissect the basis for photoreceptor-specific expression of this gene, we have characterized the structural gene and upstream region. The human PDE6A gene consists of 22 exons spanning about 60 kb with the intron/exon junctions highly conserved in comparison to the mouse and human PDE6B genes. Using ribonuclease protection and primer extension assays, a predominant transcription start point (tsp) was identified 120 bp upstream of the initiator ATG. To begin functional analysis of the PDE6A promoter, approx 4 kb of sequence were determined upstream of the tsp. Comparison of this upstream sequence with an approximately 500 bp sequence upstream of the mouse Pde6a gene revealed five distinct segments of identity all within 100 bp upstream of the human PDE6A tsp. A TATA box adjacent to a photoreceptor-specific RET1-like binding site, an SP1 site, and two novel putative cis-element sequences were found. A consensus initiator element sequence is present at the tsp. Additionally, within a 2.5-kb segment beginning 900 bp upstream of the tsp two Alu, a MIR, an L1, and two MER repetitive elements were found. Electrophoretic mobility shift assays generate a retina-specific bandshift using a 322-bp fragment containing the putative promoter region or a multimer of the RET1-like site. DNA footprinting assays revealed footprints over the primary transcription startpoint and the RET1-like and TATA box regions. These results indicate that a 220-bp segment of the PDE6A gene upstream region is important for tissue-specific expression.
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PMID:Structure and upstream region characterization of the human gene encoding rod photoreceptor cGMP phosphodiesterase alpha-subunit. 977 Jun 45


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