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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Specific beta-adrenergic receptors present in membrane preparations of frog erythrocytes were identified by binding of (-)-[3H]dihydroalprenolol, a potent competitive beta-adrenergic antagonist. The (-)-[3H]dihydroalprenolol binding sites could be solubilized by treatment of a purified erythrocyte membrane fraction with the plant glycoside digitonin but not by treatment with a wide variety of other detergents. The binding sites appeared to be soluble by several independent experimental criteria including (a) failure to sediment of 105,000 X g for 2 hours; (b) passage through 0.22-mu Millipore filters; (c) chromatography on Sepharose 6B gels; and (d) electron microscopy. The soluble receptor sites retained all of the essential characteristics of the membrane-bound sites, namely rapid and reversible binding of beta-adrenergic agonists and antagonists; strict stereospecificity toward both beta-adrenergic agonists and antagonists; appropriate structure-activity relationships; saturability of the sites at low concentrations of ligand; no affinity for alpha-adrenergic drugs, nonphysiologically active catechol compounds, and catecholamine metabolites. Based on gel chromatography in the presence of detergent, the molecular weight of the soluble receptor is estimated to be no greater than 130,000 to 150,000. Equilibrium binding studies indicated a KD for the soluble receptor of 2 nM. Hill coefficients (nH) of 0.77 and curved Scatchard plots suggested the presence of negatively cooperative interactions among the solubilized receptors in agreement with previous findings with the membrane-bound sites. Kinetic studies indicated an association rate constant K1 = 3.8 X 10(6) M-1 min-1 and a reverse rate constant k2 = 2.3 X 10(-3) min-1 at 4 degrees. The kinetically derived KD (k2/k1) of 0.6 nM is in reasonable agreement with that determined by equilibrium studies. The soluble receptors were labile at temperature greater than 4 degrees but could be stabilized with high concentrations of EDTA. Guanidine hydrochloride and urea produced concentration-dependent losses of binding activity which were partially reversible upon dialysis. Trypsin and phospholipase A both degraded the soluble receptors but a variety of other proteases and phospholipases as well as DNase and RNase were without effect. Experiments with group-specific reagents indicated that free lysine, tryptophan, serine, and sulfhydryl groups may be important for receptor binding. These studies suggest that the receptor is probably a protein which requires lipids for functional integrity. Data obtained with the solubilized binding sites are consistent with the contention that these sites represent the physiologically relevant beta-adrenergic receptors which have been extracted from the membranes with full retention of their properties.
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PMID:Solubilization and characterization of the beta-adrenergic receptor binding sites of frog erythrocytes. 0 47

In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1.
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PMID:Chemical modifications of ribonuclease U1. 1 50

The action of Armillaria mellea protease has been evaluated on a number of polypeptide substrates. It has been shown to split the Pro7-Lys8 bonds in both native and oxidised lysine-vasopressin and the Ser11-Lys12 bond in glucagon. No other splits were detected in these substrates. The enzyme also caused extensive degradation of S-carboxymethyl lysozyme, S-carcoxymethyl pepsinogen and oxidised ribonuclease. A. In each case the only new amino-terminal residue to appear was lysine. A. mellea protease was inhibited by the chelating agents 1,10-phenanthroline, alpha, alpha'-bipyridine and imidazole. The pK1 values (negative log10 of concentration required for 50% inhibition) for these three inhibitors were 3.9, 3.4 and 1.1, respectively. Lysine, S-2-aminoethylcysteine and short chain aliphatic amines also proved to be relatively good inhibitors of A. mellea protease while arginine was a poor inhibitor.
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PMID:Specificity and inhibition studies of Armillaria mellea protease. 2 49

The allosteric model for ribonuclease activity by Walker, Ralston & Darvey [(1975) Biochem.J. 147, 425--433; (1976) Biochem.J. 153, 329--337] involves the binding of a large number of molecules of substrate or substrate analogue to a series of allosteric sites on the enzyme. In the present paper, the nature of these allosteric interactions is investigated. The effects of ionic strength pH carbamoylation of lysine to homocitrulline and of deamidation of glutamine and asparagine on plots of velocity versus substrate concentration are examined and evidence is presented that the allosteric transition involves an electrostatic interaction between the negatively charged substrate molecules and the cationic groups on the enzyme.
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PMID:The nature of the allosteric interactions of ribonuclease and its ligands. 2 30

The ribonuclease A derivative Npi-[13C1]carboxymethyl-histine-119 ribonuclease prepared by using [13C1]bromoacetate as alkylating reagent has been investigated with high resolution 13C NMR spectroscopy. In the 13C NMR spectra two carbon resonances of relatively high intensity appear which can be assigned to carboxyl groups attached to His-119 and Met-30, their intensity ratio being 10 : 1. The pH dependence of the carbon resonance of the carboxy-methyl group bound to the Npi of His-119 differs in the absence and presence of Cyd-2'-P, thus indicating that the catalytically inactive derivative does bind nucleotides. A mechanism of the alkylation reaction at pH 5.6 is proposed in which the epsilon-amino group of Lys-41 acts as the binding site for the carboxyl group of bromoacetate pushing the bromomethylene group towards the Npi of His-119 or the Ntau of His-12.
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PMID:13C NMR investigations on Npi-[13C1]carboxymethyl-histidine-119 ribonuclease. 3 23

Ethyl bromoacetimidate was designed as a potential reagent for cross-linking protein NH2 groups with a vicinal nucleophile. The chemical properties of this compound were studied by model reactions with small molecules. Ethyl bromoacetimidate amidinates lysine residues in ribonuclease at pH 9. In a second step, at lower pH values, one of the bromoacetamidino groups bound to the enzyme alkylates a proximal histidine residue. This substitution is pH-dependent with a sharp optimum at 5.6, the same as was earlier observed for alkylation of histidine-119: histidine-12 by halogenoacetates and halogenoacetamides. A common mechanism is suggested for all these types of alkylation. Ethyl bromoacetimidate thus appears as a short-distance crosslinker which can be used, for example, to explore chemically the microenvironment of an essential lysine residue of an enzyme within the active site.
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PMID:Ethyl bromoacetimidate, a NH2-specific heterobifunctional reagent. Model reactions with ribonuclease. 4 7

The role of procapsids during foot-and-mouth disease virus multiplication was studied on infected BHK-21 cells. Purified virus and procapsids were obtained by treating the infected cytoplasmic extracts with RNase and EDTA. The synthesis of virus, procapsids, and total particles was determined in pulse-chase experiments. A precursor-product relationship between procapsids and virions was obtained. The results show that the rate of synthesis of total particles (virus + procapsids) was linear from the addition of the label and was identical to that corresponding to virions. Therefore, the speed of the morphogenetic process as well as the existence of a precursor pool of structural proteins was established. Furthermore, the rate of virus synthesis from procapsids was identical to the rate of synthesis of procapsids from their structural precursors. A quantitative recovery of label from procapsids into virions was obtained by the use of cycloheximide or tosyl-lysine chloromethyl ketone. Under these conditions, virus synthesis proceeds, indicating that these drugs do not affect the morphogenetic step studied in this paper.
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PMID:Morphogenesis of foot-and-mouth disease virus. I. Role of procapsids as virion Precursors. 22 34

The amino groups of ribonuclease A (RNase-A) have been methylated with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance (PMR) studies. Although enzymatic activity is lost, PMR difference spectroscopy and PMR studies of thermal denaturation show native conformation is largely preserved in methylated RNase-A. Resonances corresponding to the NH2-terminal alpha-amino and 10 xi-amino N-methyl groups are titrated at 220 MHz to obtain pK values. After correction for the effects of methylation, using values previously derived from model compound studies, a pK of 6.6 is found for the alpha-amino group, a pK of 8.6 for the xi-amino group of lysine-41 and pK values ranging from 10.6 to 11.2 for the other lysine xi-amino groups. Interactions between lysine-7 and lysine-41 or between the alpha-amino and xi-amino groups of lysine-1 have been proposed to account for deviations from simple titration behaviour. The correct continuities for the titration curves of the histidine H-2 proton resonances have been confirmed by selective deuteration of the H-2 protons. Titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A show deviations from the titration curves for the native enzyme, indicating some alteration of the active-site conformation. In the presence of phosphate, titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A indicate binding of phosphate at the active site, but these curves continue to show deviations from the titration behaviour of native RNase-A. The titration curve for the N-methyl resonance of lysine-41 is perturbed considerably by the presence of phosphate, which indicates a possible catalytic role for lysine-41.
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PMID:Proton-magnetic-resonance studies of the lysine residues of ribonuclease A. 23 43

M protein was extracted from type 24, group A streptococci with pepsin at pH 5.8 and was further purified by ammonium sulfate precipitation, ribonuclease digestion, ion-exchange chromatography, and isoelectric focusing. The purified pepsin extract of M (pep M) protein was shown to be free of nontype-specific immunoreactivity in (a) complement fixation tests with heterologous M antiserum, (b) skin tests in normal adult guinea pigs, and (c) passive hemagglutination tests for the presence of lipoteichoic acid sensitizing or antigenic activity. The pep M24 was highly immunogenic; two of three rabbits developed opsonic antibody titers of 1:256 and the third a titer of 1:32 6 wk after a single injection of 100-pg doses of pep M24 emulsified in complete Freund's adjuvant. The antisera lacked nontype-specific antibodies and produced single precipitin lines in agar gel diffusion tests against crude HC1 extracts of the homologous M protein. Thus, the type-specific antigenic determinant(s) of type 24 M protein appears to be separable from immunotoxic, cross-reactive antigens without loss of immunogenicity in rabbits. The mobility of pep M24 upon electrophoresis in 10 percent sodium dodecyl sulfate pelyacrylamide gel was consistent with an average mol wt of 33,500 daltons. Amino acid analysis demonstrated a predominance of alanine, followed by glutamic acid, lysine, leucine, and aspartic acid. Pep M24 contained an estimated six to seven methionine residues and approximately ten phenylalanine residues per molecule. No other aromatic amino acids were detected. Automatic Edman degradation of pep M24 yielded the sequence of the first 29 amino acids (the amino terminal amino acid being valine) of the amino terminal region of the molecule. The detection of only one new amino acid at each step of Edman degradation confirmed the homogeneity of the purified pep M24.
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PMID:Purification and properties of M protein extracted from group A streptococci with pepsin: covalent structure of the amino terminal region of type 24 M antigen. 32 68

EDTA binds at the active site of ribonuclease causing a selective downfield shift of the C2 proton resonance of His 12 at pH 5.5 (pH denotes an uncorrected glass electrode pH meter reading of a 2H2O solution). A dissociation constant for EDTA binding to ribonuclease of 1.70 mM was calculated from this chemical shift change. The pKa of His 12 increased from 5.79 in ribonuclease alone to 6.73 in the RNAase . EDTA complex. Compared to these effects, the other histidine residues were not significantly affected by EDTA. EDTA was shown to act as a competitive inhibitor of cytidine 2',3'-cyclic phosphate hydrolysis by ribonuclease with a Ki of 1.37 mM at pH 5.5, 25 degrees C. Molecular model building suggests that three of the four carboxyl groups of EDTA could simultaneously interact with histidine 12, lysine 41 and lysine 7. A complex of this type would account for the data described herein.
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PMID:1H NMR studies of the binding of EDTA to bovine pancreatic ribonuclease. 41 52


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