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)

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

Protein phosphorylation is considered an early cellular mechanism of signal transduction by surface immunoglobulins (sIg) and other receptors of B cells. Using intact human peripheral blood B cells of young subjects labeled with orthophosphate, increased phosphorylation levels of serine/threonine and tyrosine substrates were demonstrated on indicator phosphoproteins corresponding to the CD20 isoforms and microtubule-associated protein 2 kinase after cross-linking sIg and costimulation with phorbol diesters. By contrast, stimulated B cells from certain elderly subjects displayed substantial alterations in the phosphorylation patterns of serine/threonine or tyrosine indicator phosphoproteins. Also, age-related impairments in sIg stimulated mobilization of cytosolic protein kinase C (PKC) enzymatic activity and in cytosolic calcium [Ca2+]i responses of B cells were observed with the altered phosphorylation reactions. Comparison of the substrate phosphorylation profiles to the proliferative responses of stimulated B cells from individual elderly subjects suggested a model of signal transduction in which differing stimuli have different dependencies on phosphorylation reactions. Diminished proliferative responses after sIg ligation coincided with decreased phosphorylations of either tyrosine or serine/threonine indicator substrates. However, the decreased proliferative responses of B cells from elderly subjects with substantial reductions of tyrosine phosphorylation after sIg ligation were enhanced by the direct stimulation of serine/threonine kinase activity with phorbol diesters or CD40 ligation. Experiments with kinase inhibitors evaluated the relative dependency of different B cell stimuli on tyrosine and serine/threonine phosphorylation reactions. The proliferative responses of normal B cells to sIg ligation were quite sensitive to the tyrosine kinase inhibitor genistein whereas those observed following costimulations with phorbol diesters or CD40 ligation were more resistant. However, treatment of B cells with H7, an inhibitor of PKC activity, led to a more uniform reduction of B-cell responses after different stimuli. Results from RNase protection assays of c-myc expression also suggested that different B-cell stimuli might utilize distinct intracellular signaling pathways. Both the type of stimuli and mode of sIg ligation were important in determining the stimulated levels of c-myc mRNA expression. Thus, the current findings suggest that age-related defects are present in human B cell signaling pathways as reflected by tyrosine and serine/threonine phosphorylation reactions. Also, these age-related defects can coexist with altered mobilization of PKC enzymatic activity and with alterations in [Ca2+]i and proliferative responses.
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PMID:Signal transduction in human B cells during aging: alterations in stimulus-induced phosphorylations of tyrosine and serine/threonine substrates and in cytosolic calcium responsiveness. 180 9

Coulombic interactions between charges on the surface of proteins contribute to stability. It is difficult, however, to estimate their importance by protein engineering methods because mutation of one residue in an ion pair alters the energetics of many interactions in addition to the coulombic energy between the two components. We have estimated the interaction energy between two charged residues, Asp-12 and Arg-16, in an alpha-helix on the surface of a barnase mutant by invoking a double-mutant cycle involving wild-type enzyme (Asp-12, Thr-16), the single mutants Thr----Arg-16 and Asp----Ala-12, and the double mutant Asp----Ala-12, Thr----Arg-16. The changes in free energy of unfolding of the single mutants are not additive because of the coulombic interaction energy. Additivity is restored at high concentrations of salt that shield electrostatic interactions. The geometry of the ion pair in the mutant was assumed to be the same as that in the highly homologous ribonuclease from Bacillus intermedius, binase, which has Asp-12 and Arg-16 in the native enzyme. The ion pair does not form a hydrogen-bonded salt bridge, but the charges are separated by 5-6 A. The mutant barnase containing the ion pair Asp-12/Arg-16 is more stable than wild type by 0.5 kcal/mol, but only a part of the increased stability is attributable to the electrostatic interaction. We present a formal analysis of how double-mutant cycles can be used to measure the energetics of pairwise interactions.
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PMID:Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles. 224 51

The previously described poliovirus-associated protein kinase activity phosphorylates viral proteins VP0 and VP2 as well as exogenous proteins in the presence of Mg2+. In this paper, the effect of Zn2+ on the phosphorylation reaction and the stability of the poliovirus capsid has been studied in detail and compared to that of Mg2+. Phosphorylation patterns of viral and other proteins depend on the divalent cation present. In the presence of Zn2+, phosphorylation of capsid proteins VP2 and VP4 is significantly higher while phosphorylation of VP0 and exogenous phosphate acceptor proteins is not detected. Our results indicate the activation of more than one virus-associated protein kinase by Zn2+. The ion-dependent behavior of the enzyme activities is observed independently of whether the virus was obtained from HeLa or green monkey kidney cells. The poliovirus capsid is destabilized by Zn2+. The destabilization leads to a substantially increased permeability of virus particles to ethidium bromide and RNase, concomitant with decreased infectivity of the sample. This alteration of the poliovirus capsid structure is a prerequisite for effective phosphorylation of viral capsid proteins. The increased level of phosphorylation of viral capsid proteins results in further destabilization of the viral capsid. As a result of the conformational changes, poliovirus-associated protein kinase activities dissociate from the virus particle. High-performance liquid chromatography-purified viral protein VP2 is phosphorylated by the released enzymes on serine, threonine, and tyrosine in the presence of Zn2+. We suggest that the destabilizing effect of phosphorylation on the viral capsid plays a role in uncoating of poliovirus.
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PMID:Poliovirus-associated protein kinase: destabilization of the virus capsid and stimulation of the phosphorylation reaction by Zn2+. 254 9

A sequence similarity has been found between two segments of endothiapepsin (acid proteinase, 2APE), bovine pancreatic ribonuclease A, and peptide T, a segment of the gp120 protein of human immune deficiency virus (HIV), which has been implicated in blocking viral attachment to the T4 receptor. The two similar sequences of the acid proteinase enzyme are Leu-Ile-Asp-Ser-Ser-Ala-Tyr-Thr (residues 169-176) and Tyr-Thr-Gly-Ser-Leu-Asn-Tyr-Thr (residues 175-182). Since the X-ray crystallographic structures of the acid proteinase and ribonuclease are known, it has been possible to determine whether the three-dimensional structures of the segments are similar. Portions of both the segments of acid proteinase are directly superimposable on the structure of the RNase A 19-26 segment. The fact that the three similar sequences from two completely unrelated proteins give rise to almost identical structures raises the possibility that these segments may be involved in nucleating the folding of these proteins. In addition, this provides further support for the concept that the octapeptide sequence of peptide T of HIV, which is also similar in sequence to the 19-26 sequence of RNase A, is also structurally similar to these residues, which adopt a beta-bend conformation. Furthermore, comparison of similarities and differences in the structure of these similar sequences provides an explanation for alterations in the biological activity of various truncated or substituted derivatives of peptide T and additional confirmation of the structural requirements for peptide T in T4-receptor recognition.
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PMID:Comparative X-ray crystallographic evidence for a beta-bend conformation as the active structure for peptide T in T4 receptor recognition. 254 25

Several studies have suggested that heterogeneity exists in the type I insulin-like growth factor (IGF) receptor beta subunit. We have examined type I IGF receptor mRNA transcripts by ribonuclease (RNase) protection assay to determine if the heterogeneity could result from alternative splicing of the gene. An area that corresponded to the nucleotide sequence just upstream of the region encoding the transmembrane domain of the beta subunit was identified as being a potential site of alteration in the transcript. Since the 5' and 3' ends were known, polymerase chain reaction was used to clone a cDNA that included this region. Analysis revealed that an alternate type I IGF receptor mRNA transcript with a 3-base pair deletion could account for the results of the RNase protection assay. The deletion changes the amino acid sequence at position 899 substituting Arg for a Thr-Gly. Furthermore, this alternate transcript was ubiquitously found in tissue and cell line RNAs. Although the identified transcript cannot fully account for the documented heterogeneity in type I IGF receptor beta subunit sizes, the results suggest that a form of the beta subunit with an alternate primary sequence may exist.
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PMID:Identification of an alternate type I insulin-like growth factor receptor beta subunit mRNA transcript. 255 27

The first and last four residues of alpha-helices differ from the rest by not being able to make the intrehelical hydrogen bonds between the backbone greater than C=O groups of one turn and the greater than NH groups of the next. Physico-chemical arguments and statistical analysis suggest that there is a preference for certain residues at the C and N termini (The C- and N-caps) that can fulfil the hydrogen bonding requirements. We have tested this hypothesis by constructing a series of mutations in the two N-caps of barnase (Bacillus amyloliquefaciens ribonuclease, positions Thr 6 and Thr 26) and determining the change in their stability. The N-cap is found to stabilize the protein by up to approximately 2.5 kcal mol(-1). The presence of a negative charge of the N-cap adds some 1.6 kcal mol(-1) of stabilization energy because of the interaction with the macroscopic electrostatic dipole of the helix.
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PMID:Capping and alpha-helix stability. 281 29

The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Amino acid sequence of human tumor derived angiogenin. 286 94

Ribonuclease A was studied by two-dimensional 1H NMR spectroscopy. 10 out of 12 alanine and 9 out of 10 threonine spin systems as well as all valine [9], leucine [2] and isoleucine [3] spin systems were identified from the correlated spectroscopy (COSY) and relayed coherence transfer spectroscopy (RCT). Sequence-specific assignments were obtained from nuclear Overhauser effect spectra for proton resonances of 21 amino acid moieties. 2' and 3'-pyrimidine-nucleotide-RNase-A complexes were also investigated by two-dimensional NMR. We were able to monitor structural changes in the active center, the vicinity of the active center and in regions far from the catalytic region. Chemical shift changes of resonances of protons near Thr-45 reflected the binding of the same moiety. This in turn is also dependent on the position of the nucleotide phosphate group. Binding of 2' nucleotides led to characteristic changes in protein regions not affected by the binding of 3' nucleotides. These results are interpreted in terms of structural differences between the 2' and 3'-nucleotide-RNase-A complexes; the structure of the complex of the native 3' nucleotide inhibitor being more closely related to that of the free protein.
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PMID:Two-dimensional 1H NMR investigation of ribonuclease A and ribonuclease-A--pyrimidine-nucleotide complexes. 299 93

The oncogene product p53, isolated from SV3T3 cells where it forms a complex with simian virus 40 large tumor antigen (T antigen) in the nucleus, has been found to be phosphorylated at at least four distinct sites on the 390 amino acid protein. Separation of tryptic phosphopeptides has permitted identification of two sites as Ser-312 and Ser-389, and permitted analysis of the types of phosphate bonds. The peptide containing Ser-312 separates electrophoretically into three charged forms; two are resistant to dephosphorylation by both alkaline phosphatase and alkaline hydrolysis, suggesting a phosphodiester. The carboxyl-terminal phosphopeptide containing Ser-389 was alkaline phosphatase-resistant and liberated four ribonucleoside monophosphates upon base or RNase hydrolysis, suggesting that Ser-389 may be covalently linked to RNA. Phosphorylation of Ser-389 decreased markedly at the nonpermissive temperature in simian virus 40 tsA58-transformed cells, indicating a dependence on native T antigen function and a possible role in transformation by T antigen. Two additional phosphorylation sites, one involving serine and one involving threonine, probably reside in the amino-terminal segment of p53 and appear to be peptide-phosphate monoesters.
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PMID:Mapping of phosphomonoester and apparent phosphodiester bonds of the oncogene product p53 from simian virus 40-transformed 3T3 cells. 300 31


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