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)

Previous work demonstrated that 12(S)-HETE [12(S)-hydroxyeicosatetraenic acid], a lipoxygenase metabolite of arachidonic acid, stimulates the surface expression of integrin alpha v beta 3 on mouse lung vascular endothelial cells (CD clone 3) in a post-transcriptional and protein kinase C (PKC)-dependent fashion. In this study we examined the effect of 12(S)-HETE on the expression of integrin receptors alpha v beta 3 and alpha 5 beta 1 in a different clone of a mouse endothelial cell population derived from lung microvasculature (designated CD clone 4). The results indicated that 12(S)-HETE transcriptionally activates the gene expression of integrin alpha v as assessed by quantitative reverse transcription/polymerase chain reaction/Southern hybridization, RNase protection assay, solution hybridization, and northern blotting. The induction of alpha v mRNA occurred within 1 hour, peaked at approximately 4 hours (2- to 4-fold increase), persisted for up to 16 hours, and thereafter gradually declined. The PKC activator phorbol 12-myristate 13-acetate (PMA) induced the alpha v mRNA, in a similar way. 12(S)-HETE treatment did not, in contrast, alter the mRNA levels of integrin subunit alpha 5 or beta 1. The induction of alpha v mRNA appeared to be protein synthesis-independent, since cycloheximide did not alter the 12(S)-HETE effect. 12(S)-HETE also did not appear to alter the mRNA half-life of alpha v. On the other hand, 12(S)-HETE-induced increase in alpha v mRNA levels was PKC-dependent, since pretreatment of CD clone 4 cells with calphostin C significantly inhibited 12(S)-HETE-increased alpha v mRNA. Nuclear runoff experiments revealed that the increase in alpha v mRNA results from an enhanced gene transcription. Facilitated alpha v gene transcription resulted in an increased surface expression of alpha v beta 3 protein, which resulted in an increased cell adhesion to vitronectin. The above observations, in conjunction with our previous experimental data, suggest that 12(S)-HETE may employ diverse mechanisms to stimulate the integrin alpha v beta 3 expression in vascular endothelial cells, which could play important roles in tumor cell adhesion, angiogenesis, hemostasis, and many other vascular events.
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PMID:Transcriptional activation of endothelial cell integrin alpha v by protein kinase C activator 12(S)-HETE. 759 4

Ribonuclease A (RNase A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are selectively taken up and degraded by isolated rat liver lysosomes by very similar processes. The uptake and degradation of both of these proteins are stimulated by the heat shock cognate protein of 73 kDa and ATP/Mg2+. Both binding and uptake of RNase A and GAPDH by lysosomes are saturable, and uptake of RNase A and GAPDH requires a protease-sensitive component within the lysosomal membrane. GAPDH competes for binding and uptake of RNase A by lysosomes and vice versa while another protein, ovalbumin, does not compete. RNase S-peptide (amino acids 1-20 of RNase A) also competes for RNase A binding and uptake by lysosomes, while RNase S-protein (amino acids 21-124 of RNase A) does not compete. The uptake of RNase A by lysosomes appears to involve an intermediate step in which approximately 2 kDa of the polypeptide's COOH terminus remains outside lysosomes while the remainder is inside the lysosomal lumen.
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PMID:Selective binding and uptake of ribonuclease A and glyceraldehyde-3-phosphate dehydrogenase by isolated rat liver lysosomes. 792 57

Derivatives of ribonuclease A (RNase A) with modifications in positions 1 and/or 7 were prepared by subtilisin-catalyzed semisynthesis starting from synthetic RNase 1-20 peptides and S-protein (RNase 21-124). The lysyl residue at position 1 was replaced by alanine, whereas Lys-7 was replaced by cysteine that was specifically modified prior to semisynthesis. The enzymes obtained were characterized by protein chemical methods and were active toward uridylyl-3',5'-adenosine and yeast RNA. When Lys-7 was replaced by S-methyl-cysteine or S-carboxamido-contrast, the catalytic properties were only slightly altered. The dissociation constant for the RNase A-RI complex increased from 74 fM (RNase A) to 4.5 pM (Lys-1, Cys-7-methyl RNase), corresponding to a decrease in binding energy of 10 kJ mol-1. Modifications that introduced a positive charge in position 7 (S-aminoethyl- or S-ethylpyridyl-cysteine) led to much smaller losses. The replacement of Lys-1 resulted in a 4-kJ mol-1 loss in binding energy. S-protein bound to RI with Ki = 63.4 pM, 800-fold weaker than RNase A. This corresponded to a 16-kJ mol-1 difference in binding energy. The results show that the N-terminal portion of RNase A contributes significantly to binding of ribonuclease inhibitor and that ionic interactions of Lys-7 and to a smaller extent of Lys-1 provide most of the binding energy.
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PMID:Interaction of semisynthetic variants of RNase A with ribonuclease inhibitor. 800 61

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex. We have substituted the wild-type residue at position 13, methionine (Met), with norleucine (Nle), where the only covalent change is the replacement of the sulfur atom with a methylene group. The thermodynamic parameters associated with the binding of this variant to S-protein, determined by titration calorimetry in the temperature range 10-40 degrees C, are reported and compared to values previously reported [Varadarajan, R., Connelly, P. R., Sturtevant, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426] for other position 13 analogs. The differences in the free energy and enthalpy of binding between the Met and Nle peptides are 0.6 and 7.9 kcal/mol at 25 degrees C, respectively. These differences are slightly larger than, but comparable to, the differences in the values for the Met/Ile and Met/Leu pairs. The structure of the mutant complex was determined to 1.85 A resolution and refined to an R-factor of 17.4%. The structures of mutant and wild-type complexes are practically identical although the Nle side chain has a significantly higher average B-factor than the corresponding Met side chain. In contrast, the B-factors of the atoms of the cage of residues surrounding position 13 are all somewhat lower in the Nle variant than the Met wild-type.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Thermodynamic and structural consequences of changing a sulfur atom to a methylene group in the M13Nle mutation in ribonuclease-S. 803 93

Bovine seminal ribonuclease (BS-RNase) is an unusual homolog of RNase A. Isolated from bulls as a dimer, BS-RNase has special biological properties including antispermatogenic, antitumor and immunosuppressive activities. The structural bases for these properties are unknown. Four forms of BS-RNase were isolated after folding and air oxidation of the denatured and reduced protein produced in Escherichia coli: two dimers (M = M and M x I, where x signifies an active site composed of residues from both subunits) and two monomers (M and I). Considerable ribonuclease activity was generated by air oxidation of an equimolar mixture of two inactive mutant proteins ([H12D]BS-RNase and [H119D]BS-RNase) prepared by site-directed mutagenesis. This activity came from a dimer (M x I) with a composite active site. 1H-NMR spectroscopy revealed that this dimer contained one correctly folded subunit (M), and one incorrectly folded subunit (I). Form I, which is a poor catalyst, was activated by ribonuclease S-protein, suggesting that the C-terminal portion of I was not folded properly. Electrospray-ionization mass spectrometry and sulfhydryl group titration indicated that I contains a single oxidized sulfhydryl group, which cannot participate in a disulfide bond. These results show that quaternary structure in BS-RNase is attained by the initial formation of two monomers, M and I, which then combine with another M to form M = M and M x I, respectively. Adventitious oxidation can thus lead to the formation of a misfolded but active enzyme (M x I).
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PMID:A misfolded but active dimer of bovine seminal ribonuclease. 807 30

A fusion protein (FP) comprised of the RNase A S-peptide and human epidermal growth factor was shown to form a stable noncovalent catalytically active complex with the RNase A S-protein at a stoichiometric ratio 1:1 with Kdiss = 5.0 x 10(-7) M. The S-protein complex with FP exhibits the pyrimidine specificity toward substrates in both reactions catalyzed by RNase S, transesterification and hydrolysis. The fusion protein can be determined specifically and quantitatively in the presence of S-protein by RNase activity assays. The possibility of effective purification of S-peptide-containing proteins by affinity chromatography on an S-protein-Sepharose column has been demonstrated.
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PMID:Formation and properties of S-protein complex with S-peptide-containing fusion protein. 811 57

As a model for the reaction of chemically reactive quinone metabolites with cellular proteins in vivo, the reactions of benzoquinone (BQ; 1-64 mol/mol of protein) with bovine pancreatic ribonuclease A (RNase), reduced RNase, S-(amidomethylated) reduced RNase, and reduced guanidinated RNase were investigated. The reaction stoichiometry and products were characterized by means of HPLC, UV-vis spectrophotometry, electrospray mass spectrometry, amino acid analysis, alkaline permethylation analysis, and measurement of the covalent binding of [14C]BQ to protein. Native RNase and S-(amidomethylated) reduced RNase show no reaction with BQ over 60 min at pH 7.4-9.6, whereas reduced RNase, which has 8 free SH groups/mol, reacts rapidly with exactly 24 molecules of BQ, of which 8 become covalently bound to protein-SH groups while 16 are reduced to hydroquinone. Half of the latter is formed via BQ oxidation of the initially formed S-(2,5-dihydroxyphenyl)cysteine moieties. Michael addition of a second protein nucleophile to each resulting S-(2,5-quinoyl)cysteine moiety, followed by reoxidation of that addition product by BQ, generates the second group of 8 molecules of HQ and results in cross-linking. Reduced guanidinated RNase, in which most of the lysines are blocked by guanidination with O-methylisourea, also reacts rapidly with BQ, but only ca. 16 equiv are consumed; of these, 8 become covalently bound to protein-SH groups while the others are reduced to HQ. Thus, even though the lysine residues in native RNase and S-(amidomethylated) reduced RNase do not react with BQ, they may react with (2,5-quinonyl)-S-protein moieties.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Covalent binding of benzoquinone to reduced ribonuclease. Adduct structures and stoichiometry. 819 6

Frequency-domain fluorescence resonance energy transfer and anisotropy measurements were performed to characterize conformational dynamics of an analog of the RNase S-peptide (residues 1-20). Trp was used as a donor by replacing Phe 8, and a dansyl acceptor group was introduced at position 1 or 18. The distance-distribution parameters, half width of the distribution, end-to-end diffusion coefficient, and to some extent anisotropy decays were sensitive to changes in the S-peptide conformation. The observed mean distance of about 13-14 A between residues 1 and 8 in the presence of 50% TFE and when bound to RNase S-protein is in reasonable accord with the X-ray structure of RNase. The mean distance of 9.3 A between residues 8 and 18 in the presence of 50% TFE is, however, significantly smaller than 15.3 A found for the S-protein complex. The half-width of the distance distribution increased from about 9 to 18 A for residues 1-8 and from about 6 to 14 A for segment 8-18 with the loss of helical structure. The half-widths of 9 A in the case of 1-8 segment when peptide is helical suggests the presence of considerable conformational heterogeneity. Also, the 14 A half-width for segment 8-18 when it is random-coil is smaller than that expected for a random coil 11-residue segment. The donor-to-acceptor diffusion coefficients were less than 1 x 10(-7) cm2/s at 2 degrees C for both segments and increased to 1-2 x 10(-6) cm2/s at 35 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fluorescence study of conformational flexibility of RNase S-peptide: distance-distribution, end-to-end diffusion, and anisotropy decays. 824 Nov 20

Proper expression of the human platelet fibrinogen receptor is necessary for the maintenance of normal hemostasis. This receptor is formed by the heterodimer alpha IIb beta 3, a prototypic member of the integrin family of adhesive molecules. beta 3 is also expressed in other tissues with alpha v as the vitronectin receptor. It was not possible to study the basis for tissue-specific expression of this gene, because the beta 3 gene promoter had not been isolated previously. We have now isolated a 6.0-kb human genomic DNA fragment containing 2.0 kb of sequence 5' to the beta 3 ATG start codon. This clone also contains sequence encoding the signal peptide of the immature beta 3 protein and 3.0 kb of 3' intronic sequence. Primer extension and RNase protection studies of poly A+ RNA from a human erythroleukemia (HEL) cell line indicated a major transcription start site 30 bp upstream of the ATG start codon. In an orientation-dependent manner, a 584-bp fragment 5' to the start codon promotes expression of the chloramphenicol acetyl transferase (CAT) reporter gene in K562 cells. CAT expression from this beta 3 promoter is fivefold above expression from a "promoter-less" control CAT construct. This beta 3 promoter lacks TATA and CAAT cis-acting elements, but there are two Sp1 sites flanking the transcription start site. Other potential transcription factor binding sites are also identified. Phorbol esters (TPA), which increase beta 3 transcription in K562 cells, stimulated transcription from the 584-bp 5' beta 3 region. The isolation of this beta 3 promoter region should permit a more detailed analysis of its transcriptional regulation.
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PMID:Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene. 829 29

The fused polypeptides of human epidermal growth factor and one or two S-peptide of RNase A was shown to form stoichiometric (1:1) strong noncovalent and enzymatically active complexes with S-protein of RNase A. The dissociation constants for these complexes were found to be 5.0 x 10(-7) M and 1.1 x 10(-7) M. The complexes of polypeptides with S-protein were capable to hydrolyze ribopolynucleotides, and pyrimidine-2',3'-cyclophosphates specifically, like RNase S'. A possibility was shown of effective purification of the S-peptide-containing polypeptides by affinity chromatography in which S-protein is immobilized on solid supports.
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PMID:[Formation and properties of S-protein complexes with S-peptide-containing hybrid polypeptides]. 836 97


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