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)

An in vitro system of guinea pig pancreatic lobules convenient for the study of secretory processes is described in this paper. In this system: (a) the over-all glandular architecture of the tissue is preserved: lobules remain morphologically intact through 5 hours; (b) amylase discharge from unstimulated lobules is low (similar to 4%/hour) and linear over the 5 hours tested; (c) response to carbamylcholine chloride (10-5 M) is energy-dependent, rapid, and extensive (92% discharge of amylase by 5 hours); (d) initial rates of discharge remain stable over the first 3 hours; and (e) no autoactivation of zymogens occurs in incubation medium or tissue. The activation of four zymogens, i.e. chymotrypsinogen, trypsinogen, and procarboxypeptidases A and B, was studied using the following criteria for optimal activation: (a) maximal activation attainable under experimental conditions; (b) stability at the level of maximal activation; and (c) linear relationship between amounts of protein activated and enzyme activity elicited by activation. The concentration of activators (trypsin or enterokinase) and secretory protein, the presence or agents (bovine plasma albumin or Triton X-100) which minimize adsorptive losses of secretory protein on glass or plastic surfaces, and the temperature at which activation is carried out were found to be critical and different for each of the zymogens tested. The kinetics of the appearance of three enzyme activities (amylase, lipase, and ribonuclease) and four potential proteolytic activities (chymotrypsinogen, trypsinogen, and procarboxypeptidases A and B) into the incubation medium was studied under different conditions; i.e. rest and stimulation with various secretogogues (carbamylcholine chloride, caerulein, and pancreozymin). All seven activities estimated to represent similar to 75% of the secretory protein output of the exocrine pancreas were discharged in synchrony and in constant proportions and were released from the tissue to the same extent under each experimental condition investigated.
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PMID:Studies on the guinea pig pancreas. Parallel discharge of exocrine enzyme activities. 112 25

Porcine rotaviral infectivity for continuous porcine kidney (PK-15) cells was enhanced by incorporation of pancreatic endopeptidases into the cell culture maintenance medium. Marked enhancement of infectivity was induced by trypsin, whereas elestase and alpha-chymotrypsin enhanced infectivity to a lesser extent. Bacterial protease also induced some enhancement of porcine rotaviral infectivity. A synergistic enhancement of porcine rotaviral infectivity was noticed with trypsin and alpha-chymotrypsin combined. Porcine rotaviral infectivity was not affected by incorporation of alpha-amylase, alkaline phosphatase, beta-galactosidase, carboxypeptidase-A, deoxyribonuclease, enterokinase, lipase, or ribonuclease into the maintenance medium.
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PMID:Porcine rotaviral infection of cell culture: effects of certain enzymes. 624 64

We generated a recombinant immunotoxin, named scFv(MGR6)-Cla, composed of the Fv region of an anti ErbB2 monoclonal antibody (MGR6) fused to clavin, a type 1 ribosome-inactivating protein (RIP) from Aspergillus clavatus. ErbB2 is a tyrosine kinase receptor which is overexpressed in most adenocarcinomas; clavin is a 17 kDa ribonuclease which inhibits protein synthesis by inactivating ribosomes. A recombinant DNA construct containing the cDNA of the single chain Fv fragment (scFv) of the MGR6 antibody fused to the clavin cDNA, was expressed at high levels in Escherichia coli as an insoluble fusion protein containing an N-terminal affinity tag of six consecutive histidine residues. Inclusion bodies were denatured and the recombinant fusion protein was purified under denaturing conditions by single-step purification using immobilised metal ion affinity chromatography (IMAC). The purified immunotoxin was renatured at high yield and histidine tag removed by digestion with enterokinase. The purity of the immunotoxin obtained after refolding was confirmed by SDS-PAGE, RP-HPLC, GPC-HPLC and N-terminal sequence analysis. Cell-free protein synthesis inhibition and binding assays showed that both clavin and scFv(MGR6) maintained their properties after refolding.
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PMID:Production and characterisation of a recombinant single-chain anti ErbB2-clavin immunotoxin. 985 10

A number of different immunotoxins composed of cell-specific targeting structures coupled to plant or bacterial toxins have increasingly been evaluated for immunotherapy. Because these foreign proteins are highly immunogenic in humans, we have developed a new CD30 ligand-based fusion toxin (Ang-CD30L) using the human RNase angiogenin. The completely human fusion gene was inserted into a pET-based expression plasmid. Transformed Escherichia coli BL21(DE3) were grown under osmotic stress conditions in the presence of compatible solutes. After isopropyl beta-D-thiogalactoside induction, the M(r) 37,000 His(10)-tagged Ang-CD30L was directed into the periplasmic space and functionally purified by a combination of metal ion affinity followed by enterokinase cleavage of the His(10)-Tag and molecular size chromatography. The characteristics of the recombinant protein were assessed by ELISA, flow cytometry, and toxicity assays showing specific activity against CD30(+) Hodgkin-derived cells. Specific binding activity of Ang-CD30L was verified by competition with anti-CD30 monoclonal antibody Ki-4 and commercially available CD30L-CD8 chimeric protein. Ang-CD30L showed RNase activity in vitro. The human recombinant immunotoxin showed significant toxicity toward several CD30-positive cell lines (HDLM-2, L1236, KM-H2, and L540Cy) and exhibited highest cytotoxicity against L540 cells (IC(50) = 8 ng/ml) as determined by cell proliferation assays. CD30 specificity was confirmed by competitive toxicity assays. This is the first report on the specific cytotoxicity of a recombinant completely human fusion toxin with possibly largely reduced immunogenicity for the treatment of CD30-positive malignancies.
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PMID:Human angiogenin fused to human CD30 ligand (Ang-CD30L) exhibits specific cytotoxicity against CD30-positive lymphoma. 1175 93

The C40,82A;I87E mutant of barstar, an intracellular inhibitor of the ribonuclease barnase from Bacillus amyloliquefaciens, was obtained, and its physicochemical properties were studied. It was produced as a fusion protein with thioredoxin and then cleaved from this by EKmax enterokinase. The mutant was shown by NMR to retain the spatial structure of the wild-type protein but, in contrast to barstar, does not form the homodimers characteristic of barstar in aqueous solution. The mutant protein binds barnase with the dissociation constant (6.6 +/- 1.1) x 10(-11) M and exhibits other physicochemical properties similar to those of the wild-type barstar. This allows the use of C40,82A;I87E mutant instead of wild-type barstar in investigations where the protein dimerization is undesirable. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 6; see also http://www.maik.ru.
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PMID:[I87E mutation prevents barstar dimerization]. 1558 16

Ribonuclease inhibitor (RI) is a 50-kDa cytosolic scavenger of pancreatic-type ribonucleases which inhibits ribonucleolytic activity. Expression of recombinant RI is extremely difficult to reach high levels in soluble form in the cytoplasm of Escherichia coli. Here, we utilized five N-terminal fusion partners to improve the soluble expression of RI. Among these five fusion partners which have been screened, maltose-binding protein (MBP), N-utilization substance A (NusA) and translation initiation factor 2 domain I (IF2) have greatly improved the soluble expression level of recombinant murine RI under the drive of T7 promoter, while glutathione S-transferase (GST) and small ubiquitin modifying protein (SUMO) were much less efficient. All these RI-fusion proteins remained to be highly active in inhibiting RNase A activity. Furthermore, all fusion tags can be efficiently removed by enterokinase digestion to generate native RI which results the highest yield to date (>30mg of native RI per liter culture). And a convenient two-step immobilized metal affinity chromatography (IMAC) method has been implemented in our study, comparing with the traditional RNase A affinity chromatography method.
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PMID:High level soluble production of functional ribonuclease inhibitor in Escherichia coli by fusing it to soluble partners. 2129 12

Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.
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PMID:Recombinant Human Erythropoietin with Additional Processable Protein Domains: Purification of Protein Synthesized in Escherichia coli Heterologous Expression System. 2922 55