Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.4.1 (cytosine deaminase)
 747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytosine deaminase (EC 3.5.4.1) from Salmonella typhimurium has been purified 419-fold to apparent homogeneity. SDS polyacrylamide gel electrophoresis indicated that the final cytosine deaminase preparation was homogeneous. The molecular weight of cytosine deaminase was determined to be approx. 230000 containing four identical subunits with each subunit having a molecular weight 54000. Cytosine was deaminase has a pH optimum of 7.30 to 7.50 and a temperature optimum of 45 to 50 degrees C. Cytosine was deaminated specifically; 5-fluorocytosine was deaminated to a lesser extent. The Km and V values for cytosine were 0.74 mM and 47.16 mumole/min, respectively. As effectors of enzyme activity, PPi stimulated the deamination while metal ions and orotidine monophosphate inhibited it. The physical characteristics of cytosine deaminase lend credence to its proposed salvage role in pyrimidine metabolism as indicated previously by physiological studies (West, T.P. and O'Donovan, G.A., J. Bacteriol. (1982) 149, 1171-1174).
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PMID:Purification and some properties of cytosine deaminase from Salmonella typhimurium. 675 59

Yeast cytosine deaminase (yCD) was cloned, expressed, and purified by affinity chromatography. We have characterized the products resulting from covalent attachment of 2-4 PEG chains on yCD and determined the major and minor isomers for each respective conjugate. The results show that for non-covalently associated homodimers, it is possible to characterize and deduce PEGylation levels on individual subunits through the concurrent use of size exclusion chromatography (SEC), MALDI-TOF MS, and SDS-PAGE gels. The results also show that contrary to what we expected, attaching more than two PEG chains to yCD decreased its stability. Enzymatic activity studies revealed that the fusion of an N-terminus purification tag on yCD has no significant effect on 5-fluorocytosine or cytosine affinity, with apparent turnover rates remaining within 10(5) M(-1) . s(-1). Stability studies at 37 degrees C revealed that t1/2 = 8-9 h for yCD and 2mPEG(5K)-yCD, whereas for 3-, 4mPEG(5K)-yCD and yCD/BSA, t(1/2) < 2 h. Incubation of BSA with yCD also decreased enzyme stability over prolonged incubation at 37 degrees C. This finding is important if yCD is to be used in a pretargeting strategy.
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PMID:PEGylation of yeast cytosine deaminase for pretargeting. 1585 41

Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventional chemotherapy by employing artificial antibody-enzyme constructs to convert a non-toxic prodrug into a cytotoxic agent specifically localized to the tumor site. The gpA33 antigen is a promising target for ADEPT in colon cancer, as it is expressed by >95% of human colon cancers, but is absent in all non-gastrointestinal tissues. We designed a recombinant fusion construct of a phage display-generated anti-gpA33 single chain fragment, A33scFv, with cytosine deaminase from yeast (CDy), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). The resulting construct, A33scFv::CDy, was overexpressed in Pichia pastoris and secreted into culture supernatant. The fusion protein was purified by affinity chromatography on protein L. Silver-staining after SDS-polyacrylamide gel electrophoresis confirmed molecular mass and purity. Antibody binding and specificity were quantified by flow cytometry. The complete ADEPT system was applied in vitro on gpA33-positive LIM1215 cells, assessing cell survival by a fluorescein diacetate assay. Cytotoxicity of the prodrug 5-FC after A33scFv::CDy binding was equimolar to that of 5-FU, and this effect depended specifically on both antibody and enzyme function. These results demonstrate bifunctional activity of the heterogeneous Pichia-produced A33scFv::CDy fusion protein and proof of principle for the ADEPT system proposed herein.
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PMID:Design, construction, and in vitro analysis of A33scFv::CDy, a recombinant fusion protein for antibody-directed enzyme prodrug therapy in colon cancer. 1778 29