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Purification, characterization, and kinetics of porcine recombinant dihydropyrimidine dehydrogenase.
Protein Expr Purif. 1997 Jul; 10(2):185-91.PE

Abstract

Porcine recombinant dihydropyrimidine dehydrogenase was purified from Escherichia coli cells using cell disruption, ammonium sulfate fractionation, and chromatography on DEAE-cellulose and 2',5'-ADP-Sepharose. The yield was 60% with a specific activity of 14 units/mg protein. On SDS/PAGE the purified dehydrogenase exhibits a single band, indicating that no proteolytic degradation was taking place during purification. In agreement with the native enzyme, all cofactors, FMN, FAD, NADPH, and two iron-sulfur clusters, have been found. EPR spectra of the reduced dehydrogenase obtained at pH 9.5 are characteristic for two [4Fe-4S]1+ cubanes in dipolar interaction. Quantification of the observed signals indicated 0.95 spins per subunit, showing only partially reduced iron-sulfur clusters. The kinetic parameters of the porcine recombinant enzyme are very similar to those of the native enzyme. Thus, it can be concluded that the porcine recombinant enzyme behaves like the native dehydrogenase.

Authors+Show Affiliations

Theodor-Boveri-Institut für Biowissenschaften, Universität Würzburg, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

9226714

Citation

Rosenbaum, K, et al. "Purification, Characterization, and Kinetics of Porcine Recombinant Dihydropyrimidine Dehydrogenase." Protein Expression and Purification, vol. 10, no. 2, 1997, pp. 185-91.
Rosenbaum K, Schaffrath B, Hagen WR, et al. Purification, characterization, and kinetics of porcine recombinant dihydropyrimidine dehydrogenase. Protein Expr Purif. 1997;10(2):185-91.
Rosenbaum, K., Schaffrath, B., Hagen, W. R., Jahnke, K., Gonzalez, F. J., Cook, P. F., & Schnackerz, K. D. (1997). Purification, characterization, and kinetics of porcine recombinant dihydropyrimidine dehydrogenase. Protein Expression and Purification, 10(2), 185-91.
Rosenbaum K, et al. Purification, Characterization, and Kinetics of Porcine Recombinant Dihydropyrimidine Dehydrogenase. Protein Expr Purif. 1997;10(2):185-91. PubMed PMID: 9226714.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification, characterization, and kinetics of porcine recombinant dihydropyrimidine dehydrogenase. AU - Rosenbaum,K, AU - Schaffrath,B, AU - Hagen,W R, AU - Jahnke,K, AU - Gonzalez,F J, AU - Cook,P F, AU - Schnackerz,K D, PY - 1997/7/1/pubmed PY - 1997/7/1/medline PY - 1997/7/1/entrez SP - 185 EP - 91 JF - Protein expression and purification JO - Protein Expr Purif VL - 10 IS - 2 N2 - Porcine recombinant dihydropyrimidine dehydrogenase was purified from Escherichia coli cells using cell disruption, ammonium sulfate fractionation, and chromatography on DEAE-cellulose and 2',5'-ADP-Sepharose. The yield was 60% with a specific activity of 14 units/mg protein. On SDS/PAGE the purified dehydrogenase exhibits a single band, indicating that no proteolytic degradation was taking place during purification. In agreement with the native enzyme, all cofactors, FMN, FAD, NADPH, and two iron-sulfur clusters, have been found. EPR spectra of the reduced dehydrogenase obtained at pH 9.5 are characteristic for two [4Fe-4S]1+ cubanes in dipolar interaction. Quantification of the observed signals indicated 0.95 spins per subunit, showing only partially reduced iron-sulfur clusters. The kinetic parameters of the porcine recombinant enzyme are very similar to those of the native enzyme. Thus, it can be concluded that the porcine recombinant enzyme behaves like the native dehydrogenase. SN - 1046-5928 UR - https://www.unboundmedicine.com/medline/citation/9226714/Purification_characterization_and_kinetics_of_porcine_recombinant_dihydropyrimidine_dehydrogenase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1046-5928(97)90735-X DB - PRIME DP - Unbound Medicine ER -