Abstract
The cDNA encoding human dCMP deaminase was isolated from a lambda ZAPII expression library using an antibody generated against highly purified HeLa cell dCMP deaminase. The cloned cDNA consists of 1856 base pairs and encodes a protein of 178 amino acids with a calculated molecular mass of 19,985 daltons. The sequence of several cyanogen bromide-cleaved peptides derived from HeLa cell dCMP deaminase are all contained within the deduced amino acid sequence. A zinc binding region is present in the enzyme, similar to that reported for cytidine deaminase (Yang, E. C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). Northern blot analysis revealed a predominant messenger RNA species of 1.9 kilobases. Expression of the active protein to about 10% of Escherichia coli's total protein was achieved by subcloning the open reading frame into a high expression system using the polymerase chain reaction. Polyacrylamide gel electrophoresis revealed a prominent protein band which comigrated with affinity purified HeLa dCMP deaminase, while Western blot analysis yielded an immunoreactive band which comigrated with the single immunoreactive affinity column purified dCMP deaminase band. The enzyme which possesses a kcat of 1.02 x 10(3) s-1 was purified to homogeneity in over 60% yield. The overexpression of dCMP deaminase should permit more exacting studies on the regulation of this important allosteric enzyme which provides substrate for DNA synthesis.
Highlights
The cDNA encoding human dCMP deaminase was of dUMP available for thymidylate synthase is finely conisolatedfroma XZAPII expression libraryusingan trolled by the end productsof the pyrimidine deoxynucleotide antibody generated against highly purifiedHeLa cell pathway
HeLa cell dCMP deaminase activity all contained within the deduced amino acid sequence.is highest in late S phase and subsequently declines in the A zinc binding regioins present in the enzyme, similarfollowing G2 phase [6]
Controolf deaminase from a mammalian source. Activity at this juncturein deoxyribonucleotide metabolism is determined by the ratio of dCTP to dTTP in the cell, since the enzyme is allosterically activated by dCTP andinhibited by dTTP [1].Evidence in support of this thesis was obtained recently by Xu and Plunkett [2] using an in situ assay
Summary
Color development was initiated by incubating the filters with 0.3mg/ml of nitro blue tetrazolium and 0.15 mg/ml of 5-hromo-4-chloro-3-indolylphosphate in 100mM Tris-HC1,pH 9.5, 100 mM NaCl, and 5 mMMgC12 Under these conditions, approximately 2 ng of purified HeLa dCMP deaminase could be detected by dot blotting. Frozen cells (BL21(DE3) pLys/pETCD) from 4 liters of cell culture (8-10 g) were thawed and suspended in 12 ml/g of TME containing 20 mM 2mercaptoethanol, and the protease inhibitors indicated above This mixture was sonicated 10 times for 1 min each using a probe with. Additional solid ammonium sulfate was added to a final concentration of 80% saturation, and after stirring for 10 min the precipitate was collected by centrifugation. The results of a typical purification are presented in Sequence oDf eHouxmycayntidylate
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
