ABSTRACT
The structural udp gene encoding uridine phosphorylase (UPase) was cloned from the Salmonella typhimurium chromosome and overexpressed in E. coli cells. The S. typhimurium UPase was purified to an apparently homogeneous state, and some physicochemical characteristics of the enzyme were studied. The molecular weight of one subunit of UPase is 27.5 kD, and the optimal pH for its activity is 7.2--7.4. The native S. typhimurium UPase consists of six identical subunits, and its molecular weight is about 165 kD. According to these parameters, the S. typhimurium UPase is similar to the E. coli UPase. However, these enzymes differ substantially from one another by the substrate sensitivity and sensitivity to polarity of the medium. The S. typhimurium UPase has much higher phosphorylation activity toward thymidine, deoxyuridine, and 5;-bromide- or 5;-fluoride-containing analogs of nucleosides than that of E. coli UPase.
Subject(s)
Salmonella typhimurium/enzymology , Uridine Phosphorylase/isolation & purification , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , Escherichia coli/enzymology , Hydrogen-Ion Concentration , Kinetics , Salmonella typhimurium/genetics , Substrate Specificity , Uridine Phosphorylase/genetics , Uridine Phosphorylase/metabolismABSTRACT
5,5-Bis(hydroxymethyl)-2-oxo-[1-(2-trifluoromethyl)-3,3,3- trifluoropropionamido)-1-trifluoromethyl-2,2,2-trifluoroethyl- 1,3,2-dioxaphosphan (CA-423) is an in vitro inhibitor of the Escherichia coli uridine and thymidine phosphorylases. Unlike widely studied nucleoside analogues, this compound binds to the enzymes irreversibly. Its LD50 in mice was 40 mg/kg. Due to the involvement of pyrimidine phosphorylases in carcinogenesis and the relatively low toxicity of CA-423, it is promising for anticancer therapy.
