RESUMEN
Two methods for measurement of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) mRNA expression were compared. Although the relative mRNA levels compared to beta-actin measured with competitive template RT-PCR were different from the data obtained with a TaqMan based PCR, a significant correlation between the two assays was found.
Asunto(s)
Fluorouracilo/farmacología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Actinas/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Regulación de la Expresión Génica , Humanos , Timidilato Sintasa/metabolismoRESUMEN
Catalytic properties of Escherichia coli polyphosphate kinase (EC 2.7.4.1), a promising enzyme for use in ATP regeneration (Hoffman, et al., 1988, Biotechnol. Appl. Biochem. 10, 107-117), are reported here. E. coli polyphosphate kinase (PPK) is broadly active in the pH range 5.5 to 8.5, having an optimal Vmax at pH 7.2. The Km values for the substrates, ADP and polyphosphate (Pn), change little in the same pH range. The optimal concentration range for the Mg2+ activator is 1-20 mM, with an activity maximum at 10 mM Mg2+. In addition to Mg2+, Mn2+ and Co2+ can serve as activators of E. coli PPK, whereas Zn2+ and Cu2+ are highly inhibitory. E. coli PPK is most active with Pn substrates of chain length greater than 132 phosphoryl units. The enzyme activity decreases with decreasing Pn chain length and approaches zero (less than 1%) at a chain length less than or equal to 5. Equilibrium yields of ATP of greater than 85% are readily attained at substrate concentrations below 1 mM. An operational equilibrium constant for the PPK reaction, defined as [ATP]/[ADP][Pn], was determined to be 7.5 (+/- 3.4) x 10(5) M-1. The data presented here serve as a base of information from which assessments of the suitability of E. coli PPK for specific ATP regeneration applications can be made.