Dihydrofolate Reductase human, >=80 % (SDS - PAGE ), recombinant, exprimé en E. coli, > = 1 unité/mg de protéines

Non disponible en dehors du Royaume-Uni et de l'Irlande

Application

Dihydrofolate Reductase human has been used:to investigate the stable expression of green fluorescent protein and the targeted disruption of thioredoxin peroxidase-1 gene in Babesia bovisto study the structural analysis of human dihydrofolate reductase as a binary complexto study its in vitro kinetic assay for the enzyme inhibition study

Human dihydrofolate reductase has been used in a study to investigate the stable expression of green fluorescent protein and the targeted disruption of thioredoxin peroxidase-1 gene in Babesia bovis. Human dihydrofolate reductase has also been used in a study to investigate the structural analysis of human dihydrofolate reductase as a binary complex.

Biochem/physiol Actions

Dihydrofolate reductase (DHFR) is a key enzyme in thymidine synthesis. It catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). At a much lower rate, it catayzes the conversion of folate to THF. Since thymidine is a necessary substrate for DNA synthesis, DHFR is a target for anticancer drug development. Methotrexate is the prototype dihydrofolate reductase inhibitor. The enzyme from Sigma has been used in the inhibitory studies of Leishmaniasis donovani pteridine reductase 1 (PTR1). The enzyme has also been used as a positive control to measure the DHFR activity of a protein, MS0308, purified from Mycobacterium smegmatis.

The human DHFR gene, as well as DHFR genes in other mammalian species, overcome the inhibitory effects of methotrexate by a mechanism of gene amplification or by amino-acid mutagenesis. Dihydroflate reductase (DHFR) catalyzes the NADPH dependent reduction of dihydrofolate (DHF) to tetrahydrofolate (THF) and, at a much lower rate, the conversion of folate to THF. The reaction product, THF, is an essential cofactor in the conversion of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) by thymidylate synthetase. It is a key enzyme in thymidine synthesis. Therefore, DHFR is a critical enzyme in DNA synthesis and has become a target for drug development and cancer therapy. The variations between DHFR from different sources have enabled the development of species selective DHFR inhibitors, such as trimethoprim (antibacterial and antifungal), pyrimethamine (antiprotozoal), and methotrexate; MTX (antineoplastic, antipsoriatic, and anti-inflammatory).

Km^5,6NADPH 0.16 mM 7,8-dihydrofolate 0.03 mM 8-methylpterin 0.13 mMKi⁷Folate 2.6x10-5 mMMethotrexate 6.1-9x10^-9

General description

Human DHFR is an 186 amino acid protein with an apparent molecular weight of 25 kDa. It is 30% homologous to the E. coli protein and up to 70% homologous to vertebrate proteins.

Packaging

0.25 unit in glass bottle

Physical form

Solution in 10 mM Tris pH 8, 1 mM EDTA, 0.5 mM DTT, 5 µM NADPH, protease inhibitors, and 50% glycerol.

Unit Definition

One unit will convert 1.0 µmole of dihydrofolic acid to tetrahydrofolic acid in 1 minute at pH 7.5 at 22 °C.