Add time:07/23/2019         Source:sciencedirect.com

Dihydrofolate reductase was purified from mouse liver and spleen, and certain physical and kinetic properties were compared. The Chromatographic properties of the two enzymes on Sephadex G-75 were similar. Both enzymes exhibited two broad double pH optima, one between pH 4.5 and 5.5, and another between pH 7.5 and 8.5. The two enzymes were activated to a similar degree by KCl, urea and guanidine-HCl; methylmercuric bromide did not activate either enzyme. The presence of 5 × 10−6M of either substrate, dihydrofolate or NADPH, protected the enzymes against thermal denaturation. The rate of reduction of dihydrofolate was 27–29 times that of folate for each of the enzymes. From the titration of the enzymes by the stoichiometric inhibitor, methotrexate, turnover numbers of 940 were calculated for both the liver and spleen enzymes.The liver and spleen enzymes were both inactivated by an antibody prepared against the murine L1210 lymphoma enzyme. Crude liver extracts were found to contain a fraction which bound antibody and prevented inactivation of enzyme activity. The I50 values for a series of 2,4-diaminopyrimidine and 4,6-diaminotriazine inhibitors were determined and no significant differences were noted when the purified enzymes were tested. These studies show that when purified preparations of dihydrofolate reductase from mouse liver and spleen are compared, no differences in physical and kinetic properties are demonstrable, and stress the importance of using purified enzymes in measuring inhibition produced by analogs.

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