Rapid separation of methotrexate from folate reductase

ANALYTICAL

BIOCHEMISTRY

53, 647449 (1973)

SHORT COMMUNICATIONS

Rapid

Separation

of Methotrexate

from

Folate

Reductase

To measure the activity of folate reductase (also called dihydrofolate reductase) in normal or neoplastic cells following the administration of methotrexate (4 amino, 10 methylpteroylglutamate, MTX) , a specific inhibitor of this enzyme, it is first necessary to separate the inhibitor from the enzyme. Although chromatographic methods for such separation have been described (1, 2)) in a recent report from this laboratory (3)) we showed that if a swamping concentration of pteroylglutamate (PGA) and the anion exchanger Dowex 2-X8 was added to a cell extract containing MTX inactivated enzyme, complete recovery of reductase activity could be obtained. In that report, however, we failed to indicate the importance of time in displacing the MTX from the enzyme. In this communication, we wish to briefly describe the method with special note of this important step. Lysates of hematopoietic or leukemic cells are prepared as previously described (3)) but are frozen in 0.1 M instead of 0.05 M sodium citrate. To separate methotrexate from folate reductase, 30 pmoles of PGA contained in a 10 ,~.l volume is added to 0.8-1.0 ml of lysate and the mixture is incubated 5 min at room temperature. At pH above 7, MTX is a competit.ive inhibitor of folate reductase (4) and by mass action the great excess of PGA will displace the drug from the enzyme during this time. Following this incubation period, 300 mg of Dowex 2-X8 is added to the mixture and shaken intermittently for 30 min at room temperature. This anion exchanger now binds the displaced MTX and excessPGA in solution and removes the PGA from the enzyme. After the resin is centrifuged, the supernatant solution is assayed for enzyme activity. We have used a previously described radioenzymatic assay for this purpose (5). Before using the resin, we found it best to wash it with 0.1 N HCl and then deionized water. It is blotted dry with filter paper just prior to use. TabIe 1 shows that: (a) the addition of PGA and Dowex 2-X8 to leukemic lysate does not affect enzyme activity ; (b) if the Dowex 2-X8 647 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

648

SHORT

Recovery

of Activity

of Folate

COMMUNICATIONS

TABLE Keductase

(1) (2) (3) (4)

Leukemic Leukemic Leukemic Leukemic

Iysate lysate lysate lysate

+ MTX + MTX

(5)

Leukemic

lysate

+ MTX

as nmoles

Inactivation

Treatment. lysate

Preparation

0 Expressed

1 After

PGA

reduced

by Methotrexate

of

None PGA + Dowex 2-X8 None PGA + Dowex 2-X8 immediately PGA + 5 min incubation + Dowex 2-X8 per mg protein

Enzyme activity” 0.34 0.32 0 0 0.32

per hr.

is added to MTX-inactivated enzyme immediately after the PGA, there is no recovery of enzyme activity; and (c) if the PGA is incubated with the lysate for 5 min before the addition of the Dowex, there is complete recovery of enzymic activit,y. The data illustrated in Fig. 1 show that in one experiment 86% of the MTX-inactivated folate reductase in the lysate of chronic myelogenous leukemia cells could be recovered after 1 min, and 95% recovered after 5 min of incubation with PGA before the addition of the exchange resin. In a similar experiment using MTX-inactivated folate reductase from chicken liver, only 67% of the activity was recovered after 1 min incubation, but recovery appeared to slowly increase to 87% as the incubation with PGA continued up to 20 min.

x-z .----.

-

INACTIVATED CYL AEDUCTASE INACTIVATED CHICKEN LIVER REDUCTASE

I 5 IO TIME INCUBATED WITH PGA BEFORE ADDING DOWEX 2-X6

FIG. 1. The recovery of MTX-inhibited was added to a lysate of leukemic cells chicken liver to inactivate the previously then treated with PGA and Dowex 2-X8

20

folate reductase activity. or partially purified folate measured enzyme activity as described in the text.

Su5cient MTX reductaze from and the mixture

649

SHORT COMMUNICATIONS

We have found either no, or only slight, decrease in protein concentration of the lysates after the Dowex 2-X8 treatment and this may be the result of varying degrees of resin hydration which dilutes the lysate rather than to adsorption of protein. ACKNOWLEDGMENTS The technical assistance of Zoltan Rosenberg is gratefully acknowledged. This work was supported by grant number CA 08976 from the National Cancer Institute, and by a Career Investigator Award, I-423, from the New York City Health Research Council. REFERENCES J. R., CASHMORE, A., FINK, M., CALEBRESI, P., AND LEFKOWITZ, E. (1965) Clin. Pharmacol. Ther. 6, 763. BERTINO, J. R., CASHMORE, A. R., AND HILLCOAT, B. L. (1970) Cancer Res. 3Q, 2372. RQTHENBERG, S. P. (1969) Cancer Res. 29, 2047. BERTINO, J. R., BOOTH, B. A., BIEBER, A. L., CASHMORE, A., AND SARTORELLI, A. C. (1964) J. Biol. Chem. 239, 479. RQTHENBERG, S. P. (1966) Anal. Biochem. 16, 176.

1. BERTINO, 2. 3. 4. 5.

SHELDON Division of Hematology Department of Medicine New York Medical College New York 10029 Received July 14, 19Y??; accepted

February

9, 197s

P. ROTHENBERG