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Stereochemical Characteristics of the Folate-Antifolate Transport Mechanism in L1210 Leukemia Cells¹

Memorial Sloan-Kettering Cancer Center, New York, New York 10021

The rate of influx, extent of concentrative uptake, and the rate of efflux (loss) by active transport in L1210 leukemia cells has been compared for the pteridine antifolates, aminopterin and methotrexate, eight related quinazoline analogs, and two pyrimidine derivatives. The data reveal a difference in the stereochemical specificity for influx and efflux. Influx is preferential in the order pteridine, quinazoline, and pyrimidine. Influx of aminopterin is more rapid than that of methotrexate. L-Glutamylquinazolines were taken up faster than L-aspartylquinazolines, but influx of a D-glutamylquinazoline was slower than the corresponding D-glutamylquinazoline was slower than the corresponding D-aspartyl derivative. Influx of the quinazolines was faster when there was a methyl- or chloro- substitution at position 5. Influx of the pyrimidines was also faster when a methyl group was at position 6. Michaelis constants (K_m) for influx of the various analogs varied from 1.42 x 10^-6 M to over 10^-4 M. Individual V_max values were essentially the same (1.87 to 2.22 nmoles/min/g dry weight). The relationship between the values for initial velocity of influx (v), the K_m and V_max obtained with each analog are in agreement with that predicted by the Michaelis-Menten equation and is consistent with the notion that differences in rates of influx are attributable to differences in the affinity of the carrier for the system. Efflux is preferential in the order pteridine, pyrimidine, and quinazoline. Efflux of aminopterin and methotrexate occurs at the same rate. Both aspartyl- and glutamylquinazolines efflux at about the same rate, but the D-aspartyl and D-glutamyl forms efflux more rapidly than the corresponding L forms. A methyl, and particularly a chloro, substitution at position 5 of the quinazoline reduces the rate of efflux. The extent of concentrative uptake observed for each analog directly reflects the relative magnitude at which the influx and efflux processes operate and may be the physiological parameter most relevant to therapeutic efficacy.

¹ This work supported in part by Grant CA-08748 from the National Cancer Institute and Grant BC-108 from the American Cancer Society.

Received 6/20/73. Accepted 11/ 7/73.