A system of H(3)-amethopterin uptake, physiologically and kinetically resembling active transport, has been described in Diplococcus pneumoniae. Uptake by this system has a pH optimum near 6.0, is temperature-dependent, requires a readily available source of energy, and conforms to Michaelis-Menten kinetics. The system showed a K(m) of 0.9 x 10(-6)m and a V(max) of 1.9 x 10(-13) moles per min per mg (dry weight). Both folate and H(2)-folate compete with H(3)-amethopterin for the same system, but to a limited degree. The intracellular concentration of H(3)-amethopterin accumulated at equilibrium was 1.06 x 10(-9) moles/ml or fivefold the external concentration when the latter was limiting, but at least 60-fold the internal concentration attained solely by diffusion in the same time interval at 0 C.

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