Abstract
Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (Kd) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. Kd measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca2+, Mg2+, and ethylenediammonium2+ ions). Monovalent cations (e.g., Na+, K+, and Tris+) were also effective, but only at concentrations in the millimolar range. The Kd for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl2. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.
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