Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

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Abstract
L. 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.degree. C was time and concentration dependent and at low levels (1-10 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 potassium phosphate buffer, and it varied by less than 2-fold when measured over a range of pH values (5.5-7.5) or in buffered salt solutions of differing ionic compositions. 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 (10-50 .mu.M) 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. Thus, the folate transport protein of L. casei may contain both a substrate- and cation-binding site and folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites may indicate that folate is transported across the cell membrane via a cation-folate symport mechanism.

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