Kinetic evidence for two interconvertible forms of the folate transport protein from Lactobacillus casei.
Lactobacillus casei cells contain a folate transport protein which exhibits a high affinity for folate. The dissociation constant (KD) for folate derived from binding parameters at the steady state (at 0 degree C) is 0.4 nM at pH 7.5 and 0.1 nM at pH 6.0. In the present study, folate binding to this protein at pH 7.5 (and 0 degree C) was shown to follow second-order kinetics and to proceed with an association constant (k+1) of 4.9 X 10(7) liter/mol per min. K+1 was not affected by preincubation conditions which alter the energetic state of the cell. Measurements on the extent of binding showed further that (at 0 degree C) essentially all unoccupied folate-binding sites reside at or are readily accessible to the outer surface of the membrane. In contrast, after saturating the binding site with [3H]folate, the first-order rate constant (k-1) for dissociation of the bound substrate (at 0 degree C) was found to vary substantially with the conditions employed. k-1 was 0.028/min in freshly harvested cells, but it increased by 2.8-fold in cells preincubated at 23 degrees C for 60 min and by 5.4-fold in isolated membranes. In addition, the faster rate observed in preincubated cells (k-1 = 0.077/min) returned to a slower rate after brief exposure of the cells to pH 6.0 (k-1 = 0.041/min), glucose (k-1 = 0.050/min), or both (k-1 = 0.012/min). k-1 was twofold lower at pH 6.0 than at pH 7.5 and was less dependent on the preincubation conditions, although it also increased substantially (5.5-fold) when the cells were converted to plasma membranes. The proposed explanation for these results is that folate transport protein of L. casei exists in two forms which can be distinguished by the accessibility of the binding site to the external medium and whose amounts are dependent upon the presence of bound folate, the pH, and the energetic state of the cell. It is suggested that these forms are transport proteins with binding sites oriented towards the inner and outer surfaces of the membrane.
D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D058973:Folate Receptors, GPI-Anchored; D005492:Folic Acid / Q000378:metabolism*; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000378:metabolism*; D011956:Receptors, Cell Surface; D014316:Tritium
D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D011956:Receptors, Cell Surface; D014316:Tritium; D005492:Folic Acid