Abstract
Human KB cells produce two immunologically cross-reactive folate-binding proteins: a particulate cell-associated protein which is solubilized by Triton X-100, and a soluble protein which is released into their growth medium. This compartmentation of these two folate-binding proteins provides a convenient system for studies of their biochemical relationship. The two folate-binding proteins behave similarly to the purified particulate and soluble folate-binding proteins of human milk in analysis by radioactive folate binding, Sephacryl S-200 gel filtration profiles, polyacrylamide gel electrophoresis in either Triton X-100 or sodium dodecyl sulfate, and in Triton X-100 binding based on sucrose density gradient ultracentrifugation in H2O and D2O. The two folate-binding proteins were endogenously labeled by pulsing methionine-starved KB cells with [35S]methionine, and each protein was purified to apparent homogeneity by affinity chromatography at different times during the chase with nonradioactive methionine. The time course of the changes in specific activity (moles of [35S]methionine per mole of folate-binding protein) revealed a more rapid initial rate of synthesis and an earlier maximum in specific activity for the cell-associated folate-binding protein than for the soluble folate-binding protein released into the growth medium. Differences in the levels and specific activities of the two folate-binding proteins of cells exposed to cycloheximide compared with simultaneous controls after pulsing with [35S]methionine suggest that, whereas the cell-associated folate-binding protein is probably produced by de novo protein synthesis, the soluble folate-binding protein seems to be produced from a cellular pool of an already synthesized protein. These results combined with the immunologic cross-reactivity of the two folate-binding proteins strongly suggest a precursor-product relationship between them.
Highlights
Human KB cells produce twiommunologically cross- Soluble andparticulate(membrane-associated)forms of reactive folate-binding proteins: a particulate cell-as- high affinity folate-binding proteins have been identified in sociated protein which is solubilized by Triton X-100, human tissues
[35S]methionine,and each protein was purified to ap- 100, but with an apparent M, of approximately 40,000 on parent homogeneity byaffinity chromatography at diSf-DS-PAGE [1].The high apparent M, of the M-FBP from ferent times during the chase with nonradioacmtiev-e human milk is largely due to Triton X-100 binding (l), and thionine
A very small quantity of high M, M-FBP and a large quantity of Soluble folate-binding proteins (S-FBP) that eluted at an apparent M, of
Summary
Human KB cells produce twiommunologically cross- Soluble andparticulate(membrane-associated)forms of reactive folate-binding proteins: a particulate cell-as- high affinity folate-binding proteins have been identified in sociated protein which is solubilized by Triton X-100, human tissues. The two folate-binding proteins werendogenously FBP from human milk is alsoa glycoprotein with an apparent labeled by pulsing methionine-starved KB cells with M , of 160,000 bygel filtration in the presence of Triton X-
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