HIGH-AFFINITY FOLATE BINDING IN HUMAN MAMMARY-GLAND

High-affinity H-3-folate binding in Triton X-100 solubilized human mam mary gland tissue displayed characteristics, e.g. apparent positive co operativity and increasing affinity with decreasing concentration of f olate binding protein, shown to be typical of specific folate binding. Radioligand dissociation was slow at pH 7.4. A major fraction of the bound radioligand dissociated rapidly at pH 3.5, while a residual bind ing of 20% persisted even after prolonged dialysis at pH 3.5. Gel chro matography revealed two major folate binding proteins (M(r) almost-equ al-to 100 kDa and 25 kDa). However, only one single band was detectabl e on SDS-PAGE immunoblotting. The highest folate binding activity per g protein was associated with the upper triglyceride-containing layer of the 1000 g supernatant of the homogenate. The folate binding protei n extracted from this layer had a low cross-reactivity (<5%) with rabb it antibodies against 25 kDa human milk folate binding protein. The fo late binding protein in the 1000 g pellet and the aqueous phase of the 1000 g supernatant was present at a low concentration and had a cross -reactivity of 100%.