Human erythrocyte membranes contain high-affinity folate-binding proteins (FBPs) which on solubilization with detergents resolve into apparent 160 000 Mr moieties on Sephacryl S-200 gel filtration in Triton X-100. These FBPs share antigenic and ligand binding characteristics with particulate FBPs from other human tissues. During studies to define the vectorial orientation of these FBPs on the erythrocytes, we trypsinized intact cells with 250 μg trypsin per ml packed cells and quantitatively analysed the remaining cell-associated FBPs as well as the products of proteolysed FBPs in the supernatant. Incubation of intact cells with trypsin resulted in a dose-dependent decrease in their capacity to bind 125I-labelled pteroyglutamate (histamine derivative); at 250 μg/ml trypsin, folate binding was decreased by 77% compared to nontrypsin-treated control cells. While trypsinized cells contained proportionately lower quantities of apparent 160 000 Mr FBPs than untreated control cells, the supernatant of trypsinized cells (soluble phase) contained a single species of Mr = 40 000 which retained folate binding capacity. The sum of FBPs in trypsin supernatant and trypsin-treated cells was 87% of that found in untreated cells. Analysis of solubilized particulate erythrocyte FBPs and soluble (trypsin product) FBPs by sucrose density gradient ultracentrifugation in H2O and 2H2O above the critical micellar concentration of Triton X-100 revealed that apparent 160 000 Mr FBPs were detergent-binding (hydrophobic) species (which sedimented at Mr = 40 000 in H2O) while soluble FBPs (also sedimenting at Mr = 40 000) were hydrophilic and did not bind Triton X-100. These are the first data which show that hydrophobic FBPs can be directly converted to hydrophilic FBPs by a trypsin-mediated proteolytic event. The trypsin-sensitive site is likely to be at the junction between the detergent-binding site and the major body of the protein (Mr = 40 000) containing the folate binding site.
- (Human erythrocyte)
- Folate binding protein
- Hydrophilic folate binding protein
- Hydrophobic folate binding protein
ASJC Scopus subject areas
- Cell Biology