Bovine beta-lactoglobulin receptors on transformed mammalian cells (hybridomas MARK-3): characterization by flow cytometry.
Flow cytometry was used to demonstrate the presence of beta-lactoglobulin (betaLG) receptors on living murine hybridoma MARK-3 cells using a fluorescein isothiocyanate-betaLG conjugate (FITC-betaLG: molar ratio of 5:1). A site occupation curve was produced using a shift in the mean channel fluorescence at various concentrations of FITC-betaLG. The binding of labelled ligand was concentration dependent and was inhibited by unlabelled betaLG. The on-rate constant was 3.2x10(2) M(-1) min(-1) and the off-rate constant was 0.002 min(-1). Scatchard plot analysis gave a dissociation constant (K(d)) of 44+/-21x10(-7) and 39+/-24x10(-5) M (n=3). Flow cytometry indicated that at least 15% of the FITC-betaLG were internalized for 5 min and that internalization was temperature- and time-dependent. The internalization was confirmed by 3-D fluorescence microscopy (CELLScan system).
Palupi
N S
NS
Laboratory of Medical Biochemistry-Prof. NABET, School of Medicine, University of Henri Poincare-Nancy I, 54505, Vandoeuvre, France.
Franck
P
P
Guimont
C
C
Linden
G
G
Dumas
D
D
Stoltz
J
J
Nabet
P
P
Belleville-Nabet
F
F
Dousset
B
B
D000818:Animals; D002417:Cattle; D002460:Cell Line; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006825:Hybridomas / Q000737:chemistry*; D007782:Lactoglobulins / Q000378:metabolism*; D051379:Mice; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000378:metabolism
D005456:Fluorescent Dyes; D007782:Lactoglobulins; D011956:Receptors, Cell Surface; D016650:Fluorescein-5-isothiocyanate