AVIDIN-BIOTIN INTERACTIONS AT VESICLE SURFACES - ADSORPTION AND BINDING, CROSS-BRIDGE FORMATION, AND LATERAL INTERACTIONS

Densely packed domains of membrane proteins are important structures i n cellular processes that involve ligand-receptor binding, receptor-me diated adhesion, and macromolecule aggregation. We have used the bioti n-avidin interaction at lipid vesicle surfaces to mimic these processe s, including the influence of a surface grafted polymer, polyethyleneg lycol (PEG). Single vesicles were manipulated by micropipette in solut ions of fluorescently labeled avidin to measure the rate and give an e stimate of the amount of avidin binding to a biotinylated vesicle as a function of surface biotin concentration and surface-grafted PEG as P EG-lipid. The rate of avidin adsorption was found to be four times les s with 2 mol% PEG(750) than for the unmodified surface, and 10 mol% PE G completely inhibited binding of avidin to biotin for a 2-min incubat ion. Using two micropipettes, an avidin-coated vesicle was presented t o a biotinylated vesicle, In this vesicle-vesicle adhesion test, the a ccumulation of avidin in the contact zone was observed, again by using fluorescent avidin. More importantly, by controlling the vesicle memb rane tension, this adhesion test provided a direct measure of the spre ading pressure of the biotin-avidin-biotin cross-bridges confined in t he contact zone. Assuming ideality, this spreading pressure gives the concentration of avidin cross-bridges in the contact zone. The rate of cross-bridge accumulation was consistent with the diffusion of the li pid-linked ''receptors'' into the contact zone. Once adherent, the mem branes failed in tension before they could be peeled apart. PEG(750) d id not influence the mechanical equilibrium because it was not compres sed in the contact zone, but it did perform an important function by e liminating all nonspecific adhesion. This vesicle-vesicle adhesion exp eriment, with a lower tension limit of 0.01 dyn/cm, now provides a new and useful method with which to measure the spreading pressures and t herefore colligative properties of a range of membrane-bound macromole cules.