Figure 4.

Functional reconstitution of glycerol transport activity in GlpF and AqpZ + GlpF proteoliposomes. GlpF (100 μg), AqpZ + GlpF (100 μg each), or no protein was mixed with pure phospholipid, and proteoliposomes were formed and equilibrated with glycerol (855 mosM). A concentration gradient for glycerol was then imposed by rapidly mixing 100 μl of the equilibrated proteoliposome suspension (1 μg of protein) with an equal volume of isoosmotic solution containing sucrose as a nonpermeant osmolyte in a stopped-flow apparatus. Release of glycerol from the proteoliposomes creates an osmotic gradient producing water efflux, and vesicle shrinkage was measured by light scattering. The average kinetics of 5–10 measurements were normalized and fitted to a characteristic single-order exponential equation dependent on the time course of glycerol efflux. Notice the different time scales in plots.