ERYTHROCYTE-MEMBRANE LATERAL STEROL DOMAINS - A DEHYDROERGOSTEROL FLUORESCENCE POLARIZATION STUDY

Structural domains of cholesterol and their regulation in the erythroc yte membrane are poorly understood. Dehydroergosterol fluorescence pol arization change was used to continuously monitor the kinetics of ster ol exchange and sterol domain size in erythrocyte ghost membranes. Dir ect correlation between molecular sterol exchange and steady-state deh ydroergosterol fluorescence polarization measurements was obtained wit hout separation of donor and acceptor membranes. Three important obser vations were made. First, sterol exchange between small unilamellar ve sicles (SUV) with the same cholesterol/phospholipid ratio as the eryth rocyte membrane palmitoyl-2-oleoylphosphatidylcholine/cholesterolc = 1 :1) was resolved into three kinetic cholesterol domains: 23 +/- 9% of total sterol was rapidly exchangeable, with t(1/2) = 23 +/- 6 min; 59 +/- 9% of total sterol was slowly exchangeable, with t(1/2) 135 +/- 3 min; and 19 +/- 9% of total sterol was essentially nonexchangeable, wi th a t(1/2) of days. Second, the substitution of erythrocyte ghosts fo r SUV as an acceptor significantly altered the kinetic parameters of s terol exchange from donor SUV, graphically showing that both the prope rties of the acceptor and spontaneous desorption of cholesterol from t he donor SUV influenced spontaneous cholesterol transfer. Third, studi es of exchange between erythrocyte ghosts revealed multiple kinetic po ols of sterol differing from those in the SUV: 4 +/- 2% of total stero l was rapidly exchangeable, with t(1/2) 32 +/- 9 min; 29 +/- 3% of tot al sterol was very slowly exchangeable, with a t(1/2) 23 +/- 7 h; and a surprisingly large 67 +/- 2% of total sterol was nonexchangeable, wi th a t(1/2) of days.