DETERMINANTS OF G(I1)ALPHA AND BETA-GAMMA BINDING - MEASURING HIGH-AFFINITY INTERACTIONS IN A LIPID ENVIRONMENT USING FLOW-CYTOMETRY

G protein heterocomplex undergoes dissociation and association during its functional cycle, Quantitative measurements of alpha and beta gamm a subunit binding have been difficult due to a very high affinity, We used fluorescence flow cytometry to quantitate binding of fluorescein- labeled G(il)alpha (F-alpha) to picomolar concentrations of biotinylat ed G beta gamma. Association in Lubrol solution was rapid (k(on) = 0.7 x 10(6) M-1 s(-1)), and equilibrium binding revealed a K-d of 2.9 +/- 0.8 nM. The binding showed a complex dependence on magnesium concentr ation, but activation of F-alpha with either GDP/aluminum fluoride or guanosine 5'-O-(3-thiotriphosphate) completely prevented formation of the heterocomplex (K-d > 100 nM). The binding was also influenced by t he detergent or lipid environment, Unlabeled By reconstituted in bioti nylated phospholipid vesicles (pure phosphatidylcholine or mixed brain lipids) bound F-alpha similar to 2-3-fold less tightly (K-d = 6-9 nM) than in Lubrol. In contrast, beta gamma in ionic detergents such as c holate and cholamidopropyl)diethylammonio]-1-propanesulfonate exhibite d substantially lower affinities for F-alpha. Dissociation of F-alpha from beta gamma reconstituted in lipid vesicles was observed upon addi tion of aluminum fluoride or excess unlabeled alpha subunit, indicatin g that myristoylated alpha subunit has only a weak interaction with li pids without the beta gamma subunit, The kinetics of aluminum fluoride -stimulated dissociation were slower than those of the alpha subunit c onformational change detected by intrinsic fluorescence. These results quantitatively demonstrate G protein subunit dissociation upon activa tion ante provide a simple but powerful new approach for studying high affinity protein/protein interactions ire solution or in a lipid envi ronment.