The human erythrocyte glucose transporter is the most studied of the facilitated diffusion systems for membrane transport. An alternating conformation model has been proposed for the mechanism of the transport (Barnett et al., 1975;Lienhard et al., 1983), but little is known about the structural nature of this conformational change. The technique of time-resolved phosphorescence polarization has been used extensively to investigate the rotational dynamics of membrane proteins, particularly for the detection of conformational changes within proteins (Restall et al., 1984;Coke et al., 1986). Since existing methods do not usually allow the selective labelling of the protein under study, an important restriction in the study of protein rotational diffusion is the need for a purified membrane preparation. Moreover, if information about conformational changes is to be obtained, the probe should be bound to a known part of the protein. To overcome these problems, we have designed a method to selectively label known portions of the protein under study in its natural environment. The method involves labelling a polyclonal antibody, specific for a peptide sequence in the C-terminal region of the glucose transporter (Davies et al., 1987), with a triplet-probe, erythrosin-isothiocyanate (EITC), and then studying the phosphorescence anisotropy decay of the antibody-protein complex.The antibody was covalently labelled using 0.05 mg EITC/mg protein at a concentration of 1 mg/ml in 280 mMNaCO,/NaHCO,, pH9.2, by incubation for 2 h at room temperature in the dark. Labelled antibody was separated from the unbound probe by gel filtration on a Sephadex G-25 column equilibrated with 145 mM-NaC1, 5 mM-Naphosphate, pH 7.4 (PBS) and then incubated for 30 min in the dark with inside-out vesicles from human erythrocyte ghosts, prepared by the method of Lew et al. (1982). Unbound antibody was separated by centrifugation at 12 0001: for 15 min and the pellet resuspended in PBS. After addition of 30% (v/v) glycerol the sample cuvette was sealed and throughly flushed with nitrogen. The sample was excited by a 4 n s vertical plane-polarized laser flash at 532 nm, generated by a neodymium-YAG system fitted with a harmonic doubling crystal. Phosphorescence, emitted from both sides of the cuvette, was measured through two polarizers oriented parallel and perpendicular to the electric vector of the laser excitation, to obtain values for Ivv(t) and IVH (t), respectively. After amplification the decay signals were collected in a Datalab DL 1080 transient recorder and averaged in a DL4000 signal averager. After 512 or 1024 signals had been collected, average data were transferred to a Sharp MZ80B computer. Collection of data sets were alternated with that of the same components excited by horizontally polarized light to allow for correction of the G Corrected time-dependent anisotropy data were fit to a bi-exponential decay curve r(t) = Al* exp (-?/TI) + factor ( I H H ( t ) / z H V (t)).Abbreviations used: EITC, erythrosin-isothiocyanate; PBS, phosph...
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