Lipid domain structure correlated with membrane protein function in placental microvillus vesicles

Abstract

Membrane fluidity properties of placental microvillus membrane vesicles (MVV) were determined from fluorescence anisotropy (r), dynamic depolarization, and lifetime heterogeneity studies of diphenylhexatriene (DPH), trimethylamino-DPH (TMA-DPH), and cis- and trans-parinaric acids (c-PnA and t-PnA). Plots of r against temperature for DPH and TMA-DPH in MVV had slope discontinuities at 26.degree. (Tc, transition temperature); however, analysis of r in terms of probe rotational rate (R), limiting anisotropy (.infin.), and lifetime (.tau.) revealed that DPH reported a phase transition because of changes in r.infin., whereas the phase transition observed by TMA-DPH occurred primarily because of changes in R. Heterogeneity analysis using phase and modulation lifetimes at three frequencies showed that DPH and TMA-DPH lifetimes were homogeneous in MVV. Both long (> 25 ns) and short (< 6 ns) lifetime components were detected for c-PnA and t-PnA in MVV, corresponding to the probes in solid and fluid lipid phases. The fractional amplitude of the long lifetimes (solid phase) decreased from 0.86 to 0.12 with increasing temperature (5-55.degree. C) as the membrane passed through the phase transition, with 50% of the change occurring at 27.degree. C (c-PnA) or 33.degree.C (t-PnA). The activation energies for alkaline phosphatase, aminopeptidase M, and sodium-proton antiporter activities all showed discontinuities in the temperature range 27-31.degree. C. These results indicate (1) time-resolved fluorescence measurements are required to interpret correctly changes in steady-state anisotropy with temperature, (2) both solid and fluid lipid phases coexist in the MVV membrane over a broad temperature range centered around the phase transition temperature, and (3) membrane fluidity has an important modulating influence on microvillus membrane enzyme and transport functions.