Order measurements in plasma membranes from duchenne dystrophy fibroblasts

Abstract

Plasma membranes have been isolated using different methods from Duchenne dystrophy and control human skin fibroblasts. Fluorescence techniques were utilized to resolve the rotational properties and the degree of hindered rotation of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene in the membranes. Under specific conditions of fibroblast processing and membrane fractionation, plasma membranes from Duchenne fibroblasts showed significantly less order (0.0125 > P < 0.0025) and less hindrance to probe rotation than membranes from control fibroblasts. The order differences did not seem to be the result of heterogeneity in the membrane environment sampled by the probe. The frequency dependence of the fluorescence lifetime for diphenylhexatriene indicated no measurable contribution by a short lifetime component. Analysis of diphenylhexatriene rotation in the plasma membranes using the ‘wobbling-in-cone’ theory suggested that both the angle of probe rotation ( θ c) and the rotational rate ( D w) were important parameters in understanding the variations between Duchenne and control membranes at 16, 22 and 30°C. Electron spin resonance studies with 5′-doxylstearic acid at 25°C confirmed our fluorescence results. The segmental motion exhibited by the spin label revealed less order in the Duchenne membranes.