Sedimentation field flow fractionation of mitochondrial and microsomal membranes from corn roots

Sedimentation field flow fractionation (sed-FFF) is shown to be a valuable procedure for analysis of a wide variety of subcellular particle preparations. The principles underlying this relatively new separation procedure are described. Separation is based on differences between particles in mass and/or density. As in chromatography, the procedure involves relating on-line or off-line measurements made on the effluent from the separation chamber to the elution (retention) time. In this work effluents were monitored for absorbance at 254, 280, and/or 320 nm; collected fractions were assayed for protein content, total ATPase activity, and/or marker enzyme activities and, when appropriate, were examined by electron microscopy. The ratio of the absorbances at 254 and 320 nm was found to provide a sensitive measure of partial resolution of subcellular particles. Preparations containing all of the subcellular particles of corn roots (exclusive of nuclei, cell walls, and ribosomes), and fractions thereof enriched in mitochondria, microsomes, Golgi membranes, or plasma membranes, were examined by sed-FFF. The subcellular particles appear to remain largely intact. All of the particles observed had a mass less than 2 X 10(11) g/mol. All of the preparations were grossly heterogeneous with respect to effective mass distribution. This is due in part to heterogeneity with respect to the organelle of origin. In microsome preparations, components of low, medium, and high density were present in the unretained peak; the retained region had comparatively more high density particles. Plasma membrane preparations had a very wide effective particle mass distribution. The observations suggest that, in addition to its utility for analytic purposes, sed-FFF is likely to prove useful for micro-preparative fractionation of some subcellular particle preparations. Sed-FFF and density gradient centrifugation can be utilized as complementary methods.