Isolation and characterization of lamellar body material from rat lung homogenates by continuous linear sucrose gradients.

Abstract

A technique is described for isolating lamellar body material from rat lung. Membranes with relative densities ranging between 1.050 and 1.074 g/ml were isolated by centrifugation of crude lung homogenates upward through continuous linear sucrose gradients at 40,000 rpm (199,000 g) for 3 hr. Their protein and lipid content was characteristic of that of lamellar bodies. They were free of contaminating microsomal and mitochondrial marker enzymes but contained enzyme activities associated with lysosomes and Golgi complex. Longer or repeated centrifugation resulted in a reduced yield and an apparent transformation of some of the material to lower densities. Electron microscopy revealed that most of the images represent disrupted rather than intact lamellar bodies. Other methods for preparation of lamellar bodies entail either sedimentation or pelleting at interfaces between sucrose solutions. Such preparations are often contaminated with endoplasmic reticulum membranes and have apparently lost the more fragile bodies. The present technique reveals the heterogeneous nature of lamellar body material and should be useful in a search for lamellar body precursors and in the investigation of the mechanisms by which surfactant is synthesized or assembled.