Journal of Lipid Research, Vol 22, 1277-1285, Copyright © 1981 by Lipid Research, Inc.
Isolation and characterization of lamellar body material from rat lung homogenates by continuous linear sucrose gradients
H Gilder, RH Haschemeyer, GF Fairclough Jr and DC Mynarcik
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.
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