Isolation of the early phase of chylomicron formation in intestinal epithelial cells of rats.
Lipid is first observed electron microscopically in the rough endoplasmic reticulum of intestinal epithelial cells during active lipid absorption. We have been able to isolate this subcellular fraction by using discontinuous sucrose gradients of 0.25/0.86/1.11 M sucrose. A preliminary low speed centrifugation of mucosal homogenate removed the heavier subcellular organelles. The resulting supernatant was centrifuged at 5.25 x 10(6) x g.min. The pellet from this centrifugation was placed on top of the gradient and the fractions isolated at the density interfaces after centrifugation at 25.5 x 10(6) x g.min. The isolated fractions were characterized enzymatically and electron microscopically. Electron microscopically, the fractions were predominantly composed of rounded vesicles decorated with ribosomes. Most contained lipid droplets whose diameters were 453 nm in the lighter membranes and 245 nm in the membranes isolated from the heavier density region. The vesicles contained NADPH cytochrome c reductase and glucose-6-phosphatase activity indicative of the presence of microsomes. Contamination with other subcellular organelles was minimal. These studies demonstrate a method which enables the isolation of vesicles containing chylomicron-sized particles which are from the earliest phase of chylomicron formation. Isolation of chylomicrons from these vesicles will enable a better understanding of the maturation process of chylomicrons as they traverse the intestinal epithelial cell.
Turner
M W
MW
Department of Medicine, University of Tennessee, Memphis.
Frase
S
S
Mansbach
C M
CM
D000818:Animals; D002914:Chylomicrons / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D004848:Epithelium / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000201:enzymology / Q000648:ultrastructure; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D002914:Chylomicrons