The isolation and properties of epithelial-cell "ghosts" from rat small intestine

Abstract

1. The preparation of gram quantities of isolated epithelial-cell ;ghosts' from mucosal scrapings of rat small intestine is described. The method involves dispersing the tissue by gentle homogenization in 6% dextran in Krebs-Ringer phosphate, pH7.4, followed by filtration through nylon cloth and sedimentation by low-speed centrifuging. 2. The isolated epithelial-cell ;ghosts' contained all of the DNA, but only 52% of the protein and 53-57% of the RNA of the original homogenate. They contained most of the activity of the following enzymes found in the homogenate: aminopeptidase (71%); alkaline beta-glycerophosphatase (82%); invertase (92%); adenosine triphosphatase (93-116%); acid beta-glycerophosphatase (83%); nonspecific esterase (76%); succinate dehydrogenase (96%). Only small proportions of the total lactate-dehydrogenase (10%) and phosphoglucose-isomerase (2%) activities found in the homogenate were recovered in the isolated cell ;ghosts'. 3. The epithelial-cell ;ghost' preparation did not respire unless cofactors and substrates were added, and did not consume glucose or produce lactic acid from glucose. 4. The effect of varying the composition of the homogenization medium was studied. Concentrations of dextran (mol.wt. 15x10(4)) from 1 to 12%, solutions of dextrans (all at 6%) with mol.wt. varying between 3.6x10(4) and 2x10(6), and a solution of 8% polyethylene glycol (mol.wt. 4000) served equally well for the production of epithelial-cell ;ghosts'. Two of these solutions, however, 12% dextran (mol.wt.15x10(4)) and 6% dextran (mol.wt. 2x10(6)), were too viscous to allow the complete sedimentation of the cell ;ghosts' at low relative centrifugal forces. Omission of either Krebs-Ringer phosphate or dextran from the medium resulted in almost complete cell breakage during the homogenization. 5. The isolated cell ;ghosts' were used as a starting material for subcellular fractionation of rat intestinal mucosa by differential centrifugation. The distributions of protein and succinate-dehydrogenase activity among the fractions were compared with corresponding values in fractions isolated by differential centrifugation of mucosa homogenized in 0.3m-sucrose-5mm-EDTA, pH7.4. The method in which cell ;ghosts' were used as starting material gave a better separation and cleaner fractions than the method in which untreated mucosal scrapings were used.