Preparation of cytoplasmic bodies (cytospheres) from isolated hepatocytes and their biochemical properties.
The controlled centrifugation of isolated rat hepatocytes at 260 000 g results in the formation of membrane-bounded cell fragments that we have termed 'cytospheres'. A method is described for the isolation of these cytospheres. Cytospheres are spherical, have a mean diameter of 9.2 +/- 3.2 microns (SD) and a protein content of 225 +/- 12 mg/g wet wt. About 3% of the protein from the original isolated hepatocyte suspension is recoverable. Transmission electron microscopy (TEM) shows cytospheres to possess a trilaminar membrane, and a finely granular hyaloplasm generally devoid of organelles, filaments and microtubules. Freeze-fracture studies reveal a membrane structure typical of a plasma membrane. Ouabain and wheat germ agglutinin (WGA)-binding studies indicate that the original orientation of the plasma membrane is maintained throughout the formation of the cytospheres. The cytospheres have also been characterized biochemically. Cytospheres are enriched in the enzymes normally associated with the hyaloplasm, whereas the activities of enzymes localized in organelles are greatly diminished. Lipid analysis of the cytosphere membrane indicates that it is derived from the plasma membrane of the hepatocyte. Cytospheres are sensitive to changes in the osmolarity and ionic composition of their environment. Cytospheres should therefore prove a useful preparation for the study of hyaloplasm metabolism and of plasma membrane receptor and permeability properties.
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000648:ultrastructure; D005614:Freeze Fracturing; D008099:Liver / Q000166:cytology / Q000201:enzymology / Q000648:ultrastructure*; D008297:Male; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D051381:Rats; D011919:Rats, Inbred Strains; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014461:Ultracentrifugation
D000254:Sodium-Potassium-Exchanging ATPase