Proteolytic microdissection of smooth-surfaced vesicles of liver microsomes.
Digestion of rabbit liver microsomal smooth vesicles with Bacillus subtilis protease released proteins and peptide fragments from the vesicles, without solubilizing phospholipids and cholesterol. The proteolysis was, however, limited when about 30% of the protein had been solubilized. The same limitation was observed when the vesicles were treated with trypsin, chymotrypsin, or their combinations with the bacterial protease. The limited proteolysis was accompanied by selective solubilization of cytochrome b(5) and microsomal NADPH-specific flavoprotein, leaving the CO-binding hemoprotein and some other enzymes still attached to the vesicular membranes. Sucrose density gradient centrifugation of protease-treated vesicles indicated that all the vesicles had been attacked by the protease to similar extents. The behavior of intact and digested vesicles in dextran density gradient centrifugation suggested that the vesicles, even after proteolytic digestion, existed in the form of closed sacs which were impermeable to macromolecules such as dextran and proteases. It was concluded that only the outside surface of the vesicles is susceptible to the proteolytic action and that cytochrome b(5) and the NADPH-specific flavoprotein are located in the susceptible area.
D000818:Animals; D001412:Bacillus subtilis; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000032:analysis; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000494:pharmacology; D008099:Liver / Q000166:cytology*; D008566:Membranes / Q000187:drug effects; D008861:Microsomes / Q000032:analysis / Q000187:drug effects*; D009438:Neuraminic Acids / Q000032:analysis; D010447:Peptide Hydrolases / Q000494:pharmacology*; D010743:Phospholipids / Q000032:analysis; D011506:Proteins / Q000032:analysis; D012313:RNA / Q000032:analysis; D011817:Rabbits; D014357:Trypsin / Q000494:pharmacology
D009438:Neuraminic Acids; D010743:Phospholipids; D011506:Proteins; D012313:RNA; D002784:Cholesterol; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D014357:Trypsin