Isolation of a sodium dodecyl sulfate-insoluble transglutaminase substrate from liver plasma membranes.
Rat liver plasma membranes contain transglutaminase activity and a large molecular weight protein complex which serves as a substrate for this enzyme. When plasma membranes were solubilized in sodium dodecyl sulfate and disulfide-reducing agents the transglutaminase substrate was recovered in the detergent-insoluble fraction. The insolubility of the complex suggested that it might be further studied by adsorbing membranes onto glass slides, then extracting with the detergent and reducing agent. After extraction, dark field light microscopy revealed numerous flattened sheets which varied in size from 4 to 12 micrometers. To confirm that these structures were the large molecular weight transglutaminase substrate, the plasma membranes were solubilized in sodium dodecyl sulfate and dithiothreitol and sedimented through a sucrose gradient containing the agent. The large molecular weight substrate was the only material found at the 1.11/1.23 g/cm3 interface. Microscopic examination showed the same structures previously observed on the glass slides. We conclude that the large molecular weight transglutaminase substrate is a sodium dodecyl sulfate-insoluble, morphologically distinct, protein complex. Due to its considerable size, nondissociable nature, and association with the lateral membrane, the sodium dodecyl sulfate-insoluble transglutaminase substrate may serve as a type of skeleton or scaffolding for this plasma membrane domain.
D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D008099:Liver / Q000201:enzymology*; D008297:Male; D008853:Microscopy; D008970:Molecular Weight; D051381:Rats; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D011503:Transglutaminases / Q000302:isolation & purification*
D012967:Sodium Dodecyl Sulfate; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine