Selective release of content from microsomal vesicles without membrane disassembly. II. Electrophoretic and immunological characterization of microsomal subfractions.
Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [(3)H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [(3)H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [(3)H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.
D000818:Animals; D001345:Autoradiography; D001797:Blood Protein Electrophoresis; D002250:Carbon Radioisotopes; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000032:analysis*; D005944:Glucosamine / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007930:Leucine / Q000378:metabolism; D008566:Membranes; D008861:Microsomes / Q000032:analysis*; D008862:Microsomes, Liver / Q000032:analysis; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D051381:Rats; D012709:Serum Albumin / Q000032:analysis; D014316:Tritium
D002250:Carbon Radioisotopes; D003902:Detergents; D006023:Glycoproteins; D010455:Peptides; D011506:Proteins; D012709:Serum Albumin; D014316:Tritium; D007930:Leucine; D005944:Glucosamine