Cross-linking studies of the protein topography of rat liver microsomes.
A cleavable cross-linking reagent, dithiobis(succinimidyl propionate), DSP, was used to study the topography of the proteins in the endoplasmic reticulum membrane of rat liver. Reaction of untreated (control), phenobarbital- or 3-methylcholanthrene-induced microsomes with 0.5 mM DSP for 30 min at 0 degrees C resulted in the cross-linking of a protein with a molecular weight of about 52 000 to form an apparent dimer. In phenobarbital microsomes, a smaller amount of a 52 000-dalton protein also appeared in a dimer in the absence of DSP if N-ethylmaleimide was not included during homogenization. In phenobarbital and 3-methylcholanthrene microsomes, a 48 000-dalton protein was cross-linked by DSP to a protein of about 57 000. In all three types of microsomes, a protein with an Mr of about 52 000 was also cross-linked to a protein of about 79 000. In phenobarbital and control microsomes, cross-linking resulted in an oligomeric protein of approximate molecular weight 180 000 which contained three proteins, two with Mr of about 52 000 and one about 79 000. Under the cross-linking conditions, little or no denaturation of cytochrome P-450 and NADPH-cytochrome c reductase was observed. The aryl hydrocarbon hydroxylase activity was significantly inhibited by the bifunctional cross-linking reagent, DSP, but not by the monofunctional reagent N-succinimidyl-3-(4-hydroxyphenyl) propionate. However, attempts to regenerate the aryl hydrocarbon hydroxylase activity by cleavage of the disulfide linkage with 2-mercaptoethanol or dithiothreitol were not successful.
D000818:Animals; D003432:Cross-Linking Reagents; D007425:Intracellular Membranes / Q000032:analysis*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000032:analysis* / Q000187:drug effects; D008970:Molecular Weight; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013388:Succinimides
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D013388:Succinimides; D008748:Methylcholanthrene; C011240:dithiobis(succinimidylpropionate); D010634:Phenobarbital