title: Degradation of hepatic stearyl CoA delta 9-desaturase.
abstract: delta 9-Desaturase is a key enzyme in the synthesis of desaturated fatty acyl-CoAs. Desaturase is an integral membrane protein induced in the endoplasmic reticulum by dietary manipulations and then rapidly degraded. The proteolytic machinery that specifically degrades desaturase and other short-lived proteins in the endoplasmic reticulum has not been identified. As the first step in identifying cellular factors involved in the degradation of desaturase, liver subcellular fractions of rats that had undergone induction of this enzyme were examined. In livers from induced animals, desaturase was present in the microsomal, nuclear (P-1), and subcellular fractions (P-2). Incubation of desaturase containing fractions at physiological pH and temperature led to the complete disappearance of the enzyme. Washing microsomes with a buffer containing high salt decreased desaturase degradation activity. N-terminal sequence analysis of desaturase freshly isolated from the P-1 fraction without incubation indicated the absence of three residues from the N terminus, but the mobility of this desaturase preparation on SDS-PAGE was identical to the microsomal desaturase, which contains a masked N terminus under similar purification procedures. Addition of concentrated cytosol or the high-salt wash fraction did not enhance the desaturase degradation in the washed microsomes. Extensive degradation of desaturase in the high-salt washed microsomes could be restored by supplementation of the membranes with the lipid and protein components essential for the reconstituted desaturase catalytic activity. Lysosomotrophic agents leupeptin and pepstatin A were ineffective in inhibiting desaturase degradation. The calpain inhibitor, N-acetyl-leucyl-leucyl-methional, or the proteosome inhibitor, Streptomyces metabolite, lactacystin, did not inhibit the degradation of desaturase in the microsomal or the P-1 and P-2 fractions. These results show that the selective degradation of desaturase is likely to be independent of the lysosomal and the proteosome systems. The reconstitution of complete degradation of desaturase in the high-salt-washed microsomes by the components essential for its catalytic activity reflects that the degradation of this enzyme may depend on a specific orientation of desaturase and intramembranous interactions between desaturase and the responsible protease.
authors.affiliation: Department of Biochemistry, University of Connecticut Health Center, Farmington 06030, USA.
authors.forename: J
authors.initials: J
authors.lastname: Ozols
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors; D002458:Cell Fractionation; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D017421:Sequence Analysis; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013230:Stearoyl-CoA Desaturase / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
chemical_list: D015853:Cysteine Proteinase Inhibitors; D015842:Serine Proteinase Inhibitors; C067713:lactacystin; D013230:Stearoyl-CoA Desaturase; D002154:Calpain; D000111:Acetylcysteine