[Immunological specificity and cytoplasmic location of delta 6-desaturase in microsomal membrane].
The enzymatic properties of the three types of microsomal acyl-CoA desaturases, delta 6-, delta 9- and delta 5-desaturase, were immunologically compared using a monospecific antibody raised against the purified linoleoyl-CoA desaturase (delta 6-desaturase). By the double immunodiffusion technique, the anti-delta 6-desaturase antibody showed a single precipitin line to the purified delta 6-desaturase and microsomes treated with Triton X-100, but no line was observed with the partially purified delta 9-desaturase. The antibody even inhibited definitely delta 6-desaturase activity in microsomes, but neither stearoyl-CoA (delta 9-) nor eicosatrienoic acid (delta 5-) desaturations were inhibited. By these immunological investigations it was confirmed that terminal delta 6-desaturase is different enzyme from desaturases delta 9- and delta 5. The intramembrane localization of delta 6-desaturase in rat liver microsomes was examined by various methods, such as digestion by proteases, effect of detergents and inhibition by the antibodies against purified terminal desaturase. Exposure of the desaturase on the surface of microsomal vesicles was suggested by the fact that the enzyme activity in the intact microsomes was susceptible to tryptic digestion and considerably inhibited by anti-desaturase antibodies. When microsomes were previously treated with trypsin, the enzyme became more susceptible to the antibodies. Furthermore, it was demonstrated that the protein fragments cleaved from microsomal membranes by tryptic digestion formed a single precipitin line with the antibodies by the double immunodiffusion test. These findings suggest the presence of delta 6-desaturase on the cytoplasmic surface in the endoplasmic reticulum, since tryptic digestion liberates only the protein components situated on the surface area of membranes. In addition, desaturase activity in the intact microsomes was not stimulated by addition of the detergent, indicating the further outside location of the active site of the enzyme in microsomal vesicles. The previous exposure of microsomes to a low concentration (0.05%) of sodium deoxycholate, which destroys the permeability barrier for macromolecules whichout membrane disassembly, did not increase the susceptibility to tryptic digestion and the antibodies. These results show that delta 6-desaturase is not present in a latent state in the membrane.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D003593:Cytoplasm / Q000201:enzymology*; D000090943:Delta-5 Fatty Acid Desaturase; D044943:Fatty Acid Desaturases / Q000032:analysis* / Q000276:immunology; D005779:Immunodiffusion; D050602:Linoleoyl-CoA Desaturase; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013230:Stearoyl-CoA Desaturase; D013501:Surface-Active Agents / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D000906:Antibodies; D000090943:Delta-5 Fatty Acid Desaturase; D013501:Surface-Active Agents; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase; D050602:Linoleoyl-CoA Desaturase; C029627:delta-9 fatty acid desaturase; D014357:Trypsin