Topography of rat hepatic microsomal enzymatic components of the fatty acid chain elongation system.

The orientation of the condensing enzyme, the beta-hydroxyacyl-CoA dehydrase, and the trans-2-enoyl CoA reductase within the rat liver microsomal membrane was investigated by the use of impermeant inhibitors of enzyme activity: trypsin, chymotrypsin, subtilisin, mercury-dextran, and anti-beta-hydroxyacyl-CoA dehydrase IgG. The activity of the condensing enzyme was inhibited more than 70% by various proteases and was completely inhibited by 80 microM mercury-dextran. Similar results were obtained for the trans-2-enoyl-CoA reductase activity. On the other hand, in the absence of detergent, proteases inhibited beta-hydroxyacyl-CoA dehydrase activity by 25-40%, while in the presence of detergent the inhibition increased to 65-90%. Furthermore, anti-beta-hydroxyacyl-CoA dehydrase IgG, which in the absence of detergent produced no inhibition, in the presence of detergent inhibited beta-hydroxyacyl-CoA dehydrase activity by more than 80%; under identical conditions, preimmune IgG caused a 13% inhibition. Microsomes used throughout this study displayed greater than 90% latency with respect to mannose-6-phosphatase activity, indicating that the microsomes were intact. Latency was not affected by the proteases, by mercury-dextran, or by the presence of the enzyme assay components. These results suggest that both the condensing enzyme and the reductase are present on the cytoplasmic surface of the membrane, whereas the beta-hydroxyacyl-CoA dehydrase is embedded in the microsomal membrane.