Fig. 6. Km and Vmax of microsomal DHS-C26 synthase activity. (A) Microsomes were lysed in 1% digitonin. A 100 µg aliquot of solubilized microsomal extract was assayed in the presence of 5 nmol of [3H]DHS, 200 nmol of C26-CoA and variable amounts (5–1500 nmol) of cold DHS or PHS in the presence of 1% digitionin. The products were extracted and analysed by TLC in solvent 1. [3H]DHS-C26 was quantitated by radioscanning and expressed as a percentage of total radioactivity in the corresponding lane. (B) The data of (A) were used to calculate enzymatic activities by calculating the total amounts of DHS-C26 made during incubations. DHS or PHS concentrations of the assays were expressed as mol/mol ratios of [long chain bases]/[total phospholipids + detergent]. The data were then transformed in double reciprocal plots according to Lineweaver–Burk. [Assays contained 120 nmol of phospholipids (estimated based on the amount of microsomal protein) and 820 nmol of detergent.]