The Occurrence of Phenylalanine Ammonia-Lyase and Cinnamic Acid /^-Hydroxylase on the Endoplasmic Reticulum of Cell Suspension Cultures of Glycine max

Subfractionation of Microsomes, Cell Suspension Culture, PAL, Cinnamic Acid p-Hydroxylase 1. The time course of activity of soluble and microsomal phenylalanine ammonia-lyase (PAL) was studied in dark grown cell cultures of soybean (Glycine max). A distinct activity increase of PAL in the soluble and microsomal fraction occurred prior to the stationary phase of the cell culture. Cinnamic acid p-hydroxylase and NADH : cytochrome c reductase, too, exhibited maximal activity in the log phase, 5 days after the transfer of soybean cells to fresh culture medium. 2. Upon subfractionation of the once washed microsomal fraction by sedimentation velocity centrifugation on a sucrose gradient, membranes of the endoplasmic reticulum could be separated from fractions containing mainly membranes from the Golgi apparatus or plasma membranes, respectively. PAL and cinnamic acid p-hydroxylase were found in fractions of endoplasmic reti­ culum whereas no activity of either enzymes could be detected in fractions containing Golgi ap­ paratus or plasma membranes. 3. Repeated washing of microsomal fractions led to a residual membrane-bound PAL re­ presenting about 1% of the total PAL activity of the cells. This residual membrane-bound activity could be solubilized almost completely by Triton X-100 or digitonin at concentrations of 0.5 — 5%.