Phosphatidylglycerol Synthesis in Castor Bean Endosperm KINETICS

The synthesis of phosphatidylglycerol in castor bean (Ricinus communis var. Hale) endosperm tissue was found to be located in both the endoplasmic reticulum and mitochondrial fractions separated on sucrose density gradients. The enzyme of both fractions attained maximum activity at 5 mM Mn'+, 0.075% Triton X-100, and pH 7.3. The addition of dithiothreitol produced little effect, but sulfhydryl inhibitors reduced activity in both systems. Cytidine diphosphate-diglyceride exhibited an apparent Michaelis constant for the endoplasmic reticulum enzyme of 2.8 AM and for the mitochondrial enzyme of 2.0 ,M; the maximum reaction rate was achieved at about 20 ,uM. For the second substrate, glycerol-phosphate, the apparent Michaelis constant for both fractions was about 50 gM and maximum velocity was reached at 400 juM. The specific activity of the mitochondrial enzyme was generally twice that of the endoplasmic reticulum. The purification of a membrane fraction derived from the endoplasmic reticulum of castor bean endosperm was recently described (15). Several enzymes involved in phospholipid synthesis (phosphatidic acid phosphatase and enzymes synthesizing phosphatidylcholine, phosphatidylserine, and phosphatidylinositol) were present in this fraction and absent from all other organelles recovered in sucrose gradients (14, 15, 20). This is convincing evidence that in this tissue, as proposed on different grounds for several others (8, 9, 12, 18, 19), the endoplasmic reticulum plays a vital role in the synthesis of phospholipids for organelle membranes. It also provides a biochemical basis for the finding of Kagawa et al. (11) that, following a pulse with "C-choline, radioactive lecithin first appeared in the endoplasmic reticulum of the endosperm and then passed into the membrane of the mitochondria and glyoxysomes. In mammalian systems, the synthesis of phosphatidylglycerol occurs only in the mitochondria (8, 19), although the synthesis of other phospholipids is associated with the endoplasmic reticulum (5, 8, 19). Only a few investigations have been made on the intracellular location of phosphatidylglycerol synthesis in plants; activity was reported in a microsomal fraction from spinach leaves (17) and in mitochondria from cauliflower (7), but these were not shown to be the exclusive sites of synthesis. 'This work was supported by the Atomic Energy Commission Contract AT-104/3. 2 Present address: Department of Botany, University of Wyoming, Laramie, Wyo. 82071 This paper reports an investigation of phosphatidylglycerol synthesis in the organelles separated from whole homogenates of castor bean endosperm. MATERIALS AND METHODS Growth Conditions. Ricinus communis var. Hale seeds were grown for 4 to 4.5 days as previously described (15). Homogenization. Endosperm tissue was chopped with a razor blade (15), and 5 ml of a 270g supernatant were layered directly onto the surface of a sucrose gradient. Organelie Isolation. Separation of organelles was achieved by centrifuging the sample at 20,000 rpm for 4 hr in a SW-27 rotor in a Beckman L2-65B preparative ultracentrifuge. The gradients consisted of: (a) 6 ml of 60%, (w/w) sucrose cushion; (b) 20 ml of a 32 to 60% (w/w) linear sucrose gradient; (c) 5 ml of 20% sucrose; and (d) 5 ml of sample in a 37.5 ml centrifuge tube. All sucrose solutions were 1 mm with respect to EDTA (pH 7.5). Enzyme Assays. Assays were as previously described for catalase (16), fumarase (21), NADH-cytochrome c reductase (22), triose-phosphate isomerase (2), and lecithin synthesis (15). Glycerol-P incorporation into chloroform-soluble products was based on the assay described by Marshall and Kates (17). The reaction mixture contained in a final volume of 0.2 ml: 30 mm MgCl,, 0.15% (w/w) Triton X-100, 0.25 M tris-HCl (pH 7.3), and 0.1 mm CDP-diglyceride (Serdary Research Laboratories; London, Ontario, Canada). The reaction was initiated by the addition of 5 ul of U-'4C-sn-glycerol-P (0.25 ,uc; 16 nmoles). In some of the studies described below, 5 mM MnCl2 was used in place of MgCl, and 0.075% Triton X-100 instead of 0.15%. The reaction was stopped after 1.5 hr at 30 C by the addition of 2 ml of methanol, and the unreacted glycerol-P was removed as described previously for untreated water-soluble substrates in lecithin synthesis (15). Chromatography. Identification of the chloroform products of the reaction was accomplished by TLC (1, 10) on Eastman silica gel sheets. The systems used were: (a) silica gel impregnated with 0.4 M boric acid by floating a thin layer sheet face down on a solution of the acid for 1 hr. The solvent system consisted of chloroform-methanol-water-concentrated ammonium hydroxide (70:30:3:2, v/v); (b) 1 mm Na,CO, impregnated plates; chloroform-methanol-acetic acid-water (50:25: 7:3, v/v); (c) untreated silica gel sheets developed in two dimensions (1) with chloroform-methanol-7 N ammonium hydroxide (65:30:4, v/ v) followed by chloroform-methanolacetic acid-water (170: 25:25:4, v/v).