Biosynthesis of Cytidine Diphosphate Diglyceride by Cauliflower Mitochondria

Abstract

The presenice of plhosphatidyl inlositol and phosphatidyl glycerol in plant tissue is well authenticated. but the pathways oif biosyntlesis are unknown. Cytidine diphosphate diglyceride has been established as an internmediate in the biosynthesis of phosphatidyl inositol in chicken liver (4) and in the biosynthesis of phosplhatidyl glycerol also in chicken liver (3). The enzymic formation of cytidine diphosphate diglyceride from CTP and phosphatidic acid in guinea pig liver (2) and embryonic chiick brain (5) has been described in detail. From the point of view of comparative biochemistry we have been interested to see if comparable reactions take place in plant tissues. Cauliflower (Brassica cauliflora) was purchased at local markets. The cauliflower inflorescence was cut up and 100 g homogenized in a mortar and pestle with 100 ml of a solution 0.5 M with respect to sucrose and 0.01 M with respect to tris-HOCl, pH 7.5. The homogenate was pressed through cheesecloth. Sixty ml of the homnogenate was centrifuged at 800 X g for 10 minutes and the pellet discarded. The supernatant was centrifuged at 18,000 X g for 30 minutes. The supernatant was decanted and the pellet resuspended in 10 nil of the homogenizing mediumii. All preparative procedures were at 00. In the formation of cytidinie conitaininig lipids the reaction mixture contained 1.30 nil of the enzyme source (homogenate, mitochondria, or supernatant) in the sucrose-tris mediumi. 30 unioles MgCL2, and 0.306 m,umole CTP-14C (222,000 dpm), in a final reaction volume of 1.52 ml. Reactions were stopped by the addition of 4.5 ml of methanol/chloroform (2/1) which was 0.1 N with respect to HCl. Chloroform and aqueous layers were separated essentially by the method of Bligh and Dyer (1). The residue was treated a second time by the Bligh and Dyer procedure. Chloroform extracts and washings were combined as were the aqueous fractions. An aliquot from the chloroform layer was dried in a scintillation counter vial, the scintillation fluid added and the sample counted. The aqueous fractions from the reaction mixtures as described above were chromatographed on paper uising propanol/water/conc. NH40Il (66/33/1) adjusted to pH 3.7 with HCI as solvelnt. This solvenit gave good separation of CMP and CTP wlhiclh were visualized by spraying with a molybdate reagent consisting of 5.0 ml 30 % perchloric acid, 10 ml 1 N HICl, 25 ml 4 % ammoniuiiimolybdate and 60 ml water. After spraying, the paper was dried in a hot air stream and the spots then visualized by ultraviolet irradiation. Wlheni radioactive samples were clironiatograplied strips of the filter paper were cut anid counted in the scintillationi counter. Figure 1 .shows the incorporation of radioactivity into lipid as a function of time in the 3 fractions of cauliflower. The supernatant is equivalent to the homogenate since there was little volume change after centrifuging down the mitochondria. The mitochondrial fraction was resuspended in 10 ml of the homogenizing medium and so it is 6 times concentrated witlh respect to the original homogenate. Even when this concentration factor is taken into accounit, it is apparent that the ability to utilize CTP in the formation of lipid soluble compound is mainly located in the mitochondria. The expected product of the reaction is cytidine disphosphate diglyceride: phosl)iatidic acid + CTP -CDP-diglvceriide + PI'P.