Characteristics and subcellular localization of phospholipase d and phosphatidic Acid phosphatase in mung bean cotyledons.
Mixed micelles of (32)P-labeled phosphatidylcholine or phosphatidic acid (PA) and the nonionic detergent octylphenol polyethylene oxide (NP-40 Nonidet) were used to assay the activities of phospholipase D and PA phosphatase in crude extracts of mung bean (Vigna radiata) cotyledons. Together these enzymes degrade phosphatidylcholine to free choline, inorganic phosphate, and sn-1,2-diacylglycerol. Both enzymes have pH optima around 5.0. The enzymes are present in fully imbibed cotyledons and increase in activity during seedling growth. Fractionation of cotyledon extracts on sucrose gradients showed that the cells contain two PA phosphatases. One enzyme with a pH optimum of 7.5 has the same distribution on sucrose gradient as the endoplasmic reticulum marker enzyme NADH-cytochrome c reductase. The other, PA phosphatase, with a pH optimum of 5.0, was present in a protein body-rich fraction and in the load portion of the gradient. Fractionation of broken protoplasts on Ficoll gradients (a method which allows for the isolation of a high proportion of intact protein bodies) indicates that most of the cellular phospholipase D and PA phosphatase (pH 5.0) are associated with the protein bodies. Using column chromatography (DEAE-cellulose and Sephadex G-200), PA phosphatase (pH 5.0) was found to be a different enzyme from the major acid phosphatase in the cotyledons. Apparent molecular weights of phospholipase D and PA phosphatase were 150,000 and 37,000, respectively. The activity of phospholipase D was not affected by free choline, but was markedly inhibited by the choline analog and plant growth retardant isopropyl 4'-(trimethylammonium chloride-5'-methylphenyl piperidine-1-carboxylate (AMO 1618). The finding that these acid hydrolases are located in the protein bodies supports the conclusion that protein bodies form the general lytic compartment in the storage parenchyma cells.
Herman
E M
EM
Department of Biology C-016, University of California, San Diego, La Jolla, California 92093.
Chrispeels
M J
MJ