Microsomal Phosphatidate Phosphatase in Maturing Safflower Seeds

Abstract

An assay system comprising sodium phosphatidate, phosphatidylcholine, and bovine serum albumin has been developed for the reproducible determination of phosphatidate phosphatase activity in maturing seeds of safflower (Carthamus tinctorius L.). The activity was detected in both membrane and soluble fractions, and the microsomal phosphatidate phosphatase was characterized. The optimum pH for Pi release was 6.7, and the activity depended on the concentration of Mg2 . Phosphatidylcholine and bovine serum albumin stimulated the phosphatase reaction. This phosphatase was highly specific for phosphatidate; lysophosphatidate, and water-soluble phosphate esters did not serve as substrate. The specific activity was approximately 20 nanomoles per minute per milligram of protein, which was close to that of glycerol-phosphate acyltransferase and higher than that of diacylglycerol acyltransferase. Furthermore, the activity per seed was enough to account for the rate of triacylglycerol accumulation in vivo. The step of diacylglycerol formation by phosphatidate phosphatase does not appear to be rate-limiting for triacylglycerol synthesis during seed maturation.Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) is a key enzyme of glycerolipid biosynthesis, and catalyzes the hydrolysis of phosphatidate to 1,2-diacylglycerol and Pi. The diacylglycerol product is not only the direct precursor of triacylglycerol but also a substrate for the synthesis of membrane glycerolipids. Although this enzyme was first demonstrated in plants by Kates (19) In mammalian tissues, the phosphatidate phosphatase reaction is considered to be the rate-limiting step of triacylglycerol synthesis (2). We will discuss whether this is also the case for maturing safflower seeds.To assay the activity of phosphatidate phosphatase with good reproducibility, we have developed an effective phosphatidate substrate, the preparative procedure for which is also described. MATERIALS AND METHODS Plant Material and Preparation of MicrosomesMaturing seeds of safflower (Carthamus tinctorius L.) were harvested 14 to 15 d after flowering when '4C-labeled acetate was most rapidly incorporated into diacylglycerol (15), and cotyledons were homogenized in 10 mm Tris-HCl (pH 7.0) containing 0.4 M sorbitol. The homogenate was centrifuged at 5000g for 10 min, and then the supernatant obtained was centrifuged at 100,000g for 1 h. The precipitate was suspended in the same buffer and stored at -20°C. Protein was determined by the method of Lowry et al. (21)

Identification and characterization of nonsedimentable lipid-protein microvesicles.

“…Phospholipase D activity has been detected in isolated fractions of endoplasmic reticulum, Golgi membranes, tonoplast, and plasmalemma (9)(10)(11)(12), and it has been proposed that the membrane-associated form ofthe enzyme participates in lipid turnover (13). Phosphatidic acid phosphatase has been found on endoplasmic reticulum and in association with other isolated membrane fractions (14)(15)(16), and there are reports of lipolytic acyl hydrolase being associated with microsomal membranes (17,18). Moreover, a recent study has indicated that phospholipase D, phosphatidic acid phosphatase, and lipolytic acyl hydrolase are all enriched in the sedimentable material obtained after partial solubilization ofmicrosomes in Triton X-100, suggesting that they are tightly associated with the membranes (19).…”

The action of phosphatidate phosphatase on the fatty-acid composition of safflower triacylglycerol and spinach glycerolipids

“…Phosphatidylcholine (1-palmitoyl-2-oleoyl) was synthesized from oleic anhydride and 1-palmitoyl-sn-glycero-3-phosphocholine which had been enzymically prepared from phosphatidylcholine (dipalmitoyl) by the action of phospholipase Az (Ichihara et al 1987). Phosphatidates corresponding to these phosphatidylcholines were prepared by the pro-cedure described in Ichihara et al (1989). Sodium phosphatidates (dilauroyl and dimyristoyl) were obtained from Sigma Chemical Co. (St. Louis, Mo., USA).…”

“…Safflower (Carthamus tinctorius L.) plants were grown in the University field. Maturing seeds were harvested in the middle stage of development (14-15 d after flowering) when triacylglycerol is rapidly synthesized and accumulated (Ichihara and Noda 1980), and microsomes were prepared by differential centrifugation (Ichihara et al 1989). Spinach (Spinacia oleracea L.) leaves, obtained from a local market, were homogenized in 10 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris)-HC1 (pH 7.8) containing 0.4 M sorbitol and 0.2 mM disodium ethytenediaminetetraacetate (EDTA-Na2) in a Waring blender.…”

“…Since this enzyme produces 1,2-diacylglycerol, the immediate precursor of triacylglycerol, by hydrolyzing phosphatidate, its substrate specificity and selectivity for phosphatidates differing in the acyl moiety must be taken into account as to the regulation of FA composition at positions sn-1 and 2 of triacylglycerol. A recent study (Ichihara et al 1989) has indicated that maturing safflower seeds have a membrane-bound, Mg 2 +-dependent, and a soluble, Mg 2 +-dependent phosphatidate phosphatase. In the present paper, I report the substrate specificity and selectivity of the microsomal enzyme which is responsible for triacylglycerol formation in the endoplasmic-reticulum membrane (Styrene and Stobart 1987).…”

Identification, Purification, and Characterization of Monoacylglycerol Acyltransferase from Developing Peanut Cotyledons

“…Following lipid extraction by the modified method of Bligh and Dyer (23), the lower chloroform-soluble materials were separated by TLC on 250 m silica gel G plates either using petroleum ether/diethyl ether/acetic acid (70:30:1, v/v) or chloroform/ methanol/water (98:2:0.5, v/v) as the solvent system (24). The lipids were visualized with iodine vapor, and the spots of DAG scraped off for determination of radioactivity by liquid scintillation counting.…”