Action and Inhibition of Endogenous Phospholipases during Isolation of Plant Membranes

Abstract

Endogenous phospholipase D and phosphatidic acid phosphatase activities were demonstrated in membrane fractions isolated from soybean (Glycine max L.) hypocotyls. Phospholipase D activity was distributed widely among different membrane fractions while phosphatidic acid phos-phatase was found predominantly in membranes equilibrating in lower sucrose densities. Phospholipase D action was unaffected by ethylenedia-minetetraacetic acid, sodium salt or ethylene glycol-bis(fl-aminoethyl ether)-N,N'-tetraacetic acid but was prevented by a mixture of 4% choline and 4% ethanolamine. Phosphatidic acid phosphatase was inhibited by 10 millimolar glycerol 1-phosphate or by homogenization media prepared with coconut milk as a solvent instead of water. Under fuily protected conditions the phospholipid composition of soybean membrane fractions remained unchanged for at least 1 hour at 20 C. Membranes prepared under protected conditions had low phosphatidic acid contents and the phospho-ipid compositions closely resembled those of corresponding animal membranes. Quantitative comparisons of the phospholipid compositions of different membrane fractions have been complicated by poor recoveries of individual phospholipids and degradative alterations (22, 31).As a part of ongoing investigations of the in vitro interactions of plant membranes with plant growth hormones, we were prompted to undertake a detailed investigation of the stability of isolated plant membranes in cell homogenates. We found that phospholipids of membranes from soybean hypocotyls under investigation were surprisingly susceptible to degradation. Both phospholipase D and phosphatidic acid phosphatase appeared to contribute. Subsequently, a procedure was developed to inhibit phospholipid degradation by these enzymes. The procedure kept membranes stable even at room temperature so that physiological effects of hormones could be studied with less drastically altered membrane preparations. The phenomenon of rapid phospholipid degradation we describe here is probably common for isolated plant membranes with some clear exceptions (9, 10). Fast growing plant tissues generally have high phospholipase D activities (33). Thus the procedures described to protect plant membranes against phos-pholipid degradation should be of general interest. MATERIALS AND METHODS Plant Material. Fungicide-treated (Orthocide 75) soybean seeds (Glycine max L. cv. Wayne) were soaked in water overnight and planted in moist Vermiculite. After 4 days growing in the dark, 1 Recipient of an award from the Max Kade Foundation. the hypocotyls (about 1 cm long) were used for membrane isola-tions. Membrane Isolations. The isolation medium contained 0.1 M K-phosphate (pH 6.5), 20 mM EDTA2 or EGTA, and 0.5 M sucrose in freshly prepared and filtered coconut milk. Hypocotyls were homogenized for 45 sec with a Polytron 20 ST (Kinematica, Lucerne, Switzerland) operating at about 5,000 rpm. Membranes from about 80-g bean hypocotyls were isolated as described (40). Four membrane fractions were collected finally from a discontin-uous coconut milk-sucrose gradient and designated A, B, C, and D. (40). For protection against lipid degradation 4% choline (w/v), 4% ethanolamine (v/v), and 10 mm glycerol-1-P were added to all solutions. Assays. Membrane pellets of the individual gradient fractions were resuspended with an all-glass homogenizer of the Potter-Elvehjem type in either 6 ml (experiment in Fig. 1) or 18 ml (all other experiments) ofassay buffer. The standard incubation buffer was 50 mm Na-acetate (pH 5.5) with or without 4% choline (w/v) and ethanolamine (v/v). The final assay volume for a single determination was 1 ml or 3 ml, respectively. Glycerol-l-P and EDTA or EGTA was added to an equal volume of resuspended membranes in the same buffer as used for incubation so that the final concentration was 10 mm for each compound. In some earlier experiments glycerol-1-P was replaced by 10 mM ATP. In assays containing coconut milk, the coconut milk was buffered and added to an equal volume of resuspended membranes. The incubation was started by transferring the membrane suspensions from an ice-water bath to a 20 C water bath. The assays were stopped by addition of I ml of chloroform to each tube with rapid mixing. Then 2 ml of chloroform-methanol (1:1, v/v) were added for the first extraction step. The phosphatidic acid phosphatase assay was as described (26) but in 50 mm Na-acetate (pH 5.5) at 20 C for 2 hr. A 50-ml volume of membrane suspension was added to a final reaction volume of 250 ml. The substrate concentration was 2 mm. Protein was estimated by the Lowry et al. procedure (25). Lipids. Lipids were first extracted with an equal volume of chloroform-methanol (2:1). Then 0.2 ml 2 M KCI was added for each 3-ml original water phase and the mixture was extracted twice with two-thirds of the initial volume chloroform-methanol (2:1, v/v). The procedure was convenient in that large volumes of solvent were avoided and could be carried out in a test tube by taking up the lower phase with long tip Pasteur pipette. The recovery for phospholipids was greater than 95% and 70 to 75% for lysophospholipids. Lipids were dried in vacuo and redissolved 2Abbreviations: PA: phosphatidic acid; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; PI: phosphatidyl-inositol; LPC: lysophosphatidylcholine; LPE: lysophosphatidylethanola-mine; EDTA: ethylenediaminetetraacetic acid, sodium salt; EGTA: eth-ylene glycol-bis(J?-aminoethyl ether)-N,N'-tetraacetic acid; acetate: 50 mM sodium acetate (pH 5.5); t: time. 933 www.plantphysiol.org on February 25, 2019-Published by Downloaded from