Plant Physiol. (1974) 53, 699-704 The Role of Galactolipids in Spinach Chloroplast Lamellar Membranes I. PARTIAL PURIFICATION OF A BEAN LEAF GALACTOLIPID LIPASE AND ITS ACTION ON SUB- CHLOROPLAST PARTICLES" 2 Received for publication October 30, 1973 and in revised form January 3, 1974 MARK M. ANDERSON,' RICHARD E. MCCARTY, AND ELIZABETH A. ZIMMER Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14850 ABSTRACT sis, such as fulfilling a structural requirement or even partici- pating directly in a reaction essential to photosynthesis. In an A galactolipid lipase has been isolated and partially purified effort to elucidate the possible role(s) of galactosyl diglycerides from the chloroplast fraction of the primary leaves of Phase- in photosynthesis, we isolated and partially purified a galacto- olus vulgaris var. Kentucky Wonder. The lipase hydrolyzed lipid lipase (galatosyl diglyceride acyl hydrolase) from primary monogalactosyl diglyceride rapidly and phosphatidyl choline bean leaf chloroplasts. The lipase was then used to deacylate relatively slowly. Triolein and p-nitrophenyl stearate were not the lipids in spinach chloroplasts and subchloroplast particles hydrolyzed. so that their ultrastructure and biochemical capabilities could Spinach subchloroplast particles were excellent substrates be determined as a function of galactolipid content. for the lipase. Initial rates of fatty acid release from sub- Galactolipase activity was first detected in extracts of Pha- chloroplast particles at 30 C by the lipase as high as 60 micro- seolus multifloris primary leaves by Sastry and Kates (19). equivalents per minute per milligram protein were observed. More recently Helmsing (9) has reported the purification of a At completion of the reaction, about 2.7 microequivalents of galactolipase from bean leaf homogenates. In this communica- fatty acid were liberated per milligram of chlorophyll in the tion, we report the isolation and partial purification of a ga- subehloroplast particles, indicating that major amounts of lipid lactolipase from the chloroplast fraction of the primary leaves in the particles were rapidly attacked by the lipase. of Phaseolus vulgaris var. Kentucky Wonder. The properties of The treatment of subehloroplast particles with the lipase re- this enzyme differ somewhat from those of previously reported sulted in a rapid inhibition of light-dependent electron flow. galactolipases (9, 19). Furthermore, we found that spinach sub- This inhibition was largely prevented when the incubation was chloroplast particles which have the same membrane polarity carried out in the presence of high concentrations of defatted as chloroplasts (13) were excellent substrates for the galacto- bovine serum albumin. These results suggest that when precau- lipase, suggesting that the acyl ester linkages of the galacto- tions are taken to prevent the binding of fatty acids to the sub- lipids in chloroplast membranes are exposed to the external chloroplast particles, large amounts of lipid may be removed medium. We have also characterized some of the effects of without a marked effect on electron flow. galactolipase treatment on light-dependent electron flow in subchloroplast particles. MATERIALS AND METHODS Galactolipase Purification. Pole bean seeds (Phaseolus vul- garis var. Kentucky Wonder) were soaked in tap water for 24 Galactolipids (mono- and digalactosyl diglycerides) com- hr and were germinated in moist vermiculite at room tempera- prise about 80% of the nonpigmented lipids in the chloroplasts ture. Following germination, the seedlings were illuminated of higher plants and algae (23). The reason for this preponder- for 16 hr per day with fluorescent light (intensity, about 1.2 X ance of galactosyl diglycerides, which is not found in most 10' ergs/ cm2' sec). After 15 to 20 days, the primary leaves were other biological membranes, has been largely unexplained. It harvested and stored at 4 C in the dark for 24 to 48 hr to re- is possible that these lipids have a unique role in photosynthe- move starch. About 150 g of leaves were homogenized in a Waring Blendor for 30 sec with 800 ml of cold 0.4 M sucrose which also contained 0.1 M potassium phosphate buffer (pH I This work was supported by National Institutes of Health Pre- 6.0). The homogenate was filtered first through three and then doctoral Training Grant ST GM 00824-10 to M. M. A., by Re- eight layers of cheesecloth. The filtrate was centrifuged at search Grant GB-30597X from the National Science Foundation, 3300g for 15 min at 4 C, and the resulting pellet, which was and by Research Career Development Award GM-14,877 to R. rich in was a E. M. chloroplasts, resuspended in small volume of su- 2This material is from a dissertation submitted by M. M. A. to crose-Pi buffer. The Chl concentration in the suspension was the Graduate School of Cornell University in partial fulfillment of adjusted to 3 mg per ml with sucrose-Pi buffer. the requirements of the degree of doctor of philosophy. The chloroplast suspension was added dropwise to 17 vol- 'Present Address: Department of Bio-organic Chemistry, Re- umes of rapidly stirred acetone at -17 C (21). The tempera- search Laboratories, Albert Einstein Medical Center, York and ture of the acetone during addition was kept below -10 C. Tabor Roads, Philadelphia, Pennsylvania 19141. After the addition was completed, the stirring was stopped, and 699 700 ANDERSON, McCARTY, AND ZIMMER Plant Physiol. Vol. 53, 1974 the precipitated chloroplast residue was allowed to settle for chloroplast particles were prepared by exposure of chloroplasts 2 to 3 min. The acetone was decanted and the gummy green to sonic oscillation (13). Bovine serum albumin was defatted precipitate was transferred to a glass plate at room tempera- either by the procedure of Chen (5) or by two extractions of ture. The precipitate was kneaded with a spatula to remove as the dry protein with 25 ml of 95% ethanol per g, followed by much acetone as possible and was then allowed to stand until dialysis of solutions of the extracted bovine serum against 5 the odor of acetone could barely be detected (about 3 hr). The mM Tricine-NaOH (pH 8.0) at 4 C for 12 to 18 hr. Bovine residue was gently resuspended in 100 ml of 50 mm potassium serum albumin concentrations were determined spectrophoto- phosphate buffer (pH 7.0) which also contained 1 mm EDTA metrically (5). Analytical polyacrylamide gel electrophoresis per 250 g of leaves used. After 20 min at room temperature, was performed according to Davis (7) except that proteins the suspension was centrifuged at 4 C for 10 min at 12,000g. were stained with Coomassie blue (6). Chlorophyll (3) and The supernatant solution was stored at 4 C. The pellet was ex- protein (22) were estimated by reported procedures. The light- tracted twice more as described above, first with one-half, and dependent reduction of methyl viologen and K3Fe(CN)0 by then with one-fourth the volume of P-EDTA' used in the first chloroplasts was followed by determining oxygen concentra- extraction. The combined supernatant solutions (crude ex- tions with a Clark electrode. tract) were assayed for lipase activity as soon as possible since Materials. Kentucky Wonder bean seeds were purchased sometimes much of the activity was lost in 2 or 3 days. from Agway, Inc., Syracuse, N.Y. Reagents and their vendors The crude extract was placed in a water bath at 68 C and include: rhodamine 6G, Allied Chemical; egg lysolecithin, was brought to 65 C with continuous stirring. After 2 min P-L Biochemicals; phosphatidyl choline and N-(morpholino) at 65 C, the beaker was plunged into an ice bath and was ethane sulfonate, General Biochemicals. stirred until the temperature reached 4 C. The precipitate was removed by centrifugation at 4 C for 10 min at 10,000g. The RESULTS supernatant solution (heated extract) was stored at 4 C. Galactolipase Purification. As may be seen in Table I, the Solid (NH,)2S04 was slowly added with continuous stirring to the heated extract until 65% of saturation at 4 C was ob- purification procedure resulted in about an 80-fold increase of hydrolysis with a recovery of tained. The mixture was stirred at 4 C for 15 min and was then in the specific activity MGDG activity. Very similar results were ob- centrifuged at 12,000g for 10 min at 4 C. The pellet was re- about 80% of enzyme tained when subchloroplast particles, rather than partially suspended in a small volume of P-EDTA buffer and insoluble MGDG, were used as the substrate. material was removed by centrifugation at 3,000g for 10 min purified at 4 C. The supernatant solution (ammonium sulfate fraction) The increase in total enzyme units which usually occurred the crude extract was heated to 65 C (Table I) is of in- was stored at 4 C. when since it suggests the presence of a heat-labile inhibitor Prior to chromatography on Sephadex G-100, the ammo- terest nium sulfate was concentrated to 5 to 10 ml in an ul- of the lipase. Furthermore, incubation of the crude extract with fraction (Table trafiltration apparatus with an Amincon UM-10 membrane trypsin caused a 70% activation of the lipase activity of under positive nitrogen pressure. The Sephadex G-100 column II). Trypsin caused an inhibition rather than an activation, more purified fractions. Since the lipase is had a bed volume of 100 ml/40 mg of protein and was equili- lipase activity in brated with P-EDTA at room temperature. The sample was slightly sensitive to trypsin, it is probable that the actual stimu- eluted from the column in 2-ml fractions. Fractions with the lation of lipase activity in the crude extract is somewhat highest lipase specific activity were combined and stored at greater than 70%.