Plant Physiol. (1974) 53, 699-704

The Role of Galactolipids in Spinach Chloroplast Lamellar Membranes

I. PARTIAL PURIFICATION OF A BEAN LEAF GALACTOLIPID 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 ) 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 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 . 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%. Attempts to isolate the heat and trypsin- -20 C. sensitive component in the crude extract, which inhibits lipase Galactolipid Preparation. Chloroplasts, prepared according activity, were unsuccessful. However, it seems clear that an to McCarty and Racker (15) from 500 g of spinach, were inhibitor is present and its removal during the purification of the suspended in cold, deionized water at a Chl concentration of 2 procedure may account for at least part of the increase to 3 mg per ml. Lipids were extracted as described by Zill and specific activity of the lipase. to Harmon (25) and were chromatographed on silicic acid The Sephadex G-100 fraction was found be polydisperse columns (19). Further purification of the galactolipids was by analysis with analytical polyacrylamide gel electrophoresis. achieved by chromatography of the lipids, dissolved in chloro- One major band (relative mobility, 0.4), three minor bands were detected. form-methanol (1:1, v/v), on a 2 X 28 cm column of Sephadex and several very faint bands of poor mobility LH-20 equilibrated with the same solvent. Fractions from this column were assayed for acyl ester content (20). Thin layer Table I. Partial Purification of Galactolipid Lipase chromatography after Nichols (17) was used to ascertain the The crude extract was prepared from the chloroplast fraction purity of the lipids. MGDG was generally contaminated with (0.7 g of Chl) isolated from 475 g primary leaves. Activity was some yellow-brown pigments, but was free of other acyl lipids. determined with MGDG as the substrate in a reaction mixture Assays and Preparations. Galactolipase activity was assayed which contained in 0.25 ml, 0.25 ,Amole MGDG (based on its acyl by following the release of fatty acids from a variety of sub- ester content), 0.4% Triton X-100, and lipase fractions equivalent strates including subchloroplast particles, MGDG, DGDG, to between 1 and 20 jug of protein. After 20 min at 30 C, the reac- and certain phospholipids, as described previously (2). When tion was stopped by the addition of 1 ml of 0.1 N HCI in 95% etha- purified lipids were used as substrates, 0.4% Triton X-100 was nol. usually added to disperse the lipids. One unit of galactolipase is defined as that amount of enzyme which catalyzes the Fraction ProteinTotal TotalUnits ActivitySpecific Recoveryof Units formation of 1 tcmole of fatty acid per min at 30 C. Sub- units/mg mg protein % 4Abbreviations: P-EDTA: a solution containing 50 mm potas- Crude extract 941 40.8 0.04 - sium phosphate buffer (pH 7.0) and 1 mM EDTA; MGDG: mono- Heated extract 751 50.2 0.07 123 galactosyl diglyceride or 1,2-diacyl[,8-D-galactopyranosyl(1'-*3]-sm- Ammonium sulfate 83.6 31.9 0.38 78 glycerol; DGDG: digalactosyl diglyceride or 1, 2-diacyl [o-D-galacto- Sephadex G-100 eluate 10.1 34.7 3.44 85 pyranosyl-(1'~6')-p-D-galactopyranosyl(1'-* 3)] -sm-glycerol. Plant Physiol. Vol. 53, 1974 PARTIAL PURIFICATION OF GALACTOLIPASE 701

Table 11. Effects of Trypsini onl Lipase Activity plast particles is arranged so that its acyl linkages can be at- Lipase fractions (51-53 ,ug of protein) were incubated at room tacked by the lipase. temperature in 1.0 ml of a mixture which contained 50 mm potas- It was puzzling that lipids in subchloroplast particles were sium phosphate buffer (pH 7.0), 2 mm EDTA, and 30,g of trypsin. hydrolyzed at much faster rates than the purified substrate- After 20 min, 100,ug of trypsin inhibitor were added. Trypsin in- MGDG. However, this can be explained in part by the fact hibitor was added to the controls prior to the trypsin. Aliquots of that Triton X-100, which was used to disperse the MGDG in the incubation mixtures were assayed for lipase activity with sub- the assay mixture, inhibits the lipase. Triton X-100 was nec- chloroplast particles as substrate (50 ,Ag of Chl per 0.25 ml of 20 essary to obtain reasonable rates of hydrolysis of MGDG by mm potassium phosphate buffer [pH 7.0]), incubated for 10 min the lipase (Fig. 3), with optimal activity at 0.4% Triton X-100. at 30 C. Higher concentrations gave less enhancement. At 0.4% Triton X-100, all of the MGDG in the reaction mixture was dispersed. Specific Activity In contrast, all concentrations of Triton X-100 tested inhibited Fraction Control plus Trypsin jAeq fatty acid/min-mg protein Crude extract 0.45 0.78 Ammonium sulfate 5.8 3.3 Sephadex G-100 eluate 7.6 5.4

The lipase activity was associated with the major band. Unfixed gels were sliced into 4-mm sections just after electrophoresis was completed, and the sections were homogenized in 1 ml of 20 mm potassium phosphate buffer (pH 7.0) which also con- tained 0.5 mg of bovine serum albumin. After removal of the gel pieces by centrifugation, the supernatant solutions were assayed for galactolipase activity. Marked lipase activity could be detected in only one region of the gel and this region cor- responded reasonably well with the major stained band. Attempts to purify the enzyme further, including preparative polyacrylamide gel electrophoresis, DEAE-cellulose and CM- cellulose ion exchange chromatography and acetone fraction- ation resulted in large losses in activity and little apparent 10 15 20 purification. It is quite possible that highly purified lipase TIME IN MINUTES preparations are unstable. Although the Sephadex G-100 FIG. 1. Time course of galactolipase-catalyzed hydrolysis of lipids fraction is polydisperse, it is significant that the specific activity in subchloroplast particles. The reaction mixture (0.25 ml) con- of the lipase in this fraction is over 20-fold higher than that of tained: 5 ,moles of potassium phosphate buffer (pH 7.0), subchloro- the homogeneous galactolipase prepared by Helmsing (9). plast particles equivalent to 50 ,ug of Chl, and Sephadex G-100 Properties of the Galactolipase and Its Action on Subehloro- lipase fraction equivalent to 1 ug of protein. The assay temperature plast Particles. Since we wished to use the enzyme for a study was 30 C. of the function of galactolipids in chloroplast-catalyzed re- actions, it was desirable that the enzyme rapidly deacylate these lipids in chloroplasts and subchloroplast particles. Furthermore, the enzyme preparation should be relatively specific for galactolipids and should be free of contaminating 60[ whose actions might alter chloroplast activities. Figure 1 shows that subchloroplast particles are excellent substrates for the 3-- lipase. The reaction is not linear with time and, as seen in I-- 0 Figure 2, the initial rate of fatty acid release from subchloro- U plast particles by the lipase increased linearly with increasing concentration of subchloroplast particles. Amounts of sub- 30[ chloroplast particles equivalent to greater than 0.2 mg of 0LIU- Chl per reaction could not be used because Chl interferes with $A the colorimetric assay for fatty acid used in this study. Thus, although subchloroplast particles (or chloroplasts) can be utilized as convenient substrates for the lipase, the concentra- tion of substrate is limiting and maximal velocities are diffi-

cult to ascertain. It is quite clear, however, that the rate of fatty 0 100 200 acid release from subchloroplast particles by the lipase is ,iGRAMS CHLOROPHYLL very fast (as high as 60 4moles/min mg protein), much faster than that FIG. 2. Effect of concentration of subchloroplast particles on the observed with the purified substrates of the enzyme. rate of amounts of hydrolysis of the lipids in the particles. The reaction mixture Furthermore, it is apparent that major chloroplast was identical to that given in the legend to Figure 1, except that lipids are readily deacylated by the enzyme. For example, at 1.25 ,ug of the Sephadex G-100 fraction was used, and that the completion of the action of lipase with subchloroplast particles. amount of subchloroplast particles was varied. The incubation was about 2.6 ,imoles of fatty acid had been formed per mg of carried out at 30 C for 6 min. Specific activity is defined as Aeq Chl. It must be, therefore, that much of the lipid in subchloro- fatty acid formed/min mg protein. 702 ANDERSON, McCARTY, AND ZIMMER Plant Physiol. Vol. 53, 1974 phatidyl choline was a somewhat better substrate than di- palmityl phosphatidyl choline. In view of the nonhomogeneity of the Sephadex G-100 lipase fraction used in these studies, it 6 is possible that the phospholipid lipase activity is a contam- inant. However, it is clear that activity with purified lecithin as substrate and the deacylase activity with subchloroplast particles as substrate were purified to a similar extent and that the activities chromatographed similarly on Sephadex G-100 (Table IV). Furthermore, both phospholipase and galactolipase activities were present in the major band after I- polyacrylamide gel electrophoresis. 0 No protease activity, as assayed by the method of Nelson et al. (16), could be detected in the Sephadex G-100 fraction U even when large amounts (about 20 ,[g of protein) of the en- a zyme fraction and several hours of incubation at 30 C were used. Furthermore, a and /3-galactosidase activity could not be 2 detected using the appropriate p-nitrophenyl galactosides as substrates. Temperature is a very important factor controlling the hy- drolysis of lipids in subchloroplast particles (Table V). Above 15 C, the reaction shows a Q1o of about 2. However, below this temperature, the activity decreases much more dramatically with decreasing temperature. It may be that the dramatic ~0 0.4 0.8 Table III. Hydrolytic Activity of Galactolipase with Varioius Lipids % TRITON-X-100 The reaction mixtures are identical to that given for MGDG FIG. 3. Effect of Triton X-100 on MGDG hydrolysis. The reac- hydrolysis in Table I. Each lipid was present at 0.5 ,umole per 0.25 tion mixture was similar to that described in Table I. The Sephadex ml reaction mixture. All samples were incubated for 20 min at G-100 fraction (1 ug of protein) was used. The incubation was car- 25 C with the Sephadex G-100 fraction of the lipase equivalent to ried out at 30 C for 20 min. Specific activity is defined as ,ueq fatty acid released/min-mg of protein. 6.25 ,Ag of protein. Substrate Activ-ity 8 sAeqfatty acid released 1min -mg protein p-Nitrophenyl stearate 0 Triolein 0 I-- Plant phosphatidyl inositol 0 Egg lysophosphatidyl choline 1.6 Dipalmityl phosphatidyl choline 0.7 v 4 Monogalactosyl diglyceride 6.8 0Iu ta 2 Table IV. Purificationi of Phospholipase anid Hydrolytic Activity with Suibchloroplast Particles as Suibstrate Phospholipase and fatty acid release from subchloroplast par- ticles were assayed as described in Table I and the legend to Fig. 0 1, respectively. SCP, subchloroplast particles; PC, phosphatidyl choline. % TRITON-X-100 Lipase Activity FIG. 4. Effect of Triton X-100 on the hydrolysis of lipids in sub- Activity with chloroplast particles. The reaction mixture (0.25 ml) contained sub- Fraction Phos- Sub- SCP/Activity chloroplast particles (50 mg of Chl), 20 mm potassium phosphate phatidyl chloroplast buffer (pH 7.0), 1 ,ug of the Sephadex G-100 fraction of the lipase, choline particles and the various concentrations (expressed as volume %) of Triton peq fattN acid X-100. The incubation was carried out at 30 C for 30 min. formed/mining protein Crude 0.048 0.70 14.5 lipase activity with subchloroplast particles as substrates (Fig. (NH4)2SO4 0.082 1.5 18.3 4). Cholate gave similar results. Sephadex G-100 No. 101 0.33 4.7 14.2 The galactolipase does not appear to be a nonspecific acyl Sephadex G-100 No. I1 1.07 14.7 13.7 hydrolyase. p-Nitrophenyl stearate, triolein, and phosphatidyl Sephadex G-100 No. 12 0.68 10.5 15.4 inositol were not hydrolyzed by the enzyme (Table III). How- Sephadex G-l00 No. 13 0.31 5.9 15.1 ever, dipalmityl phosphatidyl choline was hydrolyzed by the Sephadex G-100 No. 14 0.11 1.5 13.6 enzyme at a rate about one-tenth of that of MGDG hydrolysis. Although the data are not shown, DGDG was also hydrolyzed Numbers refer to fraction numbers from the Sephadex G-ICO by the lipase, but at a slower rate than MGDG. Lysophos- column. Plant Physiol. Vol. 53, 1974 PARTIAL PURIFICATION OF GALACTOLIPASE 703

Table V. Effect of Temperature onz Lipase Activity with galactolipase was to use the enzyme to deplete chloroplasts of Subchlloroplast Particles as Substrate their galactolipids and study the effects of the removal of these The reaction mixture was the same as that described in the lipids on chloroplast structure and function. To avoid compli- legend to Figure 1. The incubation time was 6 min with the Sepha- cations which might be caused by membrane stacking in chloro- dex G-100 fraction of the lipase equivalent to 6,g of protein. plasts, subchloroplast particles were used. It is evident that the treatment of subchloroplast particles with the lipase elicited a Temperature Specific Activity marked inhibition of electron flow through either both photo- systems or photosystem I alone (Table VI). The inhibition of C lieqfatty acid released/min-ig protein electron flow by the lipase appeared to be an enzymatic process 0 0.5 since inactivation of the lipase by heat also destroyed the abilitv 4 1.7 of the preparation to inhibit electron flow. Fatty acids are potent 15 17.5 inhibitors of electron flow in chloroplast (12, 14), and the 25 31.8 amounts of fatty acid released by the lipase are more than 35 60.8 sufficient to cause the marked inhibition observed. If, however, the subchloroplast particles were incubated with the lipase in the presence of high concentrations of defatted bovine serum Table VI. Inzhibition of Electroni Flow in Subchloroplast albumin, there was little loss in electron transport activity. This Particles by Galactolipase Treatment protective effect of bovine serum albumin was not caused by In the experiment in which electron flow from water to ferri- an inhibition of the lipase-catalyzed release of fatty acids from cyanide was assayed, subchloroplast particles (1.5 mg of Chl) were the subchloroplast particles. Bovine serum albumin at 30 mg/ incubated at 21 C in a reaction mixture (3.0 ml) which contained ml had little effect on the rate and extent of fatty acid release 50 mm potassium phosphate buffer (pH 7.0) and Sephadex G-100 from subchloroplast particles. Therefore, the bovine serum lipase fraction (15 jig of protein) with and without 30 mg per ml probably protects against lipase inactivation of electron flow of defatted bovine serum albumin. Ferricyanide reduction was by binding the fatty acids released by the enzyme. assayed at 21 C by following light-dependent oxygen evolution Under the conditions used in the study of the effects of lipase with 0.2-ml aliquots of the incubation mixtures. The reaction mix- treatment of subchloroplast particles on electron transport, ture (1.75 ml) contained: 25 mm Tricine-NaOH (pH 8.0), 20 mm about 2.6 ,umoles of fatty acid were liberated per mg of Chl. NaCl, 5 mM MgCl2, 3 mm NH4Cl, and 1 mm K3Fe(CN)6. In the Galactosyl monoglycerides could not be detected in lipid ex- experiment in which the photosystem I-dependent oxidation of tracts from lipase-treated subchloroplast particles. This result, diaminodurene was measured, subchloroplast particles (1 mg of coupled with the finding that water soluble forms of galactose Chl) were incubated at 25 C in a mixture (1 ml) which contained (probably galactosyl glycerides), as assayed with galactose 0.4 M sucrose, 0.02 M Tricine-NaOH (pH 8), 0.01 M NaCl, Sephadex oxidase. were released from subchloroplast particles by lipase G-100 lipase fraction (4,ug of protein) with and without 35 mg per digestion. shows that the entire galactolipid molecule can be ml of defatted bovine serum albumin. Diaminodurene oxidation removed from subchloroplast particles by the lipase treatment. was assayed at 25 C in a reaction mixture (1.75 ml) which con- Preliminary analysis of the lipid content of lipase-treated tained: 25 mm N-(morpholino)ethane sulfonate-NaOH (pH 7.0), subchloronlast particles showed that MGDG and DGlDG are 20 mm NaCl, 5 mm sodium ascorbate, 1 mm diaminodurene, 0.07 attacked mM methyl viologen and 0.02 ml aliquots of the lipase incubation by the lipase to the greatest extent. Although a more mixtures. FeCy, ferricyanide; DAD, diaminodurene; MV, methyl Drecise and detailed analysis of the linid contents is required. it was found that at least half of the MGDG and DGDG could viologen. 'he removed by the lipase without strong effects on electron Electron Flow Rates flow.

H20 - FeCy DAD -+ MXV DISCUSSION Incubation with Lipase -Bovine of the serum +Bovine -Bovine +Bovine Properties Galactolipid Lipase. Although Helmsing albumin serum serum serum (9) chose to purify the galactolipid lipase present in particle- OR albumin albumin albumin +Bovine a free supernatants of extracts of Phaseolus multifloris primary leaves, most of the activity was associated with the chloro- mmin rntoles 02 evolved or consu,ned/I:r,ng Chl plast fraction (19). Since we observed a similar distribution of 0 73 119 2380 2040 galactolipase activity in extracts of Phaseolus vlulgaris primary 5 0 98 2138 2410 leaves and since we have had experience in solubilizing pro- 10 0 92 1610 2210 teins from chloroDlasts, we decided to purifv the enzyme pres- 15 0 88 1151 2375 ent in the chloroplast fraction. Although neither repeated wash- 20 0 71 576 2460 ing of the chloroplasts or their exposure to sonic oscillation 25 0 73 336 2240 released much of the activitv, the treatment of the chloroplasts 30 0 71 240 2360 with cold acetone was quite effective. This procedure also causes the solubilization of several chloroplast membrane Pro- teins including plastocvanin (1. 10), coupling factor 1 (21). decrease in activity of the lipase reflects a change in the state Pnd ferredoxin-NADP5 reductase. Although the lipase is of the lipids within the membrane. firmlv bound to chloronlasts, it is possible that it is a soluble The lipase activity of the Sephadex G-100 fraction is quite enzvme which associates with chloroplasts on lysis of the stable on storage in P-EDTA buffer at -20 C. Even after re- cells. peated freezing and thawing, one preparation lost less than Our lipase Preparation differs from Helmsine's (9). Although half of its activity in over a year's storage. our preparation hydrolvzed monogPlactosvl diolyceride more Effects of Lipase Treatment of Subchloroplast Particles on rapidly than Pelmsing's, at least part of this difference may be Electron Transport. The primary reason for purifying the ascribed to the fact that we used Triton-X-l00 to disperse the 704 ANDERSON, McCARTY, AND ZIMMER Plant Physiol. Vol. 53, 1974 substrate. Helmsing's enzyme could also have contained a photosyntlietic efficiency and imoriphology of chloroplasts. Plant Plhysiol. 41: tightly bound inhibitor similar to that which we suspect is 1591-1600. 5. CHEN, R. F. 1967. Removal of fatty acids fr oii serum alsuinin by char-coal present in our preparations. Furthermore, rough estimates of treatment. J. Biol. Chem. 242: 173-181. the mol wt of our lipase indicate that it is about one-half 6. CHRANIBACH, A., R. A. REISFEID, AM. WYCKOFF, AND J. ZACCARI. 1967. A that of Helmsing's. Cysteine, a potent inhibitor of Helmsing's procedure for rapid and sensitive staining of protein fractionated by poly- lipase activity (9) had no effect on the activity of our prepara- acrylamide gel electrolioresis. Aiial. Bioclhem. 20: 150-1.54. 7. DA.IS, B. J. 1964. Disc electrophoresis II. Ann. 'N.Y. Acad. Sci. 121: 404-427. tion. 8. GRESSEL, J. AND Ml. AvRON. 1965. The effects of structurial degradation on the Action of the Galactolipase on Subehloroplast Particles. The coupled pliotoclieiical activities of isolate(d chloroplasts. Biochim. Biophys. rapid attack of galactolipids in subchloroplast particles is con- Acta 94: 31-41. sistent with the orientation of these lipids in 9. HELMSING, P. J. 1969. Purification and properties of galactolipase. Bioclhinm. chloroplast mem- Biophys. Acta 178: 519-533. branes proposed by Weier and Benson (23), but is not con- 10. KATOH, S. 1971. Plastocyaniin. Mletisods Enzymol. 23: 408-413. sistent with other proposals. Kreutz (I 1) suggested that the lipids 11. KRECTZ, W. 1970. X-ray stuticture research on the photosynthetic membrane. in chloroplasts form a bilayer sandwiched between protein Advan. Bot. Res. 3: 53-169. layers. 12. KROGNIANN, D. W. AND A. T. JAGENDORF. 1959. Inhibition of the Hill reaction by fatty acids and nmetal chelating agents. Arch. Biochem. Biophys. 80: Effects of exogenous (4, 8, 18) and endogenous (24) 421-430. on chloroplast photochemical activities have been reported. 13. MICCARTY, R. E. 1968. Relation of photophosphorylation to hydrogen ion However, the interpretation of these experiments must be transport. Biochem. Biophys. Res. Commun. 32: 37-43. questioned, since no attempts were to circumvent 14. MCCARTY, R. E. AND A. T. JAGENDORF. 1965. Chloroplast damage due to made in- enzymatic hydrolysis of endlogenious lipids. Planit Plsysiol. 40: 725-735. hibition of the activities by the fatty acids released by lipase. 15. MICCARTY, R. E. AND E. RACKER. 1967. The inlibition and stimulation of Furthermore, Wintermans et al. (24) showed that prolonged photophosphorylation by N, N' -dicyclohexylearbodiimide. J. Biol. Chem. aging of chloroplasts can result in inhibition of electron flow 242: 3435-3439. through mechanisms which are not related to transforma- 16. NELSON, W. L., E. I. CIACCIO, AND G. P. HESS. 1961. A rapidl metliod for lipid the quantitative assay of proteolytic enzymes. Anal. Biochem. 2: 39-44. tions. However, these authors (24) did find that the photo- 17. NICHOLS, B. W. 1963. Separation of the lipids of photosynthetic tissues: system I-dependent reduction of NADP+ from reduced di- improvements in analysis by tlhin layer chromatography. Biochim. Biophys. chlorophenolindophenol was more resistant to aging under Acta 70: 417-422. conditions in which an endogenous the 18. OKAYAMIA, J., B. L. EPEL, K. ERIXON, R. LozIER, AND W. L. BCTLER. 1971. lipase catalyzed hy- The effects of lipase on spinach and Chla,nydomoinas chsloroplasts. 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