- Email: [email protected]
[21] Algal preparations with photophosphorylation activity
242
CELLULAR AND SUBCELLULAR PREPARATIONS
[21]
with Euglena chloroplasts isolated by the procedure of Eisenstadt and Brawerman 6 (Dr. J. A. Schiff, personal communication). However, high rates of incorporation (78 #moles HCO~-/mg chlorophyll/rag) have been reported with preparations from Acetabularia mediterrania2 °
[21] Algal Preparations with Photophosphorylation Activity By PETERB~ER Most of our knowledge of the photophosphorylation processes in vitro is based on preparations from higher plants and purple bacteria. Chloroplasts isolated from algae are not usually active. The first active preparations, however, were obtained with the (prokaryotic) blue-green alga Anabaena 1,2 after disruption of the cells by mechanical means. These particles catalyzed only cyclic photophosphorylation [e.g., with Nmethylphenazinium methyl sulfate (PMS)], although Hill reactions with artificial electron acceptors were consistently observed. A gentle rupture of the cell walls with enzymes was necessary to obtain particles active in both cyclic and noncyclic photophosphorylation2 ,4 There are very few reports concerning the preparation of chloroplasts with photophosphorylation activity from eukaryotic algae. Preparations from the chrysomonad H y m e n o m o n a s yielded about 70 t~moles of ATP per milligram of chlorophyll per hour with PMS. 5 Rather high rates with this cofactor (approximately 180 t~moles ATP/mg Chl X hour) have been reported using chloroplasts from the green alga Chlamydomonas reinhardi. 6 This author has prepared chloroplasts with good yield and high phosphorylation activity from the heterokont BumiUeriopsis fili]ormis. 7 Active chloroplast preparations from other algae groups, e.g., red algae or diatoms, have not yet been reported. In this article the methodology of photophosphorylation will be described for only those algae preparations which show reasonably high lB. Petrack and F. Lipmann, in "Light and Life" (W. D. McElroy and B. Glass, eds.). Johns Hopkins Press, Baltimore, Maryland, 1961. 2W. A. Susor, W. C. Duane, and D. W. Krogmann, Rec. Chem. Progr. 25, 197 (1964). 8B. Gerhardt and R. Santo, Z. Natur]orsch. B 21, 673 (1966). 4j. Biggins, Plant Physiol. 42, 1447 (1967). 5S. W. Jeffrey, J. Ulrich, and M. B. Allen, Biochim. Biophys. Acta 112, 35 (1966). ~A. Givaa and R. P. Levine, Plant Physiol. 42, 1264 (1967). P. BSger, Z. Pflanzenphysiol. 61, 85 (1969).
[21]
ALGAL CHLOROPLAST PHOSPHORYLATION
243
photophosphorylation rates due to both cyclic and noncyclic electron transport. Particles from Anacystis nidulans (Cyanophyceae, Blue-Green Algae) a,s Preparation. Strain No. 1402-1 from the Algae Culture Collection, University of GSttingen, Germany, is grown in the medium of Kratz and Myers 9 at 30 ° with 3000 lux (fluorescent light) and gassed with C02enriched air (1.5% v/v). The once washed algae are suspended in 5% sucrose containing 20 mM Tris buffer, pH 7.6, frozen at --50 ° to --60 ° and then lyophilized for 2-3 hours. The dry powder can be stored without loss over P205 up to 3 days at 5 °. For use, it is resuspended in 40 mM MgC12 and lysozyme (Calbiochem, B grade, Los Angeles, California)--2 mg of enzyme per 0.2 mg of chlorophyll--is added. After standing for 3-5 hours at 15 °, the particles are collected by centrifugation and resuspended in 5% sucrose--20 mM Tris, pH 7.6. They may be washed once with the same medium. Photophosphorylation proceeds best when the particles are kept at room temperature. No data concerning the efficiency of cell wall lysis with this method were given by the authors2 ,8 However, lyophilization is necessary for the enzymatic action. Photophosphorylation with the same reaction mixture as described below can also be performed with lyophilized cells without lysozyme treatment. The rates are at best half of those from preparations treated with lysozyme.1° Reaction Conditions. Photophosphorylation is carried out in Warburg vessels with white light of 35,000 lux. The standard reaction mixture contains in a final volume of 3 ml (in ~moles): Tris buffer, pH 7.6, 60; MgCl~, 40; ADP, 10; inorganic phosphate, 10 (with approximately 10~ cpm 3~p); particles with 0.2 mg chlorophyll. The concentrations of some cofactors are indicated in Table I. Reaction time is 20~30 minutes; temperature, 15 °3 (or 25°s). Gas phase is prepurified nitrogen. ATP is determined by the method of Sugino and Miyoshi.1~ For ferredoxincatalyzed cyclic phosphorylation N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (Hepes) buffer, pH 7, is used. 8 Rates. Photophosphorylation rates with particles from Anacystis as reported by the Trebst group are comparable to those from spinach as far as the cyclic system with PMS and ferredoxin is concerned (Table I, part A). The rather high endogenous reaction of unwashed particles can
8H. Bothe, Thesis, Univ. of G5ttingen, Germany, 1968; Z. NaturJorsch. B 24, 1574 (1969). 9W. A. Kratz and J. Myers, Amer. J. Bot. 42, 282 (1955). loB. Gerhardt and A. Trebst, Z. NaturJorsch. B 20, 879 (1965). 11y. Sugino and Y. Miyoshi,J. Biol. Chem. 239, 2360 (1964).
244
CELLULAR AND SUBCELLULARPREPARATIONS
[21]
TABLE I Anacystis nidulans (A) ASD Phormidium luridum (B)~
PHOTOPHOSPHORYLATION WITH PARTICLES FROM
Additions Part A PMS Ferricyanide No cofactor; DCMU Fd; DCMU NADP; ( - ) F d NADP; (+)Fd Part B No cofactor NADP; ( - ) F d NADP; Fd + Fd-NADP reductase Ferricyanide
Amount added (umoles/3 ml reaction mixture)
Rates (umolesATP/Ing Chl × hour)
0.3 20.0 - - ; 3.0 X 10-8 0.10; 3 X 10-8 6.0; -6.0; 0.02 --6.0; -6.0; saturating amounts 10.0
137 27 13.0 61.5 10.5 32.0 1-2 132 125 181
Chl, chlorophyll; PMS, N-methylphenazinium methyl sulfate; Fd, ferredoxin (prepared from spinach); DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea. The first two lines represent data from B. Gerhardt and R. Santo, Z. Naturforsch. B 21, 673 (1966); the other data of part A are obtained with washed particles and are taken from R. Bothe, Z. Naturforsch. B 24, 1574 (1969), Table 3. Data in part B are from J. Biggins, Plant Physiol. 42, 1447 (1967). Ferredoxin is a crude preparation from Phormidium including the ferredoxin-NADP reductase. The amounts added are saturating for a Swiss chard chloroplast assay. be stimulated by additional ferredoxin; (this was not observed with P h o r m i d i u m particles, 4 Table I, part B). The ratio of A T P formed per two electrons transported in the noncyclic p a t h w a y (P:2e ratio) is about 0.3, and the phosphorylation rate is relatively low with ferricyanide and N A D P . Photophosphorylation with the latter is not improved by adding ferredoxin to the reaction mixture. Repeated washings with dilute buffer, however, remove ferredoxin (and ferredoxin-NADP reductase, EC 1.6.1.1, to some extent), and the reduction of N A D P can be partially restored with added ferredoxin, s I t should be mentioned that the rates m a y be improved by using shorter reaction times (see below), e.g., in the minute range. Particles from P h o r m i d i u m l u r i d u m ( C y a n o p h y c e a e ) 4,12 Preparation. The strain (P. luridum var. olivaceae Boresch) is provided by the Culture Collection of Algae, Indiana University, BloomingJ. Biggins, Plant Physiol. 42, 1442 (1967).
[21]
ALGAL CHLOROPLAST PI-IOSPHORYLATION
245
ton, Indiana, and grown in Kratz and Myers' medium 9 with fluorescent light. The culture is gassed with air supplemented with 4% C02 (v/v). Cells are washed once with 0.5 M mannitol-30 mM K phosphate, pH 6.8, and resuspended in the same medium. Egg-white muramidase (Worthington Biochemical Corp., Freehold, New Jersey) is added in a final concentration of 0.05%, and incubation is at 35 ° for 2.5 hours. About 70% of the cells lose their cell wall. The cooled mixture is passed through a column (3 X 10 cm) of loosely packed glass wool, and the eluate contains the "protoplasts" (cells minus cell wall). 12 This is centrifuged (500 g, 4 minutes), and the preparation is washed once with mannitolphosphate medium. The pellet is resuspended in 0.5 M mannitol-Tris buffer, pH 7.2-7.5. Cell-free preparations are obtained by diluting the protoplast suspension 8-fold with the standard phosphorylation reaction mixture. Stronger dilution (40-fold) should be avoided since activity will decrease, particularly that associated with the noncyclic electron flow. Reaction Conditions. Reaction is carried out in Warburg vessels with saturating white light. The final volume of 1.5 ml contains (in ~moles) : Tris.maleate, pH 7.5, 40; MgC12, 30; ADP, 5; K2H32po4, 5; particles with 50 ~g of chlorophyll and cofactors. Reaction time is 6 minutes at 25 °. Thereafter the reaction mixture is inactivated with trichloroacetic acid and ATP formation measured by the method of Avron. 13 Conditions for cyclic photophosphorylation are slightly different. Rates. Particles from this alga give the highest noncyclic phosphorylation rates so far reported for preparations from the blue-green algae (Table I, part B). No endogenous rate, i.e., without added cofactors, as described for Anacystis preparations, is observed, although addition of ferredoxin plus ferredoxin-NADP reductase has no stimulatory effect on NADP reduction (and photophosphorylation). P:2e ratios are about 0.8 and higher. Cyclic phosphorylation with high amounts of ferredoxin included in the reaction mixture was not investigated. Particles from Anabaena variabilis (Cyanophyceae) Krogmann and co-workers recently also succeeded in obtaining noncyclic phosphorylation with Anabaena24 The cells were grown according to the method of Kratz and Myers 9 and digested with lysozyme by a modified method of Biggins. ~2 The photophosphorylation methods used are similar to those described above. The reaction mixture for NADP reduction and phosphorylation contains in a final volume of 3 ml (in 13M. Avron, Biochim. Biophys. Acta 40, 257 (1960). ~4S. S. Lee, A. M. Young, and D. W. Krogmann, Biochim. Biophys. Acta 180, 130 (1969).
246
CELLULAR AND SUBCELLULAR PREPARATIONS
[21]
~moles): Tricine-NaOH (N-tris-hydroxymethylglycine), pH 7.8, 50; MgC12, 20; ADP, 10; phosphate, 3; particles with 30 ~g of Chl; NADP, 0.5; and ferredoxin plus ferredoxin-NADP reductase. Reaction time is 5 minutes at 25°; light intensity is ~140,000 lux; gas phase, air. ATP formation is assayed by the use of 32p.15 A ~atc of 107 #moles ATP/mg Chl X hour was obtained, whereas with PMS as cofactor this figure may be as high as approximately 500. Chloroplasts from Bumilleriopsis t~litormis Vischer (Xanthophyceae) 7 Preparation. This alga is a member of the coccoid Xanthophyceae; the cells, however, stick together after division and form filaments consisting of several cells even in agitated cultures. They are grown autotrophically as described previously. ~,16 Each cell has several chloroplasts, which can be released by homogenizing the cells with glass beads (0.5 ram) in a rotary shaker for 1 minute at 4000 rpm at 0°-10 ° (Braun Comp., Melsungen, Germany). The algae are washed once with 0.9% NaC1 and suspended in a freshly prepared ice-cold medium which contains (in millimolar concentration) : sucrose, 350; Tricine-NaOH (N-trishydroxymethylglycine), pH 8, 65; sodium pyrophosphate, 3.5; MgC12 1; 2-mercaptoethanol, 1.5; ascorbic acid, 0.4; EDTA, 0.45; NaOH, 6-8; and (in mg/ml) 7.7 mg of bovine serum albumin, and 35 mg of polyvinylpyrrolidone (molecular weight, approximately 24,000, FLUKA, Buchs, Switzerland). The cell number must be 1-3 >( 107 per milliliter. The homogenate is freed from the beads by a suitable sintered-glass funnel, then centrifuged at 1100 g for 2 minutes at 3 °. The supernatant solution is again centrifuged at 17,000 g for 5 minutes, and the pellet is suspended in a medium consisting of 0.44M sucrose; 8 mM Tricine, pH 8; 0.16 mM MgC12, and 0.4 mM Na pyrophosphate. Chlorophyll content is 0.5 to 1 mg/ml. Chloroplasts retain full activity for at least 1 hour when kept at 0°C. Under the light microscope, isolated chloroplasts appear as swollen vesicles with one or two strings of aggregated thylakoids traversing the vesicleY Washing with dilute buffer has little effect on activity, the phosphorylation in the noncyclic system is irreversibly decreased to some extent. Reaction Conditions. Reactions are performed in open cuvettes of approximately 1 cm light path with white light of 70,000 lux. The final volume of 1 ml contains generally (in ~moles) : Mes-NaOH (morpholinoethanesulfonie acid), pH 8, 50; sucrose, 60; MgC12, 2; Pi plus s2p (0.5-1
W. C. Duane, M. C. ttohl, and D. W. Krogmann, Biochim. Biophys. Acta 109, 108 (1965). P. BSger and A. San Pietro, Z. Pflanzenphysiol. 58, 70 (1967).
[21]
ALGAL
CHLOROPLAST
247
PHOSPHORYLATION
TABLE II PHOTOPHOSPHORYLATION WITH CHLOROPLASTS FROM Bumilleriopsis SPINACH UNDER THE SAME CONDITIONS a
filiformis AND
Rates in ~moles ATP/mg Chl × hours
BumilIeriopsis filiformis Additions
Concentration (mM)
2.3 X 10-5 ug Chl/cell
4.8 X 10-5 ~g Chl/cell
Spinach
No cofactors PMS Ferricyanide 4-Ac-Py-I~ Fd NADP;(-)Fd NADP;(T)Fd
-0. 016 1,0 1.0 0.037 0.5; - 0.5; 2.2 X 10-3
4 370 204 161 44 5 92
3 190 90 62 23 4 57
1.5 380 140 140 90 3 97
a Ferredoxins are from Bumilleriopsis and spinach, respectively, and prepared according to P. BSger, Z. Pflanzenphysiol. 61, 447 (1969). b N-Methyl-4-acetylpyridirdum iodide [see P. BSger et al., Biochemistry 6, 80 (1967) for this compound]. ~Ci), 1; A D P , 1.2; and algal chloroplasts with 8-20 #g of chlorophyll. The concentrations of some cofactors are listed in Table II. Reaction time is 3 minutes at 22 °. Anaerobic experiments are run in Warburg vessels, flushed with nitrogen. After the phosphorylation reaction the chloroplasts are denatured b y acidifying the reaction mixture with 0.4 N I I C l 0 4 ; A T P is determined by a modification of the method of Avron. 13 I t should be mentioned t h a t Mes buffer has an unfavorable p K and Hepes buffer m a y be substituted for it in case no ferredoxin is included in the reaction mixture. Rates. The phosphorylation rates obtained are as high as with spinach chloroplasts under the same conditions (Table I I ) . The endogenous rate without cofactors is low, ferredoxin has to be added to accomplish reduction of N A D P and the phosphorylation accompanied by it. The P : 2 e ratio is 0.6-0.7. Anaerobiosis has no striking effect on the noncyclic electron flow or on P M S - m e d i a t e d phosphorylation. There is A T P formation with added ferredoxin under air, but the rate is almost nil under nitrogen also when D C M U is p r e s e n t Y R a t e s (expressed as A T P formed per chlorophyll of isolated chloroplasts) v a r y inversely with the chlorophyll content of the cell (Table I I ) . Recent experiments (unpublished) indicate t h a t this is due to different ratios of chlorophyll to (photosynthetic) chloroplast enzymes. Some of 17p. BSger, Z. Pflanzenphysiol. 61, 447 (1969).
248
CELLULAR
AND
SUBCELLULAR
PREPARATIONS
[22]
these quantitative relationships have been described for Chlorella Is and E u g l e n a ? 6 In the latter, the amounts of ferredoxin and cytochrome ] (552) are constant per cell, whereas the chlorophyll content increases during the cultivation time. 18A. Pirson and P. BSger, Nature (London) 205, 1129 (1965); P. BSger, Flora 154, 174 (1964).
[22] C h l o r o p l a s t s ( a n d G r a n a ) : P h o t o s y n t h e t i c E l e c t r o n T r a n s p o r t in A l d e h y d e - F i x e d M a t e r i a l
By R. B. PARK Principle Formaldehyde and glutaraldehyde treatment of isolated chloroplasts and various algae yields chemically fixed material capable of performing photosystem I and photosystem II reactions at reduced rates and quantum efficiencies. 1-3 The fixation procedure stabilizes the material toward subsequent treatments, such as detergent extraction. 4 Treated green, red, and blue-green algae become permeable to reaction mixture components without loss of accessory pigments in the latter two groups, a,4 In proteins, glutaraldehyde reacts with e-amino groups of lysine and to some extent with tyrosine, histidine, and sulfhydryl residues2 Both interand intramolecular cross-linkages occur2 Reagents G l u t a r a l d e h y d e . Glutaraldehyde is available commercially as a technical grade aqueous solution or as purified material. Treatment of the technical grade is necessary to obtain satisfactory glutaraldehyde to preserve photosynthetic electron transport. About 20 mg of animal bone charcoal is added to 50 ml of a 20% glutaraldehyde solution. 7 This suspension is stirred at room temperature for 20 minutes. The charcoal is then removed by filtration through three layers of Whatman No. 1 filter R. B. Park, J. Kelly, S. Drury, and K. Sauer, Proc. Nat. Acad. Sci. U~S. 55, 1056 (1966). 2U. W. l~allier and R. B. Park, Plant Physiol. 44, 535 (1969). 8C. J. Ludlow and R. B. Park, Plant Physiol. 44, 540 (1969). 4If. W. Hallier and R. B. Park, Plant Physiol. 44, 544 (1969). 5A. F. S. A. Habeeb and R. Hiramoto, Arch. Biochem, Biophys. 126, 16 (1968). *F. M. Richards and J. R. Knowles, J. Mol. Biol. 37, 231 (1968). 7p. j. Anderson, J. Histochem. Cytochem. 15, 652 (1967).
CELLULAR AND SUBCELLULAR PREPARATIONS
[21]
with Euglena chloroplasts isolated by the procedure of Eisenstadt and Brawerman 6 (Dr. J. A. Schiff, personal communication). However, high rates of incorporation (78 #moles HCO~-/mg chlorophyll/rag) have been reported with preparations from Acetabularia mediterrania2 °
[21] Algal Preparations with Photophosphorylation Activity By PETERB~ER Most of our knowledge of the photophosphorylation processes in vitro is based on preparations from higher plants and purple bacteria. Chloroplasts isolated from algae are not usually active. The first active preparations, however, were obtained with the (prokaryotic) blue-green alga Anabaena 1,2 after disruption of the cells by mechanical means. These particles catalyzed only cyclic photophosphorylation [e.g., with Nmethylphenazinium methyl sulfate (PMS)], although Hill reactions with artificial electron acceptors were consistently observed. A gentle rupture of the cell walls with enzymes was necessary to obtain particles active in both cyclic and noncyclic photophosphorylation2 ,4 There are very few reports concerning the preparation of chloroplasts with photophosphorylation activity from eukaryotic algae. Preparations from the chrysomonad H y m e n o m o n a s yielded about 70 t~moles of ATP per milligram of chlorophyll per hour with PMS. 5 Rather high rates with this cofactor (approximately 180 t~moles ATP/mg Chl X hour) have been reported using chloroplasts from the green alga Chlamydomonas reinhardi. 6 This author has prepared chloroplasts with good yield and high phosphorylation activity from the heterokont BumiUeriopsis fili]ormis. 7 Active chloroplast preparations from other algae groups, e.g., red algae or diatoms, have not yet been reported. In this article the methodology of photophosphorylation will be described for only those algae preparations which show reasonably high lB. Petrack and F. Lipmann, in "Light and Life" (W. D. McElroy and B. Glass, eds.). Johns Hopkins Press, Baltimore, Maryland, 1961. 2W. A. Susor, W. C. Duane, and D. W. Krogmann, Rec. Chem. Progr. 25, 197 (1964). 8B. Gerhardt and R. Santo, Z. Natur]orsch. B 21, 673 (1966). 4j. Biggins, Plant Physiol. 42, 1447 (1967). 5S. W. Jeffrey, J. Ulrich, and M. B. Allen, Biochim. Biophys. Acta 112, 35 (1966). ~A. Givaa and R. P. Levine, Plant Physiol. 42, 1264 (1967). P. BSger, Z. Pflanzenphysiol. 61, 85 (1969).
[21]
ALGAL CHLOROPLAST PHOSPHORYLATION
243
photophosphorylation rates due to both cyclic and noncyclic electron transport. Particles from Anacystis nidulans (Cyanophyceae, Blue-Green Algae) a,s Preparation. Strain No. 1402-1 from the Algae Culture Collection, University of GSttingen, Germany, is grown in the medium of Kratz and Myers 9 at 30 ° with 3000 lux (fluorescent light) and gassed with C02enriched air (1.5% v/v). The once washed algae are suspended in 5% sucrose containing 20 mM Tris buffer, pH 7.6, frozen at --50 ° to --60 ° and then lyophilized for 2-3 hours. The dry powder can be stored without loss over P205 up to 3 days at 5 °. For use, it is resuspended in 40 mM MgC12 and lysozyme (Calbiochem, B grade, Los Angeles, California)--2 mg of enzyme per 0.2 mg of chlorophyll--is added. After standing for 3-5 hours at 15 °, the particles are collected by centrifugation and resuspended in 5% sucrose--20 mM Tris, pH 7.6. They may be washed once with the same medium. Photophosphorylation proceeds best when the particles are kept at room temperature. No data concerning the efficiency of cell wall lysis with this method were given by the authors2 ,8 However, lyophilization is necessary for the enzymatic action. Photophosphorylation with the same reaction mixture as described below can also be performed with lyophilized cells without lysozyme treatment. The rates are at best half of those from preparations treated with lysozyme.1° Reaction Conditions. Photophosphorylation is carried out in Warburg vessels with white light of 35,000 lux. The standard reaction mixture contains in a final volume of 3 ml (in ~moles): Tris buffer, pH 7.6, 60; MgCl~, 40; ADP, 10; inorganic phosphate, 10 (with approximately 10~ cpm 3~p); particles with 0.2 mg chlorophyll. The concentrations of some cofactors are indicated in Table I. Reaction time is 20~30 minutes; temperature, 15 °3 (or 25°s). Gas phase is prepurified nitrogen. ATP is determined by the method of Sugino and Miyoshi.1~ For ferredoxincatalyzed cyclic phosphorylation N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (Hepes) buffer, pH 7, is used. 8 Rates. Photophosphorylation rates with particles from Anacystis as reported by the Trebst group are comparable to those from spinach as far as the cyclic system with PMS and ferredoxin is concerned (Table I, part A). The rather high endogenous reaction of unwashed particles can
8H. Bothe, Thesis, Univ. of G5ttingen, Germany, 1968; Z. NaturJorsch. B 24, 1574 (1969). 9W. A. Kratz and J. Myers, Amer. J. Bot. 42, 282 (1955). loB. Gerhardt and A. Trebst, Z. NaturJorsch. B 20, 879 (1965). 11y. Sugino and Y. Miyoshi,J. Biol. Chem. 239, 2360 (1964).
244
CELLULAR AND SUBCELLULARPREPARATIONS
[21]
TABLE I Anacystis nidulans (A) ASD Phormidium luridum (B)~
PHOTOPHOSPHORYLATION WITH PARTICLES FROM
Additions Part A PMS Ferricyanide No cofactor; DCMU Fd; DCMU NADP; ( - ) F d NADP; (+)Fd Part B No cofactor NADP; ( - ) F d NADP; Fd + Fd-NADP reductase Ferricyanide
Amount added (umoles/3 ml reaction mixture)
Rates (umolesATP/Ing Chl × hour)
0.3 20.0 - - ; 3.0 X 10-8 0.10; 3 X 10-8 6.0; -6.0; 0.02 --6.0; -6.0; saturating amounts 10.0
137 27 13.0 61.5 10.5 32.0 1-2 132 125 181
Chl, chlorophyll; PMS, N-methylphenazinium methyl sulfate; Fd, ferredoxin (prepared from spinach); DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea. The first two lines represent data from B. Gerhardt and R. Santo, Z. Naturforsch. B 21, 673 (1966); the other data of part A are obtained with washed particles and are taken from R. Bothe, Z. Naturforsch. B 24, 1574 (1969), Table 3. Data in part B are from J. Biggins, Plant Physiol. 42, 1447 (1967). Ferredoxin is a crude preparation from Phormidium including the ferredoxin-NADP reductase. The amounts added are saturating for a Swiss chard chloroplast assay. be stimulated by additional ferredoxin; (this was not observed with P h o r m i d i u m particles, 4 Table I, part B). The ratio of A T P formed per two electrons transported in the noncyclic p a t h w a y (P:2e ratio) is about 0.3, and the phosphorylation rate is relatively low with ferricyanide and N A D P . Photophosphorylation with the latter is not improved by adding ferredoxin to the reaction mixture. Repeated washings with dilute buffer, however, remove ferredoxin (and ferredoxin-NADP reductase, EC 1.6.1.1, to some extent), and the reduction of N A D P can be partially restored with added ferredoxin, s I t should be mentioned that the rates m a y be improved by using shorter reaction times (see below), e.g., in the minute range. Particles from P h o r m i d i u m l u r i d u m ( C y a n o p h y c e a e ) 4,12 Preparation. The strain (P. luridum var. olivaceae Boresch) is provided by the Culture Collection of Algae, Indiana University, BloomingJ. Biggins, Plant Physiol. 42, 1442 (1967).
[21]
ALGAL CHLOROPLAST PI-IOSPHORYLATION
245
ton, Indiana, and grown in Kratz and Myers' medium 9 with fluorescent light. The culture is gassed with air supplemented with 4% C02 (v/v). Cells are washed once with 0.5 M mannitol-30 mM K phosphate, pH 6.8, and resuspended in the same medium. Egg-white muramidase (Worthington Biochemical Corp., Freehold, New Jersey) is added in a final concentration of 0.05%, and incubation is at 35 ° for 2.5 hours. About 70% of the cells lose their cell wall. The cooled mixture is passed through a column (3 X 10 cm) of loosely packed glass wool, and the eluate contains the "protoplasts" (cells minus cell wall). 12 This is centrifuged (500 g, 4 minutes), and the preparation is washed once with mannitolphosphate medium. The pellet is resuspended in 0.5 M mannitol-Tris buffer, pH 7.2-7.5. Cell-free preparations are obtained by diluting the protoplast suspension 8-fold with the standard phosphorylation reaction mixture. Stronger dilution (40-fold) should be avoided since activity will decrease, particularly that associated with the noncyclic electron flow. Reaction Conditions. Reaction is carried out in Warburg vessels with saturating white light. The final volume of 1.5 ml contains (in ~moles) : Tris.maleate, pH 7.5, 40; MgC12, 30; ADP, 5; K2H32po4, 5; particles with 50 ~g of chlorophyll and cofactors. Reaction time is 6 minutes at 25 °. Thereafter the reaction mixture is inactivated with trichloroacetic acid and ATP formation measured by the method of Avron. 13 Conditions for cyclic photophosphorylation are slightly different. Rates. Particles from this alga give the highest noncyclic phosphorylation rates so far reported for preparations from the blue-green algae (Table I, part B). No endogenous rate, i.e., without added cofactors, as described for Anacystis preparations, is observed, although addition of ferredoxin plus ferredoxin-NADP reductase has no stimulatory effect on NADP reduction (and photophosphorylation). P:2e ratios are about 0.8 and higher. Cyclic phosphorylation with high amounts of ferredoxin included in the reaction mixture was not investigated. Particles from Anabaena variabilis (Cyanophyceae) Krogmann and co-workers recently also succeeded in obtaining noncyclic phosphorylation with Anabaena24 The cells were grown according to the method of Kratz and Myers 9 and digested with lysozyme by a modified method of Biggins. ~2 The photophosphorylation methods used are similar to those described above. The reaction mixture for NADP reduction and phosphorylation contains in a final volume of 3 ml (in 13M. Avron, Biochim. Biophys. Acta 40, 257 (1960). ~4S. S. Lee, A. M. Young, and D. W. Krogmann, Biochim. Biophys. Acta 180, 130 (1969).
246
CELLULAR AND SUBCELLULAR PREPARATIONS
[21]
~moles): Tricine-NaOH (N-tris-hydroxymethylglycine), pH 7.8, 50; MgC12, 20; ADP, 10; phosphate, 3; particles with 30 ~g of Chl; NADP, 0.5; and ferredoxin plus ferredoxin-NADP reductase. Reaction time is 5 minutes at 25°; light intensity is ~140,000 lux; gas phase, air. ATP formation is assayed by the use of 32p.15 A ~atc of 107 #moles ATP/mg Chl X hour was obtained, whereas with PMS as cofactor this figure may be as high as approximately 500. Chloroplasts from Bumilleriopsis t~litormis Vischer (Xanthophyceae) 7 Preparation. This alga is a member of the coccoid Xanthophyceae; the cells, however, stick together after division and form filaments consisting of several cells even in agitated cultures. They are grown autotrophically as described previously. ~,16 Each cell has several chloroplasts, which can be released by homogenizing the cells with glass beads (0.5 ram) in a rotary shaker for 1 minute at 4000 rpm at 0°-10 ° (Braun Comp., Melsungen, Germany). The algae are washed once with 0.9% NaC1 and suspended in a freshly prepared ice-cold medium which contains (in millimolar concentration) : sucrose, 350; Tricine-NaOH (N-trishydroxymethylglycine), pH 8, 65; sodium pyrophosphate, 3.5; MgC12 1; 2-mercaptoethanol, 1.5; ascorbic acid, 0.4; EDTA, 0.45; NaOH, 6-8; and (in mg/ml) 7.7 mg of bovine serum albumin, and 35 mg of polyvinylpyrrolidone (molecular weight, approximately 24,000, FLUKA, Buchs, Switzerland). The cell number must be 1-3 >( 107 per milliliter. The homogenate is freed from the beads by a suitable sintered-glass funnel, then centrifuged at 1100 g for 2 minutes at 3 °. The supernatant solution is again centrifuged at 17,000 g for 5 minutes, and the pellet is suspended in a medium consisting of 0.44M sucrose; 8 mM Tricine, pH 8; 0.16 mM MgC12, and 0.4 mM Na pyrophosphate. Chlorophyll content is 0.5 to 1 mg/ml. Chloroplasts retain full activity for at least 1 hour when kept at 0°C. Under the light microscope, isolated chloroplasts appear as swollen vesicles with one or two strings of aggregated thylakoids traversing the vesicleY Washing with dilute buffer has little effect on activity, the phosphorylation in the noncyclic system is irreversibly decreased to some extent. Reaction Conditions. Reactions are performed in open cuvettes of approximately 1 cm light path with white light of 70,000 lux. The final volume of 1 ml contains generally (in ~moles) : Mes-NaOH (morpholinoethanesulfonie acid), pH 8, 50; sucrose, 60; MgC12, 2; Pi plus s2p (0.5-1
W. C. Duane, M. C. ttohl, and D. W. Krogmann, Biochim. Biophys. Acta 109, 108 (1965). P. BSger and A. San Pietro, Z. Pflanzenphysiol. 58, 70 (1967).
[21]
ALGAL
CHLOROPLAST
247
PHOSPHORYLATION
TABLE II PHOTOPHOSPHORYLATION WITH CHLOROPLASTS FROM Bumilleriopsis SPINACH UNDER THE SAME CONDITIONS a
filiformis AND
Rates in ~moles ATP/mg Chl × hours
BumilIeriopsis filiformis Additions
Concentration (mM)
2.3 X 10-5 ug Chl/cell
4.8 X 10-5 ~g Chl/cell
Spinach
No cofactors PMS Ferricyanide 4-Ac-Py-I~ Fd NADP;(-)Fd NADP;(T)Fd
-0. 016 1,0 1.0 0.037 0.5; - 0.5; 2.2 X 10-3
4 370 204 161 44 5 92
3 190 90 62 23 4 57
1.5 380 140 140 90 3 97
a Ferredoxins are from Bumilleriopsis and spinach, respectively, and prepared according to P. BSger, Z. Pflanzenphysiol. 61, 447 (1969). b N-Methyl-4-acetylpyridirdum iodide [see P. BSger et al., Biochemistry 6, 80 (1967) for this compound]. ~Ci), 1; A D P , 1.2; and algal chloroplasts with 8-20 #g of chlorophyll. The concentrations of some cofactors are listed in Table II. Reaction time is 3 minutes at 22 °. Anaerobic experiments are run in Warburg vessels, flushed with nitrogen. After the phosphorylation reaction the chloroplasts are denatured b y acidifying the reaction mixture with 0.4 N I I C l 0 4 ; A T P is determined by a modification of the method of Avron. 13 I t should be mentioned t h a t Mes buffer has an unfavorable p K and Hepes buffer m a y be substituted for it in case no ferredoxin is included in the reaction mixture. Rates. The phosphorylation rates obtained are as high as with spinach chloroplasts under the same conditions (Table I I ) . The endogenous rate without cofactors is low, ferredoxin has to be added to accomplish reduction of N A D P and the phosphorylation accompanied by it. The P : 2 e ratio is 0.6-0.7. Anaerobiosis has no striking effect on the noncyclic electron flow or on P M S - m e d i a t e d phosphorylation. There is A T P formation with added ferredoxin under air, but the rate is almost nil under nitrogen also when D C M U is p r e s e n t Y R a t e s (expressed as A T P formed per chlorophyll of isolated chloroplasts) v a r y inversely with the chlorophyll content of the cell (Table I I ) . Recent experiments (unpublished) indicate t h a t this is due to different ratios of chlorophyll to (photosynthetic) chloroplast enzymes. Some of 17p. BSger, Z. Pflanzenphysiol. 61, 447 (1969).
248
CELLULAR
AND
SUBCELLULAR
PREPARATIONS
[22]
these quantitative relationships have been described for Chlorella Is and E u g l e n a ? 6 In the latter, the amounts of ferredoxin and cytochrome ] (552) are constant per cell, whereas the chlorophyll content increases during the cultivation time. 18A. Pirson and P. BSger, Nature (London) 205, 1129 (1965); P. BSger, Flora 154, 174 (1964).
[22] C h l o r o p l a s t s ( a n d G r a n a ) : P h o t o s y n t h e t i c E l e c t r o n T r a n s p o r t in A l d e h y d e - F i x e d M a t e r i a l
By R. B. PARK Principle Formaldehyde and glutaraldehyde treatment of isolated chloroplasts and various algae yields chemically fixed material capable of performing photosystem I and photosystem II reactions at reduced rates and quantum efficiencies. 1-3 The fixation procedure stabilizes the material toward subsequent treatments, such as detergent extraction. 4 Treated green, red, and blue-green algae become permeable to reaction mixture components without loss of accessory pigments in the latter two groups, a,4 In proteins, glutaraldehyde reacts with e-amino groups of lysine and to some extent with tyrosine, histidine, and sulfhydryl residues2 Both interand intramolecular cross-linkages occur2 Reagents G l u t a r a l d e h y d e . Glutaraldehyde is available commercially as a technical grade aqueous solution or as purified material. Treatment of the technical grade is necessary to obtain satisfactory glutaraldehyde to preserve photosynthetic electron transport. About 20 mg of animal bone charcoal is added to 50 ml of a 20% glutaraldehyde solution. 7 This suspension is stirred at room temperature for 20 minutes. The charcoal is then removed by filtration through three layers of Whatman No. 1 filter R. B. Park, J. Kelly, S. Drury, and K. Sauer, Proc. Nat. Acad. Sci. U~S. 55, 1056 (1966). 2U. W. l~allier and R. B. Park, Plant Physiol. 44, 535 (1969). 8C. J. Ludlow and R. B. Park, Plant Physiol. 44, 540 (1969). 4If. W. Hallier and R. B. Park, Plant Physiol. 44, 544 (1969). 5A. F. S. A. Habeeb and R. Hiramoto, Arch. Biochem, Biophys. 126, 16 (1968). *F. M. Richards and J. R. Knowles, J. Mol. Biol. 37, 231 (1968). 7p. j. Anderson, J. Histochem. Cytochem. 15, 652 (1967).