METHODS I N ENZYMOLOGY E D I T O R S - I N - C H I E F

Sidney P. Colo wick Nathan O. Kaplan

Methods in Enzymology

Volume 148

Plant Cell Membranes EDITED BY

Lester Packer Roland Douce D E P A R T M E N T O F P H Y S I O L O G Y AND A N A T O M Y D E P A R T E M E N T D E R E C H E R C H E F O N D A M E N T A L E

U N I V E R S I T Y O F C A L I F O R N I A C E N T R E D ' E T U D E S NUCLEA1RES E T U N I V E R S I T E

B E R K E L E Y , C A L I F O R N I A D E G R E N O B L E

G R E N O B L E , F R A N C E

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

San Diego New York Berkeley Boston London Sydney Tokyo Toronto

Table of Contents

C O N T R I B U T O R S Τ Ο V O L U M E 148 xi

P R E F A C E xvii

V O L U M E S I N S E R I E S xix

Section I. Cells, Protoplasts, and Liposomes

1. Techniques of Cell Suspension Culture R I C H A R D B L I G N Y A N D J E A N - J A C Q U E S L E G U A Y 3

2. Culture and Characteristics of Green Plant Cells C O L I N D A L T O N A N D I A N M A C K E N Z I E 16

3. Preparation of Protoplasts from Plant Tissues for M I K I O N I S H I M U R A , Organelle Isolation Ι κ υ κ ο H A R A - N I S H I M U R A ,

A N D T A K A S H I A K A Z A W A 27

4. Interaction of Plant Protoplast and Liposome T O S H I Y U K I N A G A T A 34

5. Liposomes as Carriers for the Transfer and E x - M I C H E L C A B O C H E A N D pression of Nucleic Acids into Higher Plant Pro- P A U L F . L U R Q U I N 39 toplasts

6. Interspecific Transfer of Partial Nuclear Genomic O T T O S C H I E D E R 45 Information by Protoplast Fusion

Section II. Vacuoles and Tonoplasts

7. Isolation of Mature Vacuoles of Higher Plants: G E O R G E J . W A G N E R 55 General Principles, Criteria for Purity and Integrity

8. Isolation of Vacuoles and Tonoplast from Proto- A L A I N M. B O U D E T A N D plasts G I L B E R T A L I B E R T 74

9. Preparation of Tonoplast Vesicles from Isolated R O B E R T T . L E O N A R D 82 Vacuoles

10. Biochemical and Enzymatic Components of a J . D ' A U Z A C , H . C H R E S T I N , Vacuolar Membrane: Tonoplast of Lutoids A N D B. M A R I N 87 from Hevea Latex

ν

vi T A B L E O F C O N T E N T S

11. Characterization of Tonoplast Enzyme Activities B E N J A M I N J A C O B Y 105 and Transport

12. Preparation of Tonoplast Vesicles: Applications to E D U A R D O B L U M W A L D , H +-Coupled Secondary Transport in Plant Vac- P H I L I P A . R E A , A N D uoles R O N A L D J . P O O L E 115

13. Purification and Characterization of the Tonoplast S T E P H E N Κ . R A N D A L L A N D H +-Translocating ATPase H E V E N S Z E 123

14. High-Performance Liquid Chromatography for Si- Μ. H I L L , Α. D U P A I X , multaneous Kinetic Measurements of Adenine P. V O L F I N , A. K U R K D J I A N , Nucleotides in Isolated Vacuoles A N D B . A R R I O 132

Section III. Plastids

15. Isolation of Intact Chloroplasts: General Princi- D A V I D A. W A L K E R , pies and Criteria of Integrity Z O R A N G . C E R O V I C , A N D

S I M O N P. R O B I N S O N 145

16. Isolation of Plastids in Density Gradients of Per- C A R L A. P R I C E , coll and Other Silica Sols J O H N C . C U S H M A N ,

L E T I C I A R .

M E N D I O L A - M O R G E N T H A L E R ,

A N D E L L E N Μ . R E A R D O N 157

17. Purification of Chloroplasts Using Silica Sols K . W . J O Y A N D W . R . M I L L S 179

18. Separation of Chloroplasts and Cytosol from Pro- S I M O N P. R O B I N S O N 188 toplasts

19. Use of Thermolysin to Probe the Cytosolic Sur- J A C Q U E S J O Y A R D , face of the Outer Envelope Membrane from A L B E R T - J E A N D O R N E , Plastids A N D R O L A N D D O U C E 195

20. Characterization of Plastid Polypeptides from the J A C Q U E S J O Y A R D , Outer and Inner Envelope Membranes M A R Y S E A . B L O C K ,

J A C Q U E S C O V E S ,

C L A U D E A L B A N ,

A N D R O L A N D D O U C E 206

21. Isolation of Amyloplasts from Suspension Cul- T O M AP R E E S A N D tures of Soybean F R Ä S E R D. M A C D O N A L D 218

22. Isolation of Amyloplasts from Developing Endo- J A C K C . S H A N N O N , sperm of Maize (Zea mays L . ) E D G A R D O E C H E V E R R I A ,

A N D C H A R L E S B O Y E R 226

23. Isolation of Plastids from Buds of Cauliflower E T I E N N E - P A S C A L J O U R N E T 234 (Brassica οleracea L . )

24. Isolation of Membranous Chromoplasts from Daf- B O D O L I E D V O G E L 241 fodil Flowers

T A B L E O F C O N T E N T S V Ü

25. Structure and Function of the Inner Membrane S A T O R U M U R A K A M I 246 Systems in Etioplasts

26. Isolation Procedures for Inside-Out Thylakoid H A N S - E R I K Ä K E R L U N D A N D Vesicles B E R T I L A N D E R S S O N 2 5 2

27 . Characterization of Chloroplast Cytochromes D E R E K S. B E N D A L L A N D S T E P H E N A. R O L F E 259

28. Cell-Free Reconstitution of Protein Transport into M I C H A E L L . M I S H K I N D , Chloroplasts K A R E N L . G R E E R , A N D

G R E G O R Y W . S C H M I D T 274

29. Polar Lipids of Chloroplast Membranes D A V I D J . C H A P M A N A N D J A M E S B A R B E R 294

30. Isolation and Reconstitution of Thylakoid Lipids S A L L I E G . S P R A G U E A N D L . A N D R E W S T A E H E L I N 319

31 . Long-Chain Fatty Acid Synthesis and Utilization G R A T T A N R O U G H A N 327 by Isolated Chloroplasts

32 . Phosphatidylglycerol Synthesis in Chloroplast J . B R I A N M U D D , Membranes J A E N E . A N D R E W S ,

A N D S. A. S P A R A C E 338

33 . Galactolipid Biosynthesis in Chloroplast Mem- N O R A W . L E M A N D branes J O H N P. W I L L I A M S 346

34. Chlorophylls and Carotenoids: Pigments of Photo- H A R T M U T Κ . L I C H T E N T H A L E R 350 synthetic Biomembranes

35 . α-Tocopherol and Plastoquinone Synthesis in J Ü R G E N S O L L 383 Chloroplast Membranes

36. Solubilization and Reconstitution of Caroteno- P. B E Y E R 392 genie Enzymes from Daffodil Chromoplast Membranes Using 3-[(3-Cholamidopropyl)di-methylammonioj-1-propane Sulfonate

Section IV. Mitochondria

37 . Isolation of Plant Mitochondria: General Princi- R O L A N D D O U C E , pies and Criteria of Integrity J A C Q U E S B O U R G U I G N O N ,

R E N A U D B R O U Q U I S S E ,

A N D M I C H E L N E U B U R G E R 403

38. Purification of Plant Mitochondria on Silica Sol A N T H O N Y L . M O O R E A N D Gradients M I C H A E L O. P R O U D L O V E 415

39 . Isolation of Mitochondria from Leaves of C 3 , C 4 , G E R A L D E . E D W A R D S A N D and Crassulacean Acid Metabolism Plants P E R G A R D E S T R Ö M 421

viii T A B L E O F C O N T E N T S

40. Separation of Spinach Leaf Mitochondria accord- P E R G A R D E S T R Ö M A N D ing to Surface Properties: Partition in Aqueous I N G E M A R E R I C S O N 434 Polymer Two-Phase Systems

41. Isolation of Submitochondrial Particles with Dif- I A N M. M 0 L L E R , ferent Polarities A N N I K A C . L I D £ N ,

I N G E M A R E R I C S O N , A N D

P E R G A R D E S T R Ö M 4 4 2

42. Isolation of the Outer Membrane of Plant Mito- C A R M E N A. M A N N E L L A 453 chondria

43. Characterization of Channels Isolated from Plant M A R C O C O L O M B I N I 465

Mitochondria

44. Phosphoglycerides of Mitochondrial Membranes J O H N L . H A R W O O D 475

45. Ubiquinone Biosynthesis in Plant Mitochondria F R I E D H E L M L Ü T K E - B R I N K H A U S A N D H A N S K L E I N I G 486

46. Purification of Complexes I I and I V from Plant M A S A Y O S H I M A E S H I M A , Mitochondria T S U K A H O H A T T O R I , A N D

T A D A S H I A S A H I 491

Section V. Peroxisomes and Glyoxysomes

47. Isolation of Glyoxysomes and Purification of Gly- E U G E N E L . V I G I L , oxysomal Membranes T U N G K . F A N G , A N D

R O B E R T P. D O N A L D S O N 505

48. Peroxisomes and Fatty Acid Degradation B E R N T G E R H A R D T 516

49. Proteins and Phospholipids of Glyoxysomal Mem- H A R R Y B E E V E R S A N D branes from Castor Bean E L M A G O N Z A L E Z 526

Section VI. Nuclei, Endoplasmic Reticulum, and Plasma Membrane

50. Isolation of Nuclei from Soybean Suspension P A U L K E I M 535 Cultures

51. Isolation of the Plasma Membrane: Membrane D O N A L D P. B R I S K I N , Markers and General Principles R O B E R T T . L E O N A R D , A N D

T H O M A S K . H O D G E S 542

52. Preparation of High-Purity Plasma Membranes C H R I S T E R L A R S S O N , S U S A N N E W I D E L L ,

A N D P E R K J E L L B O M 558

T A B L E O F C O N T E N T S ix

53. Possible Approaches to Surface Labeling of the J . L . H A L L 568 Plasma Membrane

54. Isolation of Endoplasmic Reticulum: General J . M I C H A E L L O R D 5 7 6 Principles, Enzymatic Markers, and Endoplasmic Reticulum-Bound Polysomes

55. Phosphoglyceride Synthesis in Endoplasmic Re- T H O M A S S. M O O R E , J R . 5 8 5 ticulum

Section VII. General Physical and Biochemical Methods

56. Electron Microscopy of Plant Cell Membranes J E A N - P I E R R E C A R D E 5 9 9

57. Two-Dimensional Electrophoresis in the Analysis R . R E M Υ A N D and Preparation of Cell Organelle Polypeptides F . A M B A R D - B R E T T E V I L L E 6 2 3

58. Plant Membrane Sterols: Isolation, Identification, M A R I E - A N D R £ E H A R T M A N N and Biosynthesis A N D P I E R R E B E N V E N I S T E 6 3 2

59. Separation of Molecular Species of Plant Glyco- J . K E S S E L M E I E R A N D lipids and Phospholipids by High-Performance E . H E I N Z 6 5 0 Liquid Chromatography

60. Movement of Phospholipids between Membranes: J E A N - C L A U D E K A D E R , Purification of a Phospholipid Transfer Protein C H A N T A L V E R G N O L L E , from Spinach Leaf A N D P A U L M A Z L I A K 661

61. Pesticides and Lipid Synthesis in Plant Mem- C . A N D I N G 667 branes

62. Rapid Filtration Technique for Metabolite Fluxes Y V E S D U P O N T A N D across Cell Organelles M A R I E - J O S £ M O U T I N 6 7 5

63. Application of Nuclear Magnetic Resonance R. G . R A T C L I F F E 6 8 3 Methods to Plant Tissues

A U T H O R I N D E X 701

S U B J E C T I N D E X 7 2 9

[35] P R E N Y L Q U I N O N E S Y N T H E S I S I N C H L O R O P L A S T S 383

[35] α-Tocopherol and Plastoquinone Synthesis in Chloroplast Membranes

By J Ü R G E N S O L L

Plant prenylquinones, a-tocopherol, plastoquinone-9, and phyllo-quinone, function in chloroplasts as membrane constituents, antioxi-dants, or electron carriers. For a long time biosynthetic studies were hampered by the fact that no chemical intermediates of the biosynthetic pathway or biochemically active organelles were available. Earlier stud-ies 1 2 proposed pathways with a multitude of chemically possible interme-diates. The detailed work 3 on the chemical synthesis of prenylquinones enabled others 4 -* to work out the most probable pathway (Fig. 1) in plas-toquinone and tocopherol biosynthesis.

Chemical Synthesis of Prenylquinones

The small-scale synthesis of I V (Fig. 2) is described here4 and can be applied to the synthesis of other prenylquinones (/, / / , / / / , V, VI) without problems. In many cases methylquinones are not commercially available and have to be prepared in advance. The corresponding phenol (4 mmol) is dissolved in 10 ml methanol and oxidized by 10 mmol of Fremy's salt7

{ [ ( S 0 3 ) 2 N O ] K 2 } in 120 ml water and 4 ml sodium acetate (1 M). The reaction is allowed to continue for 30 min and the quinone is then exten-sively extracted with diethyl ether. The organic solvent is evaporated and the quinone purified by column chromatography (silica gel 60, Merck, FRG) using CHC1 3 as developing solvent. Quinone (0.7 mmol) is dis-solved in 1.5 ml benzene, 2.5 ml H 2 0 , and 250 mg Na 2S 2C>4 is added. The reduction is completed after 5 min, the quinol is washed with ice water, and dried in a desiccator. Freshly distilled BF 3-etherate (0.3 ml in 1 ml tetrahydrofuran) is added dropwise via a syringe to a solution of 1.1 mmol quinol, 200 mg A 1 2 0 3 (W-200 basic, Woelm-Pharma, FRG), and 1.1 mmol

1 W. Janiszowska and J . F . Pennock, Vitam. Horm. (N.Y.) 34, 77 (1976). 2 D. R. Threlfall and G . R. Whistance, in "Aspects of Terpenoid Chemistry and Biochemis-

try" (T. W. Goodwin, ed.), p. 335. Academic Press, London, 1971. 3 H . Mayer and O. Isler, this series, Vol. 18C, p. 241. 4 J . Soil and G . Schultz, Phytochemistry 19, 215 (1980). 5 J . Soll, M. Kemmerling, and G . Schultz, Arch. Biochem. Biophys. 204, 544 (1980). 6 S. R . Morris and D. R. Threlfall, Biochem. Soc. Trans. 11, 587 (1983). 7 H . J . Teuber and W. Rau, Chem. Ber. 86, 1036 (1953).

METHODS IN ENZYMOLOGY, VOL. 148 Copyright © 1987 by Academic Press, Inc.

All rights of reproduction in any form reserved.

384 P L A S T I D S [35]

Phytyl -PP

2-Methyl-6-phytylquinol (I)

SAM

•SAH

2 - Methyl -6 - solanesylquinol

SAM

SAH

2.3 - Dimethyl - 6 - phy tylquinol (IV 2.3- Dimethyl - 6 - solanesylquinol Plastoquinol -9

γ - Tocopherol (VIII)

-SAM

Plastoquinone - 9

α - Tocopherol (X) F I G . 1. Proposed pathway of α-tocopherol and plastoquinone-9 synthesis in spinach

chloroplasts. From the available data these are the most likely intermediates to be involved in prenylquinone synthesis. A possible bypass in tocopherol synthesis might occur which leads from I via V I I to V I I I instead of I via IV to V I I I (see text). S A M , 5-Adeno-sylmethionine; S A H , S-adenosylhomocysteine.

[35] P R E N Y L Q U I N O N E S Y N T H E S I S I N C H L O R O P L A S T S 385

λ Methyl - phytylquinols

"1 C H 3 Η Η C H 3

R 2

Η C H 3 Η C H 3

Η - 2 - M e t h y l - 6 - I Λ Η - 2 - M e t h y l - 5 - II C H 3 - 2 - M e t h y l - 3 - III Η - 2 ,3 -Dimethyl -5-IV

Ri R2 R3

C H 3 Η Η - ÖTocopherol - VII C H 3 C H 3 Η - yTocopherol - VIII

phytylquinol ™ 3 Η CH 3 - ßTocopheroi - IX C H 3 CH 3 CH 3-aTocopherol - X

C H 3 Η C H 3 - 2,5-Dimethyl - 6 - V C H 3 C H 3 C H 3 - Trimethyl-6 - V I y

F I G . 2. Nomenclature and identification of prenylquinols and tocopherols.

isophytol in 2 ml dry tetrahydrofuran. The mixture is stirred under N 2 in the dark for 35 hr. Residual B F 3 is hydrolyzed on ice, and the prenylated quinols extracted with diethyl ether, the ether solution dried, and the organic solvent evaporated. The resulting quinol (IV) is oxidized by 400 mg A g 2 0 in dry diethyl ether. Prenylquinones are purified by column chromatography (Silica-gel 60, Merck) developed with petrol (bp 60-80°) -diethyl ether, 15: 1 (system 1). Purity of the products is verified by thin-layer chromatography (precoated plates on glass, silica gel, G-1500 LS254, Schleicher and Schüll, FRG) in system 1. When the prenyl-quinones I , I I , and I I I are to be synthesized, care has to be taken to separate the isomers properly. This can be achieved by repeated thin-layer chromatography as above. The succession of prenylquinones in this thin-layer chromatography system is shown in Fig. 3A. Quinones and

origin IUI η VIV front

Β origin VII Vffl X IV VI front

F I G . 3. Separation of prenylquinones and tocopherols by thin-layer chromatography. (A) The separation of mono-, di-, and trimethylphytylquinones on precoated thin-layer plates, silica gel G-1500, with diethyl ether: petrol (1:15, ν : v) as developing solvent. The purification of prenylquinones and tocopherols using the same thin-layer plates but diethyl ether: petrol, (1:10, ν : ν) as solvent system. Rva lues and separation are variable with silica gel plates obtained from different manufacturers. Precoated plates on glass give better resolution than those on plastic or aluminum foil (see also text).

386 P L A S T I D S [35]

quinols have a tendency to oxidize and polymerize during chromatography. We have obtained the best results using the systems described; other systems caused more oxidation, decomposition, and poor separation of quinones or prenylquinone isomers. Al l products should be stored and purified in the quinone form and not in the quinol form which is more susceptible to uncontrolled breakdown. Quinones and prenylquinones are detected on thin-layer plates with fluorescence indicator at 254 nm, while quinoles and prenylquinoles are visualized with FeCl 3/2,2'-dipyridyl 0 . 1 % : 0.25% (w/w) in ethanol.

Chemicals Needed for the Enzymatic Assays

As can be seen from Fig. 1, homogentisate, polyprenyl diphosphate, and S-adenosylmethionine are necessary for the incubation. Unlabeled and labeled S-adenosylmethionine and homogentisate are commercially available, while phytyl diphosphate and solanesyl diphosphate have to be prepared. 8 - 1 0 Dry trichloroacetonitrile (15 mmol), 5 mmol ditriethylammo-nium phosphate, 1 0 and 30 ml dry acetonitrile are mixed in a round-bottom flask. Two millimoles of phytol in 15 ml acetonitrile is added dropwise over a 3-hr period. The mixture is stirred for another 12 hr, then 50 ml of acetone is added and concentrated ammonia is dropped into the solution until no further precipitation occurs. Precipitation occurs for 2 hr at 0°. The solid is repeatedly washed with 0.28 Μ ammonia in methanol to eliminate prenyl monophosphates. The resulting prenyl diphosphate is dried and used in the enzyme assay. Product analysis showed that this preparation is still heavily contaminated by inorganic phosphates which, however, do not interfere with the enzyme assays. I f further purification is desired this can be achieved by recrystallization in CHCl 3 -methanol . 8

[ 3H]Homogentisate, labeled by tri t ium exchange service, has to be purified prior to use in the following system: silica gel precoated thin-layer plates on glass and toluene/methanol/acetic acid (80/20/4 v/v/v) as developing solvent.

Preparation of Chloroplasts and Chloroplast Components

Chloroplasts are isolated from spinach leaves by standard procedures 1 1 and further purified on silica sol gradients. 1 2 Chloroplast compose. N. Joo, C . E . Park, J . K . G . Kramer, and M. Kates, Can. J. Biochem. 51, 1527

(1973). 9 R. Widmaier, J . Howe, and P. Heinstein, Arch. Biochem. Biophys. 200, 609 (1980).

1 0 G . Popjak, J . W. Cornfarth, R. H . Cornfarth, R. Ryhage, and S. de Witt Goodman, J. Biol. Chem. 237, 56 (1962).

1 1 H . Nakatani and J . Barber, Biochim. Biophys. Acta 461, 510 (1977).

[35] P R E N Y L Q U I N O N E S Y N T H E S I S I N C H L O R O P L A S T S 387

nents, e.g., envelope and thylakoid membranes and soluble chloroplast protein, are prepared as described. 1 3

Enzyme Assay for the Synthesis of Tocopherol and Its Intermediates in Chloroplasts

As outlined in Fig. 1, the synthesis of α-tocopherol comprises a number of reaction steps, catalyzed by the following enzymes: homogentisate decarboxylase-phytyltransferase; 5-adenosylmethionine: methyl-6-phy-tylquinol methyltransferase; 2,3-dimethylphytylquinolcyclase; S-adeno-sylmethionine: γ- tocopherol methyltransferase (no EC numbers available). Of the precursors and cosubstrates used only homogentisate, poly prenyl diphosphate, and S-adenosylmethionine are water soluble. Prenylquinones, prenylquinols, and tocopherols are not water soluble and it is difficult to determine their real concentration in the test. They are either added in ethanol (no more than 1% ethanol final concentration in the enzyme assay) or in diethyl ether, which is evaporated to dryness prior to the assay. Introduction of the methyl groups into the aromatic moiety is only possible at the quinol stage and not in the quinone form. 4

The same is valid for the formation of the chromanol stage ( IV —» V I I I ) . A photometrically adjusted amount of quinone (UV maxima, see Refs. 14 and 15; extinction coefficients, see Ref. 3) was dissolved in 1 ml methanol, reduced with a little solid N a B H 4 for 2 min, transferred to diethyl ether, and washed with H 2 0 . The diethyl ether is evaporated to dryness under N 2 in the reaction vials which were used later in the enzyme assay. Substrate concentrations described 4 1 4" 1 6 are 50-100 μΜ prenyl diphosphate, 100 μΜ 5-adenosylmethionine, 100-200 μΜ prenylquinol or tocopherol at pH 7.6-8.2 with MgCl 2 as cofactor (1-10 mM) and chloroplasts equivalent to 0.5-1 mg of chlorophyll. Other cofactors like cysteine, dithiothreitol (DTT), light, and M n 2 + do not seem to be necessary. 4 1 5 1 6

Increased solubilization of quinols and tocopherols can be achieved by detergents (Tween 80) which do not seem to inhibit the S-adenosyl-methionine: γ- tocopherol methyltransferase.1 7

1 2 G . Mourioux and R. Douce, Plant Physiol. 67, 470 (1981). 1 3 R. Douce and J . Joyard, Adv. Bot. Res. 7, 1 (1979). 1 4 J . Soll, G . Schultz, J . Joyard, R. Douce, and M. A. Block, Arch. Biochem. Biophys. 238,

290 (1985). 1 5 P. S. Marshall, S. R. Morris, and D. R. Threlfall, Phytochemistry 24, 1705 (1985). 1 6 B . Camara, F . Bardat, A. Seye, A. d'Harlingue, and R. Moneger, Plant Physiol. 70, 1562

(1982). 1 7 B . Camara and A. d'Harlingue, Plant Cell Rep. 4, 31 (1985).

388 P L A S T I D S [35]

Identification and Purification of Labeled Products

The intermediates obtained in tocopherol synthesis are generally purified by repeated thin-layer chromatography 4 1 8 1 9 or H P L C . 1 5 2 0 The incubation mixture is extracted with CHC1 3 : MeOH ( 1 : 2, ν : v ) . 2 1 The chloroform phase contains prenylquinols, prenylquinones, tocopherols, other lipids, and pigments. Ini t ial results have shown that prenylquinols are the products formed in this and the following reactions, i f the products are analyzed under nonoxidizing condit ions. 4 2 2 In general quinols are oxidized by air prior to thin-layer chromatography (for reasons, see above). A 25-^g aliquot of standard substances corresponding to the possible reaction products is added to the chloroform phase.

The first reaction in tocopherol and plastoquinone synthesis involves the prenylation of homogenisate with simultaneous decarboxylation (Fig. 1). The decarboxylation proceeds with stereochemical retention during the biosynthetic process. 2 3 While the chemical synthesis of monomethyl-prenylquinols using methylquinol, phytol, and B F 3 as described earlier yields a mixture of I , I I , and I I I , the enzymatic prenylation of homogenisate yields only one product ( I ) . 5 , 1 5 This is probably due to the directing influence of intermediates occurring during decarboxylation. Analysis of this reaction was done using spinach chloroplasts, [ 3H]homogentisate, and phytyl diphosphate. The products were purified by thin-layer chromatography using two different systems in succession [first run, silica gel, petrol (bp 60 -80° ) : diethyl ether, 10:1 (system I I ) ; rechromatography, cellulose plates impregnated with 7% paraffin, acetone: H 2 0 , 85 : 15 (system I I I ) , or by H P L C (Lichrosorb Si 60, 5 μπι , Merck, 0.06% dioxane in isooctane 1 5]. Substances are recovered from thin-layer plates by elution of the zones in question twice with 1 ml of methanol. The methanol is evaporated under N 2 and the residual dissolved in acetone. Only I was found to be labeled, this very specific initial reaction excludes already many further intermediates. 5 1 5 This initial step of prenylquinone formation is followed by methylation of the aromatic moiety with 5-adeno-sylmethionine as methyl group donor (see Fig. 4). Since the prenylation reaction yields only I the following methylation can occur only from two

1 8 J . Soli and G . Schultz, Biochem. Biophys. Res. Commun. 91, 715 (1979). 1 9 Η. K . Lichtenthaler, in "Lipids and Lipid Polymers in Higher Plants'' (M. Tevini and

Η. K . Lichtenthaler, eds.), p. 231. Springer-Verlag, Berlin and New York, 1977. 2 0 Η. K . Lichtenthaler and U . Prenzel, J. Chromatogr. 135, 493 (1977). 2 1 E . G . Bligh and W. J . Dyer, Can. J. Biochem. Physiol. 37, 911 (1959). 2 2 K . G . Hutson and D. R . Threlfall, Biochim. Biophys. Acta 632, 630 (1980). 2 3 R . Kriigel, Κ. H . Grumbach, Η. Κ. Lichtenthaler, and J . Retey, Bioorg. Chem. 13, 187

(1985).

[35] P R E N Y L Q U I N O N E S Y N T H E S I S I N C H L O R O P L A S T S 389

COO® • I

H 3 N - C - H I

CH2 NH2

CH 2 N^A*N H3C-S-CH2 N - ^ N ^

HO OH S - ADENOSYL - METHIONINE

C00® • I

H 3 N - C - H I CH2 NH2

I ( T j • H® S-CH2 N - ^ N ^

Λ S - ADENOSYL - HOMOCYSTEINE

F I G . 4. Methylation of 2-methyl-6-phytylquinol by S-adenosylmethionine. Reaction products are 2,3-dimethyl-6-phytylquinol and 5-adenosyIhomocysteine.

different intermediates: (1) I , (2) after cyclization of I from V I I . In vitro studies using isolated chloroplasts 4 demonstrated that methylation of I was about three times higher than from V I I . This specificity is underlined also by the finding that I I and I I I are methylated at 10 and 5% of the rate of I , respectively, with experimental conditions as described above.

The methylation product of I is I V , which is processed in a homologous sequence of events to form Χ. X formation takes place via V I I I , which is methylated to form X . Products are purified on systems I I and I I I (see Fig. 3). This sequence was supported from the data that I V is not methylated to V I . 4 1 5 In all reports known so far the cyclization of the prenylquinol to form the chromanol ring was not studied directly and it seems to be the slowest step in the α-tocopherol formation.

The reactions mentioned above described only one or two steps at a time of a series leading to α-tocopherol. I f it is necessary to look at the whole sequence, all cosubstrates have to be included (homogentisate, phytyl diphosphate, 5-adenosylmethionine). The incorporation rates vary from pmol to nmol/hr-mg chlorophyll for single reaction steps, which makes it obvious that only highly active chloroplast preparations can be used for these approaches. When these multiple step analyses are done

390 P L A S T I D S [35]

the products formed are I , I V , V I I , V I I I and X , 4 · 1 5 · 2 4 confirming the results described earlier. I t should be stressed again that it is difficult to separate the different possible isomers in tocopherol synthesis. Monomethylphy-tylquinols can only be separated by repeated thin-layer chromatography on precoated plates on glass or H P L C 4 1 5 1 9 2 0 ( X m a x , 254, X m a x n 253, \ m a x m 249 nm). Compounds I V and V are separated by simple chromatography on precoated thin-layer plates on glass using system I I . V I I I and I X should be purified as nitroso derivatives 2 5 or by H P L C . 1 5 2 0 I t is obvious from the literature and our own experience that successful separation of isomers depends strongly on the brand of chromatography plates used.

Plastoquinone Synthesis

Plastoquinone synthesis (Fig. 1) occurs essentially via reactions similar for tocopherol; homogentisate (20 μΜ) and solanesyl diphosphate (80 μΜ) are condensed to form the equivalent to I 5 (2-methyl-6-solanesyl-quinol), which is then methylated by 5-adenosylmethionine (70 μΜ) to yield plastoquinol-9 5 (2,3-dimethylsolanesylquinol) (Fig. 1). The rates of synthesis are again in the picomolar range per hr · mg chlorophyll. Purification of the incubation products is done by two successive thin-layer chromatography systems (first system; system I ; rechromatography, cellulose plates impregnated with 1% paraffin, acetone : H 2 0 , 90:10, system I V ) .

Localization of Prenylquinone Synthesis in Chloroplasts

Recently developed methods for the fractionation and purification of chloroplast components 1 3 enabled u s 5 1 4 2 4 2 6 to localize all but one enzyme in tocopherol and plastoquinone synthesis at spinach chloroplast envelopes. Thylakoids or soluble chloroplast protein had no enzymatic activi ty . Recombination of membranes with soluble chloroplast extract did not increase prenylquinone synthesis. These observations are now extended to envelope membranes from pea chloroplasts (J. Soli, unpublished). A l l test and purification conditions were essentially as described for chloroplasts. The amount of membranes used was between 50 and 100 μg protein per assay. The available data do not demonstrate the enzyme responsible for the cyclization of I V to yield V I I I . Though some enzymatic activity in plastoquinone synthesis is found associated with the thylakoid

2 4 G . Schultz, J . Soll, E . Fiedler, and D. Schulze-Siebert, Physiol. Plant. 64, 123 (1985). 2 5 S. Marcinkiewicz and J . Green, Analyst 84, 304 (1959). 2 6 J . Soll, R. Douce, and G . Schultz, FEBS Lett. \M, 243 (1980).

[35] P R E N Y L Q U I N O N E S Y N T H E S I S I N C H L O R O P L A S T S 391

membrane it is probably due to contamination of this membrane fraction by envelopes.

The envelope forms a two-membrane barrier, which surrounds the chloroplast and is present at all stages of chloroplast development. 2 7 I t is now possible to separate the two membranes into outer envelope and inner envelope membrane. 2 7 2 8 Applying these methods it is possible to align tocopherol and plastoquinone synthesis with the inner envelope membrane. 1 4 Again, all enzymes but the cyclization enzyme (IV —» V I I I ) were demonstrated.

Purification of Enzymes Involved in Tocopherol Synthesis

A membrane fraction obtained from pepper (Capsicum annuwri) chro-moplasts was shown to catalyze α-tocopherol synthesis via the same intermediates as described for chloroplasts 1 6 (Fig. 1). The methods used were essentially as above. This membrane fraction was then used as a source for the enzyme purification. A n acetone powder is obtained from the membranes at - 2 0 ° which is then solubilized in 0.1 Μ phosphate buffer (pH 7.0), 5 m M DTT, and Tween 80 (1 mg/ml), followed by ( N H 4 ) 2 S 0 4 precipitation (20-60%). The protein is purified by column chromatography 2 9: (1) blue Sepharose CL-6B, 50 m M K H 2 P 0 4 , 1 m M DTT, 1 m M E D T A , p H 6.2; (2) blue Sepharose CL-6B, 50 m M T r i s - H C l , 1 m M D T T , 1 m M E D T A , pH 8; (3) DEAE-Sephacel, 50 m M Tris-HCl, pH7.6 , 1 m M D T T e l u t e d with a gradient of 0-0.4 Μ K C l . Analysis of the active fractions obtained, by SDS-polyacrylamide gel electrophoresis Showed only one band at about 33,000 Da 2 9 (pH optimum, 8.2; Km S-adenosylmethionine, 2.5 μ Μ ; Km γ- tocopherol , 13.7 μΜ).29

Analysis of Envelope Membranes for Tocopherol and Plastoquinone

Determination should be done from fresh or deep-frozen material. Freeze-dried membranes contain less quinols and tocopherols than fresh membranes and more quinone and tocoquinone ins tead . 5 1 4 ' 3 0 3 1 (see Table I ) . Membranes are extracted either by CHCl 3 /MeOH (see earlier) or by

2 7 R. Douce, M . A. Block, A . J . Dorne, and J . Joyard, Subcell. Biochem. 10, 1 (1984). 2 8 K . Cline, J . Andrews, J . Mersey, Ε. H . Newcomb, and K . Keegstra, Proc. Natl. Acad.

Sei. U.S.A. 78, 3595 (1981). 2 9 A . d'Harlingue, F . Villat, and b. Camara, C. R. Seances Acad. Sei., Ser. 3 6, 233 (1985). 3 0 Η. K . Lichtenthaler, U . Prenzel, R. Douce, and J . Joyard, Biochim. Biophys. Acta 641, 99

(1981). 3 1 G . Schultz, Η. Bickel, Β. Buchholz, and J . Soll, in "Chloroplast Development" (G.

Akoyunoglou, ed.), p. 311. Balaban Int. Sei. Serv, Philadelphia, Pennsylvania, 1981.

392 P L A S T I D S [36]

T A B L E I

P R E N Y L L I P I D C O N C E N T R A T I O N IN C H L O R O P L A S T M E M B R A N E S F R O M S P I N A C H L E A V E S "

Envelope Inner Outer Retention Prenylquinone Thylakoid mixture envelope envelope time (min)

α-Tocopherol 1.1 2.8 6.7 9.8 5.6 a-Tocoquinone 0.24 0.2 — — 3.8 Phylloquinone K j 0.34 0.1 0.07 0.05 7.3 Plastoquinone-9 3.9* 1.2* 1.63 1.1 25.5 Plastoquinol-9 _ 1.54 1.1 11.3 Total prenylquinones 5.5 4.3 10.0 12.1 —

a Adapted from Refs. 14, 30, and 31; values are expressed in /xg/mg protein. b Represents the sum of plastoquinone and plastoquinol.3 0

hexane/acetone (10:4, v : v ) . 3 0 The lipid extract is further analyzed by HPLC (RP8, 7-jitm mesh, Merck) using methanol: water (95.7:4.3, ν : ν) as developing solvent (1.5 ml flow rate) 1 4 , 3 2 and two U V detectors set at 250 nm (to detect quinones) and 292 nm (to detect tocopherol and quinol), respectively. 1 4 3 3

Acknowledgments I would like to thank Prof. G . Schultz for his continuous support and encouragement

during the progress of this work.

3 2 Η. K . Lichtenthaler, in "Handbook of Chromatography" (Η. K . Mangold, ed.), p. 115. C R C Press, Boca Raton, Florida, 1984.

3 3 D. R. Threlfall and T . W. Goodwin, Biochem. J. 103, 573 (1967).

METHODS IN E N Z Y M O L O G Y , V O L . 148 Copyright © 1987 by Academic Press, Inc.

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