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ZEITSCHRIFT FÜR NATURFORSCHUNG

SECTION c

A EUROPEAN JOURNAL OF

BIOSCIENCES

Council

E . BüNNiNG,Tübingen A. B U T E N A N D T , München M . E I G E N , Göttingen

N . A M R H E I N , Bochum B. A . A S K O N A S , London W . B A R Z , Münster P. BÖGER, Konstanz G . B O R N KÄMM, Frei bürg D . B Ü C K M A N N , Ulm K . G . G Ö T Z , Tübingen G . GOTTSCHALK, Göttingen P. G R U S S , Heidelberg

Editorial Board

A. H A G E R , Tübingen K . H A H L B R O C K , Köln W. H A S S E L B A C H , Heidelberg P. K A R L S O N , Marburg F. K A U D E W I T Z , München J . K L E I N , Tübingen J. ST. SCHELL, Köln E. W E C K E R , Würzburg

Advisory Editorial Board

G . ISENBERG, Köln R. JAENICKE, Regensburg V . TER M E U L E N , Würzburg G . F. M E Y E R , Tübingen M . R A J E W S K Y , Essen H . SCHIMASSEK, Heidelberg D . SCHULTE-FROHLINDE, Mühlheim/R. G . S C H U L Z , Freiburg F. F. SEELIG, Tübingen

J. SEELIG, Basel H . S IMON, München W. STEGLICH, Bonn H . STIEVE, Aachen J. S U K O , Wien A . TREBST, Bochum G. WEISSENBÖCK, Köln G . W I C K , Innsbruck V . Z I M M E R M A N N , Würzburg

EDITED IN CO L L A B O R A T I O N

WITH THE INSTITUTES OF T H E M A X - P L A N C K - G E S E L L S C H A F T

Volume 41c 1986

V E R L A G D E R Z E I T S C H R I F T F Ü R N A T U R F O R S C H U N G

T Ü B I N G E N

Volume 41 c Zeitschrift für Naturforschung 1986

Contents

Conten t s of N u m b e r 1/2

O r i g i n a l C o m m u n i c a t i o n s

Identification of (/?)-Vicianin in D a v a l l i a t r i c h o -manoides Blume P. A . LizoTTE and J. E . POULTON 5

Differential Regulation of Two Genes Controlling the Biosynthesis of Isovitexin 7-O-Galactoside in Silene Plants J. M . STEYNS and J. v. B R E D E R O D E 9

Biosynthetic Capacity of Stachys Seedlings for Ver-bascoside and Related Caffeoyl Derivatives C. A N D A R Y and R. K . IBRAHIM 18

Isolation and Separation of Epidermal and Mesophyll Protoplasts from Rye Primary Leaves — Tis-sue-Specific Characteristics of Secondary Phenolic Product Accumulation M . S C H U L Z and G . WEISSENBÖCK 22

Partial Purification and Some Properties of 1-Sina-poylglucose: Choline Sinapoyltransferase ("Sina-pine Synthase") from Seeds of Raphanus sativus L . and Sinapis a l b a L . W. G R A W E and D . STRACK 28

Biosynthesis of the Furanoacetylene Phytoalexin Wyerone in V i c i a f a b a N . A . A L - D O U R I and P. M . D E W I C K 34

Host-Pathogen Interactions. X X X . Characterization of Elicitors of Phytoalexin Accumulation in Soy-bean Released from Soybean Cell Walls by Endo-polygalacturonic Acid Lyase K . R. D A V I S , A . G . D A R V I L L , P. A L B E R S H E I M , and A . D E L L 39

Inhibition of Phenylalanine Ammonia-Lyase i n v i t r o and i n v i v o by (l-Amino-2-phenylethyl)phos-phonic Acid , the Phosphonic Analogue of Phenylalanine B. L A B E R , H . - H . K I L T Z , and N . A M R H E I N 49

Flavin Nucleotide-Dependent 3-Hydroxylation of 4-Hydroxyphenylpropanoid Carboxylic Acids by Particulate Preparations from Potato Tubers J. M . B O N I W E L L and V . S. B U T T 56

Use of Immunotitration to Demonstrate Phyto-chrome-Mediated Synthesis de novo of Chalcone Synthase and Phenylalanine Ammonia Lyase in Mustard Seedling Cotyledons R. BRÖDENFELDT and H . M O H R 61

Tyrosine Biosynthesis in Sorghum b i c o l o r : Isolation and Regulatory Properties of Arogenate Dehydrogenase J. A . C O N N E L L Y and E . E . C O N N 69

Tyrosine Biosynthesis in Sorghum b i c o l o r : Characteristics of Prephenate Aminotransferase D. L . SIEHL, J. A . C O N N E L L Y , and E . E . C O N N 79

Flavonoids and Terpenoids from the Exudates of Some B a c c h a r i s Species E . W O L L E N W E B E R , I. SCHOBER, P. D O S T A L , D . H R A D E T Z K Y , F. J . A R R I A G A - G I N E R , and G . Y A T -SKIEVYCH 87

Transformation-Related Cellular Protein p53: In-creased Level in Untransformed Rat Cells Follow-ing Treatment with the Tumorpromoter, Tetra-decanoylphorbol-Acetate M . H E B E L , G . B R A N D N E R , H . K . H O C H K E P P E L , and D . G . B R A U N 94

Glycosphingolipid Analysis of Human Myeloid Leukemias (In German) B. K N I E P and P. F. M Ü H L R A D T 100

Properties of Vinorine Synthase — the R a u w o l f i a Enzyme Involved in the Formation of the Ajma-line Skeleton A . PFITZNER, L . P O L Z , and J . STÖCKIGT 103

High Performance Liquid Chromatography Coupled with Radioactivity Detection: A Powerful Tool for Determining Drug Metabolite Profiles in Biologi-cal Fluids K . - O . V O L L M E R , W . K L E M I S C H , and A . v. H O D E N BERG 115

Partial Purification and Characterization of S-Ade-nosyl-L-Methionine: Norreticuline N-Methyltrans-ferases from B e r b e r i s Cell Suspension Cultures C H I - K I T W A T , P. STEFFENS, and M . H . Z E N K 126

Contents IV

Biosynthesis of 4-Formyl-4-imidazoline-2-on, the Heterocyclic Base of Nikkomycin X R . - M . SCHMIDT, H . P A P E , and M . JUNACK 135

Metabolism of the Plant Growth Regulator (£) -[3H]2-Ethylhex-2-enoic Acid in H o r d e u m v u l g a r e B . SCHNEIDER, H. -R. SCHÜTTE, and A . PREISS 141

Metabolism and Degradation of Nicotinic Acid in Parsley ( P e t r o s e l i n u m hortense) Cell Suspension Cultures and Seedlings L . S C H W E N E N , D . K O M O S S A , and W. B A R Z 148

Basal Blotches and Blotchlessness of Poppy Flowers: A Chemogenetic Characterization (In German) H . B Ö H M 158

Chemical Investigations of Tropical Medicinal Plants, X X I . Long Chain Alky l Esters of Ferulic and p-Coumaric Acid from B a u h i n i a m a n c a H . A C H E N B A C H , M . STÖCKER, and M . A . C O N -STENLA 164

C y a n o p h o r a p a r a d o x a : Fatty Acids and Fatty Acid Synthesis i n v i t r o H . K L E I N I G , P. B E Y E R , C. SCHUBERT, B . L IEDVOGEL, and F. L Ü T K E - B R I N K H A U S 169

Degradation of N A D ( H ) by Endogenous Enzymes of Yeasts and Clostridia H.-J. SCHUETZ and H . SIMON 172

Genetic and Biochemical Studies on the Conversion of Dihydroflavonols to Flavonols in Flowers of P e t u n i a h y b r i d a G. F O R K M A N N , P. DE V L A M I N G , R. SPRIBILLE, H . WIERING, and A . W . SCHRÄM 179

Biosynthesis of Acridone Alkaloids. A Cell-free System from R u t a g r a v e o l e n s Cell Suspension Cultures A . B A U M E R T , G . SCHNEIDER, and D . GRÖGER 187

Phytoalexin Production by Isolated Soybean Proto-plasts H . M I E T H , V . SPETH, and J . E B E L 193

Composition of Lipopolysaccharides from Various Strains of R h o d o m i c r o b i u m v a n n i e l i i O. H O L S T , J. WECKESSER, B . R I E T H , and C. S. Dow 202

Plant Biochemistry of Xenobiotics. Mineralization of Chloroaniline/Lignin Metabolites from Wheat by

the White-Rot Fungus, P h a n e r o c h a e t e c h r y s o s p o -r i u m M . A R J M A N D and H . S A N D E R M A N N JR. 206

Further Studies on the Biosynthesis of Granaticin X I A N - G U O H E , C H I U - C H I N C H A N G , CHING-JER C H A N G , J . C. V E D E R A S , A . G . M C I N N E S , J . A . W A L T E R , and H . G . FLOSS 215

Minimal Time Requirement for Lasting Elicitor Effects in Cultured Parsley Cells H . STRASSER and U . M A T E R N 222

Elicitor-Stimulated Furanocoumarin Biosynthesis in Cultured Parsley-Cells: S-Adenosyl-L-Methio-nine: Bergaptol and S-Adenosyl-L-Methionine: Xanthotoxol O-Methyltransferases K . D . H A U F F E , K . H A H L B R O C K , and D . S C H E E L 228

The Topology of the Plastoquinone and Herbicide Binding Peptides of Photosystem I I in the Thyla-koid Membrane A . TREBST 240

Conten ts of N u m b e r 3


Biosynthesis of Daphnetin in D a p h n e mezereum L . ST. A . B R O W N 247

Monogalloylhamamelose from H a m a m e l i s v i r g i n i a n a (In German) G. SCHILLING and A . K E L L E R 253

Activation of Streptolysin S i n v i t r o by Oligonu-cleotides A . TAKETO and Y . TAKETO 258

Optimization of Conditions for Accurate Phospho-nate and Total Phosphorus Assay on Lipid Sam-ples, in Conjunction with Thin-Layer Chromatog-raphy V . M . K A P O U L A S and G . T H . TSANGARIS 263

Effect of the C0 2-Concentration during Growth on the Oxygen Evolution Pattern under Flash Light in C h l o r e l l a Y . SHIRAIWA and G . H . SCHMID 269

Contents V

A Large Chloroplast Thioredoxin / Found in Green Algae P. L A N G L O T Z , W . W A G N E R , and H . F O L L M A N N 275

Chlorophyll Photobleaching in Pigment-Protein Complexes R. CARPENTIER, R. M . L E B L A N C , and G . B E L L E -MARE 284

Diurnal Changes of Fructose-6-phosphate,2-kinase and Fructose-2,6-bis-phosphatase Activities in Spinach Leaves M . STITT, G . M I E S K E S , H . - D . SÖLING, H . GROSSE, and H . W . H E L D T 291

Molecular Mechanics Investigation on Conforma-tional Flexibility of 14ß Steroids in Drug-Receptor Interactions M . B O H L and M . W U N D E R W A L D 297

Alterations in the Activities of Rabbit Erythrocyte Membrane-Bound Enzymes Induced by Choles-terol Enrichment and Depletion Procedures E . K A M B E R and L . KOPEIKINA-TSIBOUKIDOU 301

Stimulation of Phosphatidylinositol Phosphorylation in the Sarcoplasmic Reticular Ca 2 +-Transport ATPase by Vanadate M . VARSÄNYI , G . B E H L E , and M . SCHÄFER 310

Relative Hypertrehalosaemic Activities of Naturally Occurring Neuropeptides from the A K H / R P C H Family G. G Ä D E 315

Notes

Influence of Temperature on the Transport of Ascor-bate across Artificial Membranes as Studied by the Spin Label Technique W. L O H M A N N , P. Z . T I A N , and D . H O L Z 348

The Influence of Spin Label on the Transport of As-corbate across Artificial Membranes W. L O H M A N N , P. Z . T I A N , and D . H O L Z 351

Effect of Magnetic Field on Ascorbic Acid Oxidase Activity, I V . S. G H O L E , P. S. D A M L E , and W. H.-P. T H I E M A N N 355

Oxygen Incorporation in Cleavage of l ßO-Labeled 13-Hydroperoxylinoleyl Alcohol into 12-Hydroxy-(3Z)-dodecenal in Tea Chloroplasts A . H A T A N A K A , T. K A J I W A R A , J. SEKIYA, and H . TOYOTA 359

New Pulvinic Acid Derivatives from P u l v e r o b o l e t u s Species (Boletales) (In German) R. M A R U M O T O , C. KILPERT, and W. STEGLICH 363

Volatiles from the Defensive Secretions of Two Rove Beetle Species (Coleoptera: Staphylinidae) K . DETTNER and G . SCHWINGER 366

Isolation of Beef Brain Phosphonolipids by Thin Layer Chromatography: Their Identification and Silicic Acid Column Chromatographie Separation M . C. MOSCHIDIS 369

Broadband, Non-Thermal Millimeter-Wave Influence on Giant Chromosomes C H R . KOSCHNITZKE, F. K R E M E R , L . SANTO, A . POGLITSCH, and L . G E N Z E L 321

The Anal Sac Secretion of Viverrids from the Genus G e n e t t a J . JACOB and H . S C H L I E M A N N 325

Some Remarks About Laser-Induced Mass Spectro-metry of Bacteria J . A L B R E C H T , E . W. SCHMID, and R. SÜSSMUTH 337

Transepithelial Cytophagy by T r i c h o p l a x adhaerens F. E . Schulze (Placozoa) Feeding on Yeast H . WENDEROTH 343

Conten t s of N u m b e r 4


Storage of Quinolizidine Alkaloids in Epidermal Tis-sues M . W I N K 375

Indole Alkaloids from O c h r o s i a e l l i p t i c a Plant Cell Suspension Cultures K . - H . P A W E L K A and J . STÖCKIGT 381

Major Indole Alkaloids Pruduced in Cell Suspension Cultures of Rhazya s t r i c t a Decaisne K . - H . P A W E L K A and J . STÖCKIGT 385

Contents VI

Determination of Hyoscyamine and Scopolamine in D a t u m i n n o x i a Plants by High Performance Liquid Chromatography K . - H . P L A N K and K . G . W A G N E R 391

Evidence for the Presence of Neutral Glycerylether Derivatives in Pollen Lipids of Pine Tree P i n u s halepensis N . K . ANDRIKOPOULOS, A . S IAFAKA-KAPADAI , N . YANOVITS-ARGYRIADIS, and C A . DEMOPOULOS

396

A Chemical Investigation of P u e r a r i a mirifica Roots J. L . I N G H A M , S . T A H A R A , and SR. Z . DZIEDZIC 403

Characterization and Properties of Different Glucosyltransferases Isolated from Suspension-Cultured Cells of D a u c u s c a r o t a E . INGOLD and H . U . SEITZ 409

Conversion Rate of Ozone with Volatile Terpenes in the Surface Region of Conifer Needles (In German) H . Russi 421

Impact of U V - B Radiation on Photosynthetic Assimilation of 1 4C-Bicarbonate and Inorganic 1 5 N -Compounds by Cyanobacteria G . D Ö H L E R , I. B I E R M A N N , and J. Z I N K 426

Pentachlorophenol Inhibits Photosynthetic Electron Flow and Quenches Chlorophyll Fluorescence after Preillumination C H . NIEHRS and J. A H L E R S 433

The Diazo Reaction of Bilirubins and Phycorubins: A Quantitative Study W . K U F E R , O. SCHMID, G. SCHMIDT, and H . SCHEER 437

Orientation Measurements on Ordered Multibilay-ers of Phospholipids and Sphingolipids from Synthetic and Natural Origin by A T R Fourier Transform Infrared Spectroscopy K . B R A N D E N B U R G and U . SEYDEL 453

The Dynamics of Bone Mineral in Some Verte-brates. F. C. M . DRIESSENS and R. M . H . VERBEECK 468

A Serum-Free i n v i t r o Culture System for Crayfish Organs G . G E L L I S S E N , M . T R A U B , and K . - D . SPINDLER 472

Characterization of a Defective Mutant of the Dahlemense Strain of Tobacco Mosaic Virus J. M . W E R T Z , P. S M I T A M A N A , and S. SARKAR 477

Electronmicroscopical Contrast by Palladium Chloride J. M . FERRER, A . T A T O , and J. C. STOCKERT 483

Notes

Cyanidin 3-Gentiobioside from Primary Leaves of Rye (Seeale c e r e a l e L.) E . B U S C H , D . STRACK, and G . WEISSENBÖCK 485

' H - N M R Studies on the Effect of Spin Label on Lipids W. L O H M A N N and B . K I E F E R 487

The Macromolecular Structure of Collagen in Ten-don Fibres of Dermatosparactic Animals E . M O S L E R , W . F O L K H A R D , W . G E E R C K E N , O. H E L L E , E . K N Ö R Z E R , M . H . J. K O C H , C H . M . L A P I £ R E , H . N E M E T S C H E K - G A N S L E R , B . N U S G E N S , and T H . N E M E T S C H E K 489

Quasisynergism as Evolutionary Advance to In-crease Repellency of Beetle Defensive Secretions K . DETTNER and R. G R Ü M M E R 493



Pyoverdine Type Siderophores from Pseudomonas a e r u g i n o s a (In German) G . BRISKOT, K . T A R A Z , and H . BUDZIKIEWICZ 497

A New Biflavone and Further Flavonoids from the Moss H y l o c o m i u m splendens R. B E C K E R , R. M U E S , H . D . ZINSMEISTER, F. H E R ZOG, and H . GEIGER 507

4-O-ß-D-Glucosides of Hydroxybenzoic and Hy-droxyeinnamic Acids — Their Synthesis and Determination in Berry Fruit and Vegetable B. SCHUSTER, M . W I N T E R , and K . H E R R M A N N 511

Chemistry and Morphology of Epicuticular Waxes from Leaves of Five E u p h o r b i a Species H . H E M M E R S , P . -G . G Ü L Z , and K . HÄNGST 521

Contents

^-Acetyl^-indolylmethylglucosinolate in Seedlings of T o v a r i a p e n d u l a Ruiz et Pav. H . SCHRAUDOLF and R. B Ä U E R L E 526

Changes in Fructose-2,6-bisphosphate Level during the Growth of Suspension Cultured Cells of C a t h a r a n t h u s roseus H . A S H I H A R A 529

Biosynthesis of Pyoluteorin: A Mixed Polyketide-Tricarboxylic Acid Cycle Origin Demonstrated by [l,2- 1 3C 2]Acetate Incorporation D. A . C U P P E L S , C . R. H O W E L L , R. D . STIPANOVIC, A . STOESSL, and J . B . STOTHERS 532

The Expression of the Isovitexin 7-O-Xylosylating Gene g X in Silene pratensis and S. d i o i c a is Re-stricted to the Petals J . STEYNS and J . v. B R E D E R O D E 537

Ergosterol as a Biochemical Indicator of Fungal In-fection in Spruce and Fir Needles from Different Sources W. F. O S S W A L D , W. H O L L , and E . F. ELSTNER 542

Plant Defense Substances X X I X . Isolation, Characterization and Synthesis of Turgorines from G/e-d i t s i a t r i a c a n t h o s L . (In German) H . SCHILDKNECHT, R. M U L E Y , G . M . K R E S B A C H , P. K U N Z E L M A N N , and D . K R A U S S 547

Preparation of Pheromones by Simple Procedures H . K . M A N G O L D and H . B E C K E R 555

Increased Lipoxygenase Activity is Involved in the Hypersensitive Response of Wheat Leaf Cells In-fected with Avirulent Rust Fungi or Treated with Fungal Elicitor C. A . O C A M P O , B . M O E R S C H B A C H E R , and H . J . G R A M B O W 559

Stimulation of Photorespiration by the Carbonic Anhydrase Inhibitor Ethoxyzolamide in C h l o r e l l a v u l g a r i s Y . SHIRAIWA and G . H . SCHMID 564

The Photosynthetic Apparatus of E c t o t h i o r h o d o -s p i r a h a l o c h l o r i s . 2. Accessibility of the Membrane Polypeptides to Partial Proteolysis and A n -tenna Polypeptide Assignments to Specific Chro-mophores R. STEINER, A . A N G E R H O F E R , and H . SCHEER 571

VII

Effects of Adenosine-3':5'-monophosphate (cAMP) on the Activity of Soluble Protein Kinases in Maize (Zea mays) Coleoptile Homogenates B. JANISTYN 579

Effects of Pyridazinone Herbicides during Chloro-plast Development in Detached Barley Leaves. III. Effects of S A N 6706 on Photosynthetic Activity and Chlorophyll-Protein Complexes G . L A S K A Y , E . L E H O C Z K I , A . L . D O B I , and L . S Z A L A Y 585

Photocontrol of Chloroplast Lipids in Fern Gameto-phytes ST. KRAISS and A . R. GEMMRICH 591

Changes in the Stoichiometry of Photosystem II Components as an Adaptive Response to High-Light and Low-Light Conditions during Growth A . W I L D , M . HÖPFNER, W . R Ü H L E , and M . RICHTER 597

The Activation of the Cytochrome P-450 Dependent Monooxygenase System by Light D. M Ü L L E R - E N O C H and H . G R U L E R 604

Specificity of Rabbit Antibodies Elicited by Related Synthetic Peptides A . CHERSI , R. A . H O U G H T E N , F. C H I L L E M I , R. ZITO, and D . CENTIS 613

Structure Investigations of Agonists of the Natural Neurotransmitter Acetylcholine, IV. X-Ray Structure Analyses of Trimethylpentylammo-nium-chloride and (4-Acetoxybutyl)trimethyl-ammonium-iodide A . G I E R E N and M . KOKKINIDIS 618

Structure Investigations of Agonists of the Natural Neurotransmitter Acetylcholine, V . Structure-Ac-tivity Correlations for Cholinergic Stimulants De-rived from Crystal Structures of Their Halides A . G I E R E N and M . KOKKINIDIS 627

Structure Investigations of Agonists of the Natural Neurotransmitter Acetylcholine, V I . X-Ray Structure Analysis of Trimethyl[2-(propionyloxy)-ethyl]ammonium-iodide (O-Propionylcholine-iodide) A . G I E R E N and M . KOKKINIDIS 641

Is the Calcium Pump Involved in Calcium Release? M . U N G E H E U E R , A . M I G A L A , and W . H A S S E L B A C H

647

Contents VIII

Selective Abolition of Sarcoplasmic Reticulum Vesi-cles' Calcium Releasing Mechanisms W. H A S S E L B A C H , M . U N G E H E U E R , A . M I G A L A , and K . RITTER 652

The Sensitivity of the Ventral Nerve Photoreceptor of L i m u l u s Recovers after Light Adaptation in Two Phases of Dark Adaptation I. C L A S S E N - L I N K E and H . STIEVE 657

Notes

Antipeptide Antibodies: Do They Distinguish H L A -Alloantigens? A . CHERSI , R. A . H O U G H T E N , D . Z E L A S C H I , and C . CENCIARELLI 668

Erratum to A . PFITZNER, L . P O L Z , and J. STÖCKIGT, Z . Naturforsch. 41c, 103-114 (1986) 671



Esters of Benzyl Alcohol and 2-Phenyl-ethanol-l in Epicuticular Waxes from J o j o b a Leaves P . - G . G Ü L Z and F.-J. M A R N E R 673

Colletruncoic Acid Methyl Ester, a Unique Meroter-penoid from C o l l e t o t r i c h u m t r u n c a t u m A . STOESSL and J. B . STOTHERS 677

Complex Flavonoids from P i t y r o g r a m m a Frond Exu-dates: Synthesis of Two Flavones with C—C-Linked Dihydrocinnamoyl Substituents M . I INUMA, K . H A M A D A , M . M I Z U N O , F. A S A I , and E . W O L L E N W E B E R 681

Occurrence of 2-(2-Hydroxy-4,7-dimethoxy-2H-l,4-benzoxazin-3-one)-ß-D-glucopyranoside in T r i t i -cum aestivum Leaves and Its Conversion into 6-Methoxy-benzoxazolinone H . J. G R A M B O W , J. L Ü C K G E , A . K L A U S E N E R , and E . M Ü L L E R 684

6-(Hydroxythio)carbonylpyridine-2-carboxylic Acid and Pyridine-2-carboxylic Acid-6-monothiocar-boxylic Acid as Intermediates in the Biosynthesis

of Pyridine-2,6-di(monothiocarboxylic Acid) from Pyridine-2,6-dicarboxylic Ac id (In German) U . H I L D E B R A N D , K . T A R A Z , and H . BUDZIKIEWICZ

691

A Furanoheliangolide in H e l i a n t h u s d e b i l i s ; Implica-tions for a Chemotaxonomy of the Genus H e l i a n thus O. SPRING, V . K L E M T , K . A L B E R T , and A . H A G E R

695

UDP-Glucose: Anthocyanidin/Flavonol 3-0-Gluco-syltransferase in Enzyme Preparation from Flower Extracts of Genetically Defined Lines of M a t t h i o l a i n c a n a R. Br. M . T E U S C H , G . F O R K M A N N , and W . SEYFFERT 699

Cyanidin 3-Oxalylglucoside in Orchids D. STRACK, E . B U S C H , V . W R A Y , L . G R O T J A H N , and E . K L E I N 707

Characterization of Glutamine Synthetase of Roots, Etiolated Cotyledons and Green Leaves from Sinapis a l b a (L.) R. MANDERSCHEID and A . W I L D 712

Are Polyphosphoinositides Involved in Signal Trans-duction of Elicitor-Induced Phytoalexin Synthesis in Cultured Plant Cells? H . STRASSER, C H . H O F F M A N N , H . G R I S E B A C H , and U . M A T E R N 717

Herbai Insecticides III. Pyrethrin I in the Essential Oil of Chrysanthemum b a l s a m i t a L . (In German) H . J. B E S T M A N N , B . C L A S S E N , U . K O B O L D , O. V O -STROWSKY, and F. K L I N G A U F 725

Interference of Dimethazone with Formation of Ter-penoid Compounds G . S A N D M A N N and P. BÖGER 729

Species-Specific Differences in Acetyl Coenzyme A Synthesis of Chloroplasts H . -J. T R E E D E , B . R I E N S , and K . - P . H E I S E 733

Nitrogen and Sulfur Starvation of the Cyanobac-terium Synechococcus 6 3 0 1 . A n Ultrastructural, Morphometrical, and Biochemical Comparison G . W A N N E R , G . H E N K E L M A N N , A . SCHMIDT, and H . -P. KÖST 741

Contents IX

Conjugated Enamino Compounds, a New Molecular Probe for the Mechanism of Photosynthetic Electron Transport T. A s A M i , N . T A K A H A S H I , and S. Y O S H I D A 751

Acyclo Nucleosides and Nucleotides: Synthesis, Conformation and Other Properties, and Be-haviour in Some Enzyme Systems, of 2',3'-Seco Purine Nucleosides, Nucleotides and 3':5'-Cyclic Phosphates, Analogues of c A M P and c G M P R . STOLARSKI, Z . K A Z I M I E R C Z U K , P. LASSOTA, and D . SHUGAR 758

Azadirachtin, a Chemical Probe for the Study of Moulting Processes in Rhodnius p r o l i x u s E . S. G A R C I A , M . U H L , and H . R E M B O L D 771

Conten t s of N u m b e r 9 / 1 0


2-Hydroxy-4-methoxy-5-methyl Pyridine N-Oxide, an A l 3 + Complexing Metabolite from Pseudomonas c e p a c i a (In German) ST. W I N K L E R , W . O C K E L S , H . BUDZIKIEWICZ, H . K O R T H , and G . PULVERER 807

Characterization of Volatile Constituents from Heterotrophic Cell Suspension Cultures of R u t a graveolens M . J O R D A N , C. H . R O L F S , W . B A R Z , R . G . B E R GER, H . K O L L M A N N S B E R G E R , and F. DRAWERT 809

Comparative Enzymatic Degradation of H 1 Subfrac-tions from Syrian Hamster Tissues E . H R A B E C , A . P L U C I E N N I C Z A K , and H . P A N U S Z

776

Polymerization of Actin in the Absence and Pres-ence of Cytochalasin B: Problems of Determining "Critical Concentration" B . F U S S M A N N and P. D A N C K E R 781

Proliferation Kinetics and Metabolie Features of i n v i t r o Grown Ehrlich Ascites Tumor Cells in the Presence of Exogenous Pyruvate W. K R O L L , ST. POSTIUS, and F. SCHNEIDER 787

Triplet-Selective Chemistry: a Possible Cause of Biological Microwave Sensitivity F. K E I L M A N N 795

Identification and Biosynthesis of Glucosylated and Sulfated Flavonols in F l a v e r i a b i d e n t i s L . V A R I N , D . B A R R O N , and R. IBRAHIM 813

Substrate-Dependent Arylsulfatase Activity in the Cyanobacterium Plectonema 73110 S. M Ü L L E R and A . SCHMIDT 820

Volatile Fragrance Compounds from the Fungus G l o e o p h y l l u m o d o r a t u m (Basidiomycotina) H.-P. H A N S S E N , V . S I N N W E L L , and W.-R. A B R A H A M 825

A n Elicitor of the Hypersensitive Lignification Response in Wheat Leaves Isolated from the Rust Fungus P u c c i n i a g r a m i n i s f. sp. t r i t i c i . I. Partial Purification and Characterization B. MOERSCHBACHER, K . H . K O G E L , U . N O L L , and H . J . REISENER 830

Notes

Interaction between Spin Labels and D P P C Vesicles W. L O H M A N N , B . KIEFER, and W. S C H M E H L 799

Aspects of Mycobacterial Response to Beryllate Ions i n v i t r o H . J . M A C C O R D I C K 802

Effect of Tuftsin on the Phagocytotic Activity of the Unicellular Tetrahymena. Does Primary Interaction Develop Imprinting? G . C S A B A , V . L Ä S Z L Ö , and P. K O V A C S 805

An Elicitor of the Hypersensitive Lignification Response in Wheat Leaves.Isolated from the Rust Fungus P u c c i n i a g r a m i n i s f. sp. t r i t i c i . II. Induc-tion of Enzymes Correlated with the Biosynthesis of Lignin B. MOERSCHBACHER, B . H E C K , K . H . K O G E L , O . OBST, and H . J . REISENER 839

Metabolie Conversions of Trichothecene Myco-toxins: Biotransformation of 3-Acetyldeoxy-nivalenol into Fusarenon-X N . C. P. B A L D W I N , B . W. BYCROFT, P. M . D E W I C K , and J . GILBERT 845

Contents X

Biochemical Properties and Crystal Structure of Ethylmethylglyoxal Bis(guanylhydrazone) Sulfate — an Extremely Powerful Novel Inhibitor of Ade-nosylmethionine Decarboxylase H . E L O , I. M U T I K A I N E N , L . A L H O N E N - H O N G I S T O , R. L A I N E , J . J Ä N N E , and P. L U M M E 851

Protein Phosphorylation — Dephosphorylation in the Cytosol of Pea Mesophyll Cells R. H R A C K Y and J . S O L L 856

Diethyldithiocarbamate, a New Photosystem I Electron Donor of Mehler-Type Hi l l Reactions B. L . U P H A M and K . K . HATZIOS 861

Photodestruction of Endogenous Porphyrins in Relation to Cellular Inactivation of P r o p i o n i b a c t e r i u m acnes T. B. M E L 0 and G . REIS/ETER 867

The Photosynthetic Apparatus of E c t o t h i o r h o d o -s p i r a h a l o c h l o r i s . 3. Effect of Proteolytic Digestion on the Photoactivity R. STEINER, B . K A L U M E N O S , and H . SCHEER 873

Phenolic Herbicides: Correlation between Lipo-philicity and Increased Inhibitory Sensitivity of Thylakoids from Higher Plant Mutants J . D U R N E R , A . T H I E L , and P. BÖGER 881

Biosynthesis and Turnover of Cell Wall Glycopro-teins during the Vegetative Cell Cycle of C h l a m y -domonas r e i n h a r d i i J . VOIGT 885

Nucleic Acid-Binding Activities of the Intermediate Filament Subunit Proteins Desmin and Glial Fibrillary Acidic Protein C. E . VORGIAS and P. T R A U B 897

Soluble and Insoluble Rat Liver Chromatin is Differ-ent in Structure and Protein Composition R. BRUST 910

Specific Binding of Calcium to Soluble Chromatin R. BRUST 917

On the Direct Observation of Water-Fluxes in Tis-sues and Leaves U . - A . HIRTH and R. L A W A C Z E C K 923

(/?)Mandelonitrile and Prunasin, the Sources of Hydrogen Cyanide in A l l Stages of Paropsis a t o -m a r i a (Coleoptera: Chysomelidae) A . NAHRSTEDT and R. H . DAVIS 928

Latitude Dependent Circadian Rhythms of Carabid Beetles

G. L E Y K and W . M A R T I N 935

Notes

New Flavonoids from the Exudate of B a c c h a r i s b i g e l o v i i (Asteraceae) F. J . A R R I A G A - G I N E R , E . WOLLENWEBER, and D. H R A D E T Z K Y 946

The C-Glycosylflavone Pattern of Passiflora i n c a r -n a t a L . H . GEIGER and K . R. M A R K H A M 949

Antiproliferative Activity of Derivatives of t r a n s -Bis(salicylaldoximato)copper(II) i n v i t r o . Some i n v i v o Properties of the Parent Compound H . E L O and P. L U M M E 951

Effect of Tumour Regression on Serum and Tissue Copper Concentration in Mice Bearing Induced Fibrosarcoma P. K . C H A K R A V A R T Y , A . G H O S H , and J . R. CHOWDHURY 956

Conten t s of N u m b e r 1 1 / 1 2

Contents of Nos 1 - 1 2 I I I - X U


Volatiles from Liquid Cultures of L e n t i n e l l u s c o c h l e a t u s (Basidiomycotina) H.-P. H A N S S E N and W . - R . A B R A H A M 959

Biosynthesis of Flavor Compounds by Microorgan-isms. 6. Odorous Constituents of P o l y p o r u s d u r u s (Basidiomycetes) R. G . BERGER, K . N E U H Ä U S E R , and F. DRAWERT

963

R h i z o m n i u m m a g n i f o l i u m and R. p s e u d o p u n c t a t u m , the First Mosses to Yield Flavone Glucuronides R. M U E S , G . LEIDINGER, V . L A U C K , H . D . Z I N S MEISTER, T. K O P O N E N , and K . R. M A R K H A M 971

Contents

Flavonoid Constituents of Rhamnus l y c i o i d e s L . M . P A ^ Ä , S. M Ä N E Z , and A . V I L L A R 976

Green Algae (Scenedesmus o b l i q u u s ) Contain Three ThioreJoxins of Regulär Size P. L A N J L O T Z , W. W A G N E R , and H . F O L L M A N N 979

A Simple and Rapid Method for Isolation of 124 kDa Oat Photochrome R. GRIMM and W. RÜDIGER 988

Investigaion of the Peptide Chain of 124 kDa Phyto-chrome: Localization of Proteolytic Fragments and Epitopes for Monoclonal Antibodies R. G R M M , F. LOTTSPEICH, H . A . W. SCHNEIDER, and W RÜDIGER 993

Isolation and Characterization of 3 Protochlorophyl-lides from Pigment Mutant C-2 A ' of Scenedesmus o b l i q m s K. KOIZABASIS and H . SENGER 1001

Phosphoenolpyruvate Carboxylase from Maize Leave*. Studies Using ß-Methylated Phosphoenolpyruvate Analogues as Inhibitors and Substrates D. H . G O N Z A L E Z and C. S. A N D R E O 1004

Purification of the Chloroplast Pyruvate Dehydrogenase Complex from Spinach and Maize Mesophyll H . -J. ^ E E D E and K. -P . H E I S E 1011

On the Pvole of Magnesium in the Reaction of the Pyruvite Kinase from S a l m o n e l l a t y p h i m u r i u m C. G A I C I A - O L A L L A and A . GARRIDO-PERTIERRA

1018

Content and Metabolism of Indole-3-acetic Acid (IAA) in Healthy and Rust-Infected Wheat Leaf Segmeits G. WIISE and H . J. G R A M B O W 1023

The Procuction of Pyrenocines A and B by a Novel A l t e r n i r i a species B. TAI and D . J. ROBESON 1032

Sensitiviiy of a Phototrophic Bacterium to the Herbi-cide Sulfometuron Methyl, an Inhibitor of Brancied Chain Amino Acid Biosynthesis I. SCHNEIDER and J . -H. K L E M M E 1037

XI

Adsorbent Culture of Tobacco Cell Suspensions with Different Adsorbents R. M A I S C H , B . K N O O P , and R. BEIDERBECK 1040

Changes in Levels of Cellular Constituents in Suspension Culture of C a t h a r a n t h u s roseus Associated with Inorganic Phosphate Depletion T. U K A J I and H . A S H I H A R A 1045

Flow Cytometric DNA-Analysis of Plant Protoplasts Stained with D A P I I. U L R I C H and W. U L R I C H 1052

Evidence for the Intercalation of Thalidomide into D N A : Clue to the Molecular Mechanism of Thalidomide Teratogenicity? H . P. K O C H and M . J. C Z E J K A 1057

Formation and Structure of Radicals from D-Ribose and 2-Deoxy-D-ribose by Reactions with SOJ Radicals in Aqueous Solution. A n i n - s i t u Electron Spin Resonance Study J. N . H E R A K and G . B E H R E N S 1062

Alkylene-bis-isothiocyanates: Novel Insect Growth Regulators G . M A T O L C S Y , I. UJVÄRY, L . M . RIDDIFORD, and K . H I R U M A 1069

C 9 Aliphatic Aldehydes: Possible Sex Pheromone from Male Tropical West African Shield Bug, S p h a e r o c o r i s annulus A . J. E . G O U G H , D . E . G A M E S , B . W. STADDON, D . W. KNIGHT, and T. O. OLAGBEMIRO 1073

Individual Variation in the Sex Pheromone Compo-nents of the False Codling Moth, C r y p t o p h l e b i a l e u c o t r e t a (Lepidoptera: Tortriciade) A . B . A T T Y G A L L E , J. S C H W A R Z , O. VOSTROWSKY, and H . J. B E S T M A N N 1077

Screening and Use of Sex Attractants in Monitoring of Geometrid Moths in Bulgaria M . A . SUBCHEV, J. A . G A N E V , O. VOSTROWSKY, and H . J. B E S T M A N N 1082

Relative Potencies of Antagonists of the Luteinizing Hormone Releasing Hormone with Lys 8 and A r g 8

and Substitutions in Positions 3 , 5, 6, 7 and 8 K . F O L K E R S , C. BOWERS, P.-F. L . T A N G , M . K O B O -TA, X . SHAO-BO, W. B E N D E R , and L . Y I N - Z E N G

1087

Contents XII

Effect of External Calcium Concentration on the In-tensity Dependence of Light-Induced Membrane Current and Voltage Signals in Two Defined States of Adaptation in the Photo-Receptor of L i m u l u s H . STIEVE, H . G A U B E , and J . K L O M F A S S 1092

Effects of Low Frequency Magnetic Fields on Chick Embryos. Dependence on Incubation Tempera-ture and Storage of the Eggs J . P. JüUTILAINEN 1111

Notes

Preliminary Studies towards a Monograph of the Liehen Family Roccellaceae Chev. VII . Second-ary Products and Relationships of the Genera Combea de Not. and Schizopelte T. M . Fries (In German) G. F O L L M A N N and M . G E Y E R 1117

Rearrangement of Glaucolide A into Vernojal-canolide 8-O-Methacrylate M . M A R T I N E Z , A . SÄNCHEZ, G . L O P E Z , and P. J O S E P H - N A T H A N 1119

Thin Layer Chromatographie and IR Spectral Evi -dence for the Presence of Phosphonolipids in Human Sperm M . C. MOSCHIDIS 1121

Evidence for the Presence of Glycerophos-phonolipids in the Land Snail E o b a n i a Ver-m i c u l a t a M . C. MOSCHIDIS 1124

A n Improved Procedure for the Quantitative Esti-mation of the Rust Fungus in Infected Plant Tissue G . WIESE, D . H U G O - W I S S E M A N N , and H . J . G R A M -BOW 1127

Low Molecular Mass Inhibitors from Calf Thymus Selective for T-Lymphocyte Proliferation H . P. MATTHIESSEN and H . R. M A U R E R 1131

The Effect of Volatile Anesthetics on Giant Neurons in the Lobula Plate in the Fly K . KIRSCHFELD 1137

Microfilament-Supported Macrovilli in the Hindgut of the Polychaete D i n o p h i l u s g y r o c i l i a t u s U . OSTER 1139

Comment on: Is there an Equilibrium between Ascorbic and Dehydroascorbic Acids? H . W . M U E L L E R 1145

R e p o r t

The Glio-Axonal Interaction and the Problem of Regeneration of Axons in the Central Nervous System — Concept and Perspectives H . W O L B U R G , J . N E U H A U S , and A . M A C K 1147

Subject Index 1157

Authors Index 1181

The Photosynthetic Apparatus of Ectothiorhodospira halochloris 2. Accessibility of the Membrane Polypeptides to Partial Proteolysis and Antenna Polypeptide Assignments to Specific Chromophores R. Steiner*, A . Angerhofer +, and H . Scheer* * Botanisches Institut der Universität München, Menzinger Straße 67, D-8000 München 19 + Physikalisches Institut, Teilinstitut 3, Universität Stuttgart, Pfaffenwaldring 57,

D-7000 Stuttgart 80 Z. Naturforsch. 41c, 571-578 (1986); received January 9, 1986

Bacterial Photosynthesis, Ectothiorhodospira, Membrane Topology, Antenna Polypeptides, Fluorescence, Circular Dichroism, Energy Transfer

E . halochloris thylakoids and spheroplasts were treated with trypsin, thermolysin or Proteinase K to determine which proteins are exposed at the different membrane surfaces. Based on SDS Polyacrylamide analysis, all 9 Polypeptides are exposed on the cytoplasmic side. Only one (28 kDa) is accessible from the periplasmic side. This Polypeptide is generally isolated as the H-subunit of the reaction centers of photosynthetic bacteria, but is in the case of E . halochloris rather isolated with the antenna (B 800/1020) (Steiner and Scheer, Biochim. Biophys. Acta 807, 278, 1983).

Proteolysis is accompanied by a shift of the absorption band at longest wavelengths from 1020 to 960 nm (B 800/960), which upon Standing is shifted further to 680 nm ("B" 800/680). The spectral changes are similar to the ones reported earlier for treatment with acid, and are also inducible with urea. The correlation of SDS-PAGE and absorption spectroscopy shows, that the chromophores absorbing at 1020 nm are transformed simultaneously with the degradation of the 6.5 kDa (= a) Polypeptide.

Introduction

Much progress has been made in recent years in understanding the topology of photosynthetic membranes in purple bacteria. The techniques used in-cluded biochemical methods of different specifities and advantages like labelling with antibodies [1], radioiodonation [2], photoaffinity labelling [3], cross-linking experiments [4—6] or proteolytic digestion [7—9]. They also included diffraction methods, like high-resolution electron microscopy [10—12] and most recently x-ray diffraction of isolated reaction centers [13].

Most of this work was focused on only a small number of closely related bacterial species. Particu-lar emphasis has been placed on the Rhodospirillales [14] e.g. Rs. r u b r u m , R p . s p h e r o i d e s , R p . c a p s u l a t a and R p . v i r i d i s . Much less is known about species

Abbreviations: Rp, Rhodopseudomonas; Rs, Rhodospiril-l u m ; E , Ectothiorhodospira; bchl, bacteriochlorophyll; SDS, sodium dodecyl sulfate; PAGE, Polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonylfluoride; EDTA, ethylendiamintetraacetate; cd, circular dichroism; LHP, light-harvesting-protein; RC, reaction center.

Reprint requests to Prof. Dr. H. Scheer.

Verlag der Zeitschrift für Naturforschung. D-7400 Tübingen 0341 -0382/86/0500-0571 S 01.30/0

from other genera [15], and their structural relations to the Rhodospirillales. We have recently begun to study the photosynthetic apparatus of E . h a l o c h l o r i s [16], an alcalophilic and extremely halophilic bacteriochlorophyll b containing organism.

The main difference as compared to e.g. R p . v i r i d i s is a second near-infrared absorption band besides the common one at 1020 nm, peaking at 800 nm with a Shoulder at 830 nm. In a previous study [17], it was

shown that the native ("high-pH") form (X m a x = 1020 and 800 nm) is reversibly transformed below pH 6.5 to a form absorbing at 960 and 800 nm ("low-pH" form). The 800/830 nm band remained unchanged by this treatment [17]. The pigments relating to the 960 nm band in the "low-pH" form are not very stable, and are oxidized irreversibly upon addition of more acid or even incubating at ambient tempera-ture. The newly formed absorption peaking at about 680 nm is typical for the Chlorophyll a-related oxida-tion products of bchl b [18]. Similar, albeit irreversible spectral changes have now been observed upon (partial) proteolysis of E . h a l o c h l o r i s membranes. Here we wish to report results pertaining to the orientation and exposition of the photosynthetic membrane proteins of this organism, and combine data from SDS-gel electrophoresis and spectroscopy,

572 R. Steiner et al. • The Photosynthetic Apparatus of Ectothiorhodospira halochloris

in order to relate spectrally distinct chromophores to certain Polypeptides.

Material and Methods

E . h a l o c h l o r i s was grown anaerobically in the medium of Imhoff and Trüper, with the differences described earlier [17]. The cells were harvested by centrifugation (14000 xg) and washed once with Tris-buffer (10 IHM, pH = 7.5). Thylakoids in which mainly the cytoplasmic surface is exposed, were prepared by the method of Feher and Okamura [19] and checked by light microscopy for homogeneity. Spheroplasts (rightside-out particles) were prepared after a modified method of Michels and Konings [20]: Harvested and washed cells were homogenized in a glass potter in Tris-buffer. The crude extract was treated with lysozyme (0.5 mg/ml) and E D T A (final concentration 5.5 raM) and stirred at room tempera-ture for about 3 hours [21]. It is necessary to check the formation of the particles by microscopy (charac-teristic swelling; addition of more lysozyme if necessary). The Suspension was then sonicated twice for one minute, centrifuged twice like chromatophores and adjusted to an absorption of 50 (1020 nm; 1 cm cuvettes).

For controlled proteolytic digestion, 500 t̂l of the membrane particles (chromatophores or spheroplasts) were incubated with increasing amounts of different proteases: Proteinase K , trypsin or ther-molysin. The digestion was stopped with PMSF (2 ml saturated Solution in acetone/0.5 ml incubation mix-ture), trypsin-inhibitor (400 mg/ml incubation buffer, added as a solid) or E D T A (1.0 M final concentration), respectiveiy. The samples were then centrifuged (14000xg) washed three times with Tris-buffer and analyzed.

SDS-gel electrophoresis was done on Polyacrylamide gels ( P A G E ) with a linear gradient (11.5-16.5% acrylamide), modified from Laemmli [22], as described earlier [17]. For calibration a Standard set of hydrophilic proteins was used (bovine serum albumin; hen egg albumin; lactoglobulin; pep-sin; trypsinogen and lysozyme) and in addition hydrophobic peptides ( R p . s p h e r o i d e s B 800/850 antenna) of known molecular weights [23]. Gels were scanned after staining with Coomassie brilliant blue G on a Scanner T C D (Vitatron).

Absorption spectra were measured on a D M R 22 (Zeiss, Oberkochen) or a ZWS II spectrophotometer

(Sigma, Berlin) connected to a BS 8000 intelligent recorder (Bryans, Mitcham). CD-spectra were ob-tained on a dichograph V (ISA, Unterhaching) equipped with a silex data handling System (Leanord, Lille) with a modified Software. Fluores-cence-emission spectra were obtained on a home-built fluorimeter equipped with a liquid-helium cryo-stat as described elsewhere [24] and are uncorrected.

A l l chemicals were reagent grade. Trypsin and Proteinase K were purchased from Merck, Darmstadt, lysozyme and trypsin inhibitor from Serva, Heidelberg, thermolysin from Boehringer, Mannheim, and the S D S - P A G E calibration set from Sigma, München.

Results

A b s o r p t i o n s p e c t r a

E . h a l o c h l o r i s has two major near infrared absorp-tions at 1020 and 800 nm. When thylakoids are treated with proteases, the 1020 nm absorption is gradu-ally transformed into a 960 nm absorption, whereas the 800/830 nm band remains unchanged (Fig. 1). Qualitatively these changes are identical irrespective of the type of protease used (trypsin, Proteinase K or

1200 1000 800 600 400

Fig. 1. Titration of E . halochloris thylakoids with increasing amounts of trypsin in 10 mM Tris buffer. pH = 8.0. The final concentration of trypsin was 100 ug/2 ml thylakoid Suspension with AI020 = 0.7/cm.

R. Steiner et al. • The Photosynthetic Apparatus of Ectothiorhodospira halochloris 573

thermolysin). The absorption changes are similar to the ones observed earlier upon lowering the pH [17], with two differences: Firstly the reaction is irreversible and thus due to a true proteolysis and not a pH-change induced by any action of the enzymes. Sec-ondly the effectiveness of proteolysis is strongly de-pendent on the membrane orientation. In contrast to the acid induced absorption change, proteolysis works with thylakoids only, but not with sphero-plasts. Similar to the pH-changes, the 960 nm form is again only metastable and transforms within a few minutes to yield the Chlorophyll <z-related oxidation-products of bchl b (Fig. 2) [18].

There is yet a third means of inducing the 1020 —» 960 nm transformation in E . h a l o c h l o r i s , e.g. by the treatment with urea. Globular, hydrophilic proteins are generally fully denatured (unfolded) by treatment with 8 M urea. The membrane proteins of E . h a l o c h l o r i s are much more stable, if judged from their absorption spectra < 900 nm. However, the 1020 nm band is again sensitive and transformed in-creasingly rapidly with increasing urea concentra-tions (4—8 M) to the 960 nm form. The absorption changes (Fig. 3) are identical to the ones induced by proteolysis or acid. Only thylakoids are susceptible to this treatment which is similar to the results ob-

1000 800 600 400 nm Fig. 2. Transformation of the B 800/960 form upon Standing to the "B" 800/680 form. Sample as in Fig. 1. The time be-tween each curve was 5 minutes.

1200 1000 800 nm

Fig. 3. Titration of E . halochloris thylakoids with increasing concentrations of urea (5.0, 5.5, . . , 7.5 M urea) in 10 m M Tris buffer, pH = 8.0.

tained upon proteolysis. We have also been unable to reverse the urea-induced transformation. Possibly the time necessary to remove the denaturant (e.g. 10 min necessary for by filtration over a short col-umn of desalting gel) is already too long to prevent the further and principally irreversible oxidation of the bchl b absorbing at 960 nm.

F l u o r e s c e n c e s p e c t r a

In order to further compare the 960 nm forms ob-tained by the different treatments (acid-, urea- or Protease), low-temperature fluorescence spectra were recorded:

The low temperature (5 K) emission spectra of all three forms show a Single band peaking at 1007 nm (5 K ) , whereas the original emission of E . h a l o c h l o r i s is at 1066 nm (Fig. 4). If the bacteriochlorophyll b chromophores absorbing at longest wavelengths are the emitters, this corresponds to Stokes-shifts of about 46 nm, for both the original chromophores (X.m a x, absorption = 1020 nm) and the modified ones (X m a x , absorption = 960 nm). The fluorescence exci-tation spectra below 920 nm are identical for all four samples with respect to band positions and intensities (Fig. 5). This is further support that the chromophores absorbing at 800/830 nm are not affected by any of these treatments. In all three forms, there is also an efficient energy transfer from the 800/830 nm absorbing chromophores to the 960 nm ones, if judged from the excitation peak around 800 nm for


i 1 1 1 1 1 1

800 900 1000 1100 1200

X / n m Fig. 4. Fluorescence emission spectra (excitation: 600 nm) of E . halochloris chromatophores at 5 K. A. Untreated chromatophores (Xm a x, absorption = 1020 nm); B. acid induced "low-pH" form (Xm a x, absorption = 960 nm); C. Protease treated (Xm a x, absorption = 960 nm); D. urea treated (X.max, absorption = 960 nm).

the 1007 nm emission. In the visible and near-UV spectral ränge, the excitation spectra are indicative of (monomeric) bacteriochlorophyll b and the absence of bacteriopheophytin b (no bands around 530 nm).

C i r c u l a r d i c h r o i s m

The three species modified by either low p H , proteolysis or urea are also identical with respect to their circular dichroism spectra. A l l three 960 nm forms show an "S"-shaped band centered around 974 nm with extrema around 990 and 940 nm (data not shown). As discussed previously for the low-pHform [17], these spectra can be rationalized by (a minimum of) two strongly interacting bchl b molecules absorbing around 960 nm.

1 1 1 1 1 1 1 1 1—1

300 400 500 600 700 800 900 1000 X/nm

Fig. 5. Excitation spectra, corresponding to the emission spectra in Fig. 4. Emission at 1020 (A) and 960 nm (B, C, D), respectively.

The 800/830 nm region shows an M-shaped signal, which is related to (a minimum of) three chromophores. The magnitude of this band is the only indi-cation, that at least some changes take place, too, in the pigments absorbing around 800 nm during the transformation of the B 800/1020 to the B 800/960 forms. The cd pattern of the latter in the 800 nm region is similar in shape, but reduced in intensity by about 50% as compared to the 800/1020 nm form.

SDS-gel e l e c t r o p h o r e s i s

In order to relate distinct changes in the Polypeptide pattern to the spectroscopic changes and hence to chromophores with distinct absorptions, the incubation with protease was followed in parallel by SDS-polyacryiamide gel electrophoresis and absorption spectroscopy. The S D S - P A G E of E . h a l o c h l o r i s membranes shows four bands in the "high-molecu-lar weight" ränge > 16 kDa (34.0, 28.0, 23.8 and 16.8 kDa). They have similar relative, but generally higher mobilities than the Polypeptides of the reaction center Polypeptides from R p . v i r i d i s (Table I)


and were therefore tentatively assigned to the R C subunits cytochrome c, H , M and L .

Table.

Rp. viridis (a) E. halochloris (c)

38.0 cytochrome c 34.0 33.0 H 28.0 27.0 M 23.8 24.0 L 16.8

9 15.2 9 14.5

11.0 (6.848, b) a-LHP 13.5 (6.5, d) 8.0 (6.138, b) ß-LHP 13.0 (6.0, d) 6.0 (4.001, b) Y-LHP 12.2 (4.5, d)

(a) SDS-PAGE data from Jay et al, 1983 [25]. (b) Data from primary structure analysis [26]. (c) Data from SDS-PAGE (this work), calibrated with

hydrophilic globular proteins. (d) Calibrated with B 800/850 subunits from Rp.

sphaeroides.

In the low-molecular weight region (< 16 kDa) two barely resolved major bands appear in addition to a weakly staining third band (Figs. 6, 7). Since they are isolated with the antenna fraction, they have been assigned to the light-harvesting complex [17]. This composition is again similar to that of R p . v i r i d i s (Table I). The only exception is that a 28.0 kDa band is isolated with the antenna from E . h a l o c h l o r i s , whereas a Polypeptide of this size is generally isolated as the "H"-subunit of the reaction center [17].

Fig. 6. SDS-PAGE of chromatophores from E . halochloris, incubated with proteinase K. 3 ml of the chromatophores (E 1020 = 50) were incubated with 0.5 mg proteinase K for 0, 5, 15, 30, 45 and 75 from left to right minutes at room temperature. See Fig. 7 for assignment of the bands.

Fig. 7. SDS-PAGE of trypsin incubate chromatophores of E . halochloris, that shows the degregation of the 6.5 kDa Polypeptide. 0.5 ml chromatophores (E 1020 = 50) were incubated with 650, 700, 750, 800, 900, 1000 u.g trypsin from left to right for 30 minutes at room temperature. The assignments of the main bands is indicated on the left margin. See text for the molecular weights.

The apparent molecular weights of these membrane proteins are shown in Table I.

When thylakoids of E . h a l o c h l o r i s are incubated with increasing amounts of proteases, the Polypeptide bands were degraded in a specific sequence. S D S - P A G E gels of the digestion with trypsin are shown in Fig. 6, but similar results are obtained with Proteinase K and thermolysin (not shown).

In the 'khigh molecular weight" region the cytochrome band disappears first, followed rapidly by H , L , and much more slowly by M . This means that in the membrane the M subunit is the most stable pep-tide of the R C (within the limits of resolution of our gels, v i z . ± 5 amino acid residues). Since the sequence of digestion is the same with all three proteases used, in a first approximation only the accessi-bility of the proteins is important rather than a distinct amino-acid sequence. It should be noted that in the bchl a containing organisms R p . c a p s u l a t a [27] and Rs. r u b r u m [28, 29] the L subunit of the R C is the most stable one. However, the assignment of R C bands by mobility alone is insufficient and further confirmation of this assignment is necessary.

The lower-molecular-weight Polypeptides of the light-harvesting complex were digested much slower than the R C Polypeptides. This different time course of the digestion has helped us to assign the fragments


to either the R C or L H Polypeptides. A gel with high resolution in this region is shown in Fig. 6. The 6.0 kDa band is least stable. It is degraded only a little to yield a band with an apparent molecular weight of 5.3 kDa, while the intensity of the 6.5 kDa band remains constant. Secondly, the 6.5 kDa band is attacked. The fate of the fastest migrating y-pep-tide (4.5 kDa) is difficult to assess quantitatively, be-cause it stains only weakly with Coomassie blue. When incubating the isolated antenna complex of E . h a l o c h l o r i s all bands disappear more or less simultaneously. This can be rationalized by the protection of the hydrophobic surfaces of the peptides in the chromatophore membrane which is lost in the solubilized complex.

Discussion

The spectroscopic results suggest, that the different treatments of E . h a l o c h l o r i s membranes, e.g. lowering of the pH below 6.5 [17], proteolysis and incubation with urea, transform the pigment ar-rangement within the antenna apparatus in the same or at least in a very similar manner. Of these reac-tions, only the one induced by acid is reversible. A reVersion may principally be possible, too, for the treatment with urea. However, the fastest time achieved for its removal (~ 10 min) is comparable to the half-life of the 960 nm chromophores and turned out to be ineffective. These results are summarized in Scheme 1.

The proteolysis has been studied in more detail. Two distinct results regarding the antenna structure within the membrane of E . h a l o c h l o r i s can be drawn. The first is a correlation of the chromophores absorbing at different wavelengths to distinct Polypeptides. The absorption shift from 1020 to 960 nm occurs simultaneous to the digestion of the 6.5 kDa (= a-) subunit, and after the proteolysis of the 6.0 kDa antenna Polypeptide and any of the reaction center Polypeptides. The chromophores absorbing at 1020 nm in the native antenna are thus either bound

urea

' low - pH f B 800/1020 « u . u u B800/960 — " B " 800/680

I high - pH .

proteolysis Scheme 1.

to the a-subunit or their spatial arrangement is at least strongly influenced by it. The role of the y -subunit is difficult to assess because it stains only weakly and unreliably. Like in R p . v i r i d i s [26] it does not carry a histidine residue (Brunisholz, unpublish-ed). Since histidine is currently assumed to be the common binding amino acid for the chromophore in the bacterial antenna Polypeptides [23, 30, 31], the y-subunit is considered a structural Polypeptide, to which the crystallinity of the membranes [10—12] in bacteriochlorophyll 6-containing bacteria may be related [26]. The chromophores absorbing around 800 nm should then be bound to the other antenna Polypeptides, most likely to the ß-subunit (6.0 kDa). However, proteolytic cleavage of a small (~ 0.5 kDa) Oligopeptide from this subunit does not affect significantly the 800/830 nm absorption.

The relative intensities of the chromophores absorbing at 1020 to the ones absorbing at 800 nm is roughly 2:1 and thus similar to the relative intensities of the chromophores absorbing at 850 and 800 nm, respectively, in type I B 800/850 antenna complexes of bchl tf-containing species, e.g. R p . s p h e r o i d e s [32]. In both complexes, the chromophores absorbing at the longest wavelengths are bound to the heavy antenna Polypeptide [23]. The most significant differ-ence is the coupling of the chromophores inferred from the circular dichroism spectra. In the B 800/850 complex of R p . s p h e r o i d e s , four excitation coupled bacteriochlorophyll a molecules were discussed as being responsible for the 850 nm absorption and orientated parallel to the a-helix of the Polypeptides, and two weakly coupled chromophores orientated perpendicular for the 800 nm absorption [33]. In E . h a l o c h l o r i s , a minimum of three strongly coupled chromophores is responsible for the 800 nm band and at least 2 for the 1020 nm absorption [17]. No data are available on their orientation with respect to the membrane.

The second aspect of the proteolytic digestion con-cerns the topology of the antenna of E . h a l o c h l o r i s . Only the H-subunit of the reaction center is digested if spheroplasts are treated with proteases. In thylakoids having mainly the cytoplasmic side exposed, all Polypeptides of the antenna (with the possible exception of the weakly staining y-subunit) and all reaction center Polypeptides are accessible to proteases. This would indicate, that only the H-subunit is spanning the photosynthetic membrane, as far as its accessibility to proteases is concerned. Similar


conclusions have been drawn from proteolytic studies with the bchl a containing Rs. r u b r u m [8, 9, 34, 35], R p . s p h e r o i d e s [36] and R p . c a p s u l a t a [5, 27], as well as with the bchl 6-containing R p . v i r i d i s [34]. In all cases, the reaction center Polypeptides are more labile than the ones related to the antennas, and only the H-subunit has always been found to be accessible from either side of the membrane. The validity of the latter results has been questioned, however, by the x-ray data of Deisenhofer et a l . [13] on R p . v i r i d i s reaction centers. The major part of the H-subunit is located on the cytoplasmic surface, with only a single a-helix spanning the membrane and less than 10 amino acid residues being exposed on the periplasmic side. From the x-ray data, it is rather the L- and M-subunits which are transmembrane Polypeptides. These discrepancies may be due to (i) a true species difference, (ii) a misassignment of the H band from S D S - P A G E derived molecular weights, or (iii) a fortuitous overlap of a large H fragment with either the M or L band. The third possibility should be indicated by an increased intensity of

either the M or L band, which was not observed. The second possibility can presently not be decided upon for the lack of sufficient sequence data. Species dif-ferences are indicated by differential proteolytic sen-sitivities of Polypeptides of the photosynthetic membranes of the species cited above. They are also supported by labeling methods exhibiting different selectivities or steric requirements, e.g. by iodination [2, 5, 25, 35] and by immunochemical data [5, 25, 39]. However, a comparison of the results obtained in different laboratories and with different biochemi-cal techniques is difficult, and comparative work with different species under otherwise identical conditions is necessary. Such work with E . h a l o c h l o r i s and the much better known R p . v i r i d i s is in progress.

A cknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft, Bonn (SFB 143). We thank H . C. Wolf (Stuttgart) and W. Rüdiger (München) for continuing support.

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