ZEITSCHRIFT FÜR NATURFORSCHUNG

SECTION C

B I O S C I E N C E S

Council

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

Editorial Board

A. H A G E R , Tübingen 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 E. W E C K E R , Würzburg

Advisory

P. B Ö G E R , Konstanz D . B Ü C K M A N N , Ulm K. G. G Ö T Z , Tübingen G. G O T T S C H A L K , Göttingen R. J A E N I C K E , Regensburg G. F . M E Y E R , Tübingen M . R A J E W S K Y , Essen

Board

H. SCHIMASSEK, Heidelberg D. S C H U L T E - F R O H L I N D E , Mülheim/R. F . F . SEELIG, Tübingen J. SEELIG, Basel H. SIMON, München W. S T E G L I C H , Bonn A. TREBST, Bochum

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

WITH T H E 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

V O L U M E 38 c 1983

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 38 c Zeitschrift für Naturforschung 1983

Contents

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

O r i g i n a l Communications

A Small-Angle-Scattering Camera without Slithigh-Error, II. (In German) H . W A W R A 1

A New Red Dipyrromethene Pigment from C a n d i d a b o i d i n i i S. L A N G , H . B E Y E R , P. S ISTIG, F . W A G N E R , W . T R O W I T Z S C H , and V . W R A Y 7

Ellagitannins from the Leaves of V i t t s v i n i f e r a (In German) C H . K A R L , G. M Ü L L E R , and P. A L S T E D P E D E R S E N 13

2-Methoxystypandrone, a New Naphthoquinone from Ehamnus f a l l a x B . (In German) H . - W . R A U W A L D and H . M I E T H I N G 17

Enzymatic Synthesis of Sinapine from 1 -O-Sinapoyl-/?-D-glucose and Choline by a Cell-Free System from Developing Seeds of Red Radish (Raphanus sativus L . var. sativus) D. S T R A C K , W . K N O G G E , and B . D A H L ­B E N D E R 21

Inhibition of Ergosterol Biosynthesis by Eta-conazole in U s t i l a g o maydis E . E B E R T , J . G A U D I N , W . M U E C K E , K . R A M ­S T E I N E R , C H . V O G E L , and H . F U H R E R 2 8

Isoelectric Focusing of the NAD(P)H-Cytochrome c Reductase Subunit of Chlamydomonas r e i n h a r d i i Nitrate Reductase E . F E R N A N D E Z and J . C Ä R D E N A S 3 5

The Effect of Silatranes on Growth, Photosynthetic Oxygen Evolution and Dark Respiration of C y c l o t e l l a c r y p t i c a (Diatomeae) (In German) R. R O T H , V . P . B A R Y S H O K , D . I. S T O M , D . W E R N E R , and M . G. V O R O N K O V 3 9

Oxygen-Evolving Extracts from a Thermophilic Cyanobacterium Synechococcus sp. S. M I Y A I R I and G. H . SCHATZ 4 4

Synthesis of 2 '- /3 '-0-Acylated Adenine Nucleotide Analogs and Their Interactions in Photophos-phorylation G. O N Ü R , G. S C H Ä F E R , and H . S T R O T M A N N 4 9

Studies on the S-State Distribution in E u g l e n a g r a c i l i s G. H . S C H M I D and P. T H I B A U L T 60

Sensitive Fluorimetric Assay for Adenosine De-aminase with Formycin as Substrate; and Sub­strate and Inhibitor Properties of some Pyra-zolopyrimidine and Related Analogues J . W I E R Z C H O W S K I and D . S H U G A R 67

Elucidation of the Different Effects of Polyamines and Other Naturally-Occurring Inhibitors of Cell Proliferation (Chalones) on T-Lymphocyte and Granulocyte Colony Growth i n v i t r o R. M A S C H L E R , C. J . S M I T H , J . C. A L L E N , and H . R. M A U R E R 74

Effect of Organic Solvents on Enzymatic Hydro-lysis of Celluloses M . K U M A K U R A and I. K A E T S U 79

Time-Resolved Fluorescence Studies of Hematopor-phyrin in Different Solvent Systems A . A N D R E O N I , R. C U B E D D U , S. D E S I L V E S T R I , G. J O R I , P . L A P O R T A , and E . R E D D I 83

On the Origin of the Non-Haemic Iron Transferrin E S R Signal: E S R Investigations on Histidine-Iron-Ascorbic Acid Systems W . L O H M A N N , D . H O L Z , B . K I E F E R , and D . S C H M I D T 90

Coupling of Monomethoxypolyethyleneglycols to Proteins v i a Active Esters E . Boccb, R . L A R G A J O L L I ,

and F . M . V E R O N E S E 94

O H Radical-Induced Main-Chain Scission of Poly-(Ribonucleic Acids) under Anoxic Conditions K . W A S H I N O , 0 . D E N K , and W . S C H N A B E L 100

Effect of Streptococcal Extracellular Nuclease on the Carrier Activi ty of R N A for Streptolysin S A . T A K E T O and Y . T A K E T O 107

D N A Content and Synthesis in Several Tissues and Variation of Moulting Hormone-Level in G r y l l u s b i m a c u l a t u s D E G (Ensifera, Insecta) F . R O M E R and I. E I S E N B E I S 112

Quasielastic Light Scattering of Isolated Chro­matin (In German) B . M E U E L and H . N O T B O H M 126

IV Contents

Physical and Chemical Analyses of the Mineral Substance during the Development of Two E x -perimental Cutaneous Calcifications in Rats: Topical Calciphylaxis and Topical Calcergy H . J . T O C H O N - D A N G U Y , G. B O I V I N , M . G E O F F R O Y , C. W A L Z E R , and C. A . B A U D 135

Long Term Cultures of Neural Retina and Pigment Epithelium from Newborn Rabbits T. T S Ü K A M O T O and H . L U D W I G 141

Notes

2^-Kaurenoic Ac id : A Diterpene as Frond Exudate on Ferns of the Genus N o t h o l a e n a E . W O L L E N W E B E R , D . M A R X , J . F A V R E - B O N V I N , and C. B R A S S Y 146

Identification of two Gossypetin Monomethyl Ethers as Yellow Flower Pigments in the Rutaceae J . B . H A R B O R N E and M . B O A R D L E Y 148

Antimicrobial Activity of N,N'-bis(decylmethyl)-a,to-alkanediamine Dioxides F . D E V I N S K Y , I. L A C K O , L . K R A S N E C , and D . M L Y N A R Ö I K 151

Fluorescence Patterns of Chromatin and Cytoplasm by Hematoxylin Solutions J . C. S T O C K E R T , M . L . M O L E R O , and R . H . E S P E L O S I N 153

On the Construction of "Optimal" Phylogenetic Trees G. D E S O E T E 156

Con ten t s o f N u m b e r 3/4

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

Composition and Accumulation of Essential Oil in M a t r i c a r i a e Radix (2. Communication) (In German) J . R E I C H L I N G , W . B I S S O N , H . B E C K E R , and G. S C H I L L I N G 159

Flavone C-Glycoside from M e t z g e r i a f u r c a t a (Hepaticae) (In German) R. T H E O D O R , R . M U E S , H . D . Z I N S M E I S T E R . and K . R . M A R K H A M 165

A New Investigation on the Composition of R h a m n i c a t h a r t i c i Cortex, IV . The Main Anthraglycosides

of the Stern Bark of Rhamnus c a t h a r t i c u s L . : Emodin-8-O-jS-gentiobioside, -glucoside and -primveroside (In German) H . - W . R A U W A L D 170

Isolation and Structure Determination of the Pre-cursors of a- and y-Irone and Homologous Com­pounds from I r i s p a l l i d a and I r i s f l o r e n t i n a W . K R I C K , F . - J . M A R N E R , and L . J A E N I C K E 179

Biosynthesis of 6a-Hydroxypterocarpans: Deute­rium N M R Evidence for Direct Hydroxylation of Pterocarpans S. W . B A N K S and P . M . D E W I C K 185

Chemotypes of M o l o p o s p e r m u m p e l o p o n n e s i a c u m ( L . ) Koch (In German) K . - H . K U B E C Z K A and I. U L L M A N N 189

Fungitoxic Isoflavones from L u p i n u s a l b u s and other L u p i n u s Species J . L . I N G H A M , S. T A H A R A , and J . B . H A R B O R N E 194

A n Improved Method of Isolation and Quantitative Determination of Vincaleucoblastine from Intact Plants and Tissue Cultures of C a t h a r a n t h u s roseus G. D O N . (In German) W . H O F M A N N , K . - H . K U B E C Z K A , and F.-C. C Z Y G A N 201

Isolation and Identification of Sterols in the Me-tabolism of the Fungus B o t r y t i s c i n e r e a (In German) P. F L E S C H , L . G W I N N E R , S. M Ü L L E R , and P . S I M O N 207

Mechanisms of Inhibition by Mevinolin (MK 803) of Microsome-Bound Radish and of Partially Purified Yeast HMG-CoA Reductase (EC.L1.1.34) T H . J . B A C H and H . K . L I C H T E N T H A L E R 212

Optically Detected Magnetic Resonance of Porphy­rin Complexes in the Bacterium Rhodopseudo-monas s p h a e r o i d e s J . B E C K , J . U . V O N SCHÜTZ, and H . C. W O L F 220

Affinity of Phycocyanin Chromopeptides to Histidyl-Sepharose Gels: A Model for Histidine-Tetrapyrrol-Interactions in Biliproteins J . R A B I E R , M . V I J A Y A L A K S H M I , and W . R Ü D I G E R 230

Contents

Sulfide Stimulation of Light-Induced Hydrogen Evolution by the Cyanobacterium Nostoc mus-c o r u m H . W E I S S H A A R and P . B Ö G E R 237

Ribulose-1,5-bisphosphate Carboxylase-Oxygenase: New Aspects Respective the pH-Dependance of the Carboxylation Reaction P. B R A U N , J . B O D E , and A . W I L D 243

Evidence for Unequal Misses in Oxygen Flash Yield Sequenee in Photosynthesis M . - J . D E L R I E U 247

Calorimeter-Fermentor Combination for Investiga-tions on Microbial Steady State Cultures H . P . L E I S E I F E R and G . H . S C H L E S E R 259

The 'Double' Michaelis-Menten Equation: Estima-tion of Parameters A . C. B O R S T L A P and P. G . D O U C E T 268

L-Usnate-Urease Interactions: Binding Sites for Polymerization B . C I F U E N T E S , I. G A R C I A , and C. V I C E N T E 273

Enhancement of i n - v i t r o Translation of Eukaryotic R N A s by Cyclic A M P M . E . J O H N and W . K N Ö C H E L 277

Characterisation of the Enzyme Intermediates of the Sarcoplasmic Transport ATPase by the Use of Inhibitors W . B O L L and M . M A K I N O S E 282

Thermal Stability of D N A Interacting with Fur-azolidone and Cu(II) Ions M . M A I T I , S. G H O S H , A . C H A T T E R J E E , and S. N . C H A T T E R J E E 290

Circular Permutation Analysis of Phage T4 D N A by Electron Microscopy G . F . G R O S S I , M . F . M A C C H I A T O , and G . G I A L A N E L L A 294

On a Quantitative Determination of Antibodies to Lipids and Proteins A . R A D U N Z 297

Effect of Sodium Cholate on the Phase Transition Temperature of Dipalmitoyl Phosphatidylcholine S. B A N E R J E E and S. N . C H A T T E R J E E 302

On the Interaction of Phospholipid Vesicles with Chaotropic Ions T H . P I G O R and R . L A W A C Z E C K 307

V

Growth Characteristics of Anaerobically Treated Early and Late S-Period of Ehrlich Ascites Tu­mor Cells after Reaeration R. M E R Z and F . S C H N E I D E R 313

Comparative Investigations on Defensive Chemistry and Gland Morphology of Abdominal Defensive Glands from Rove Beetles of the Subtribe Philon-thina (Coleoptera, Staphylinidae) (In German) K . D E T T N E R 319

Is the F l y Visual Pigment a Rhodopsin ? K . V O G T 329

Notes

The Phytoalexin Response of L a t h y r u s sativus (Grass Pea) D . J . R O B E S O N and J . B . H A R B O R N E 334

Prostaglandin Synthesis Inhibitors Reduce C a n -n a b i s and Restraint Stress Induced Increase in Rat Brain Serotonin Concentrations S. K . B H A T T A C H A R Y A and D . B H A T T A C H A R Y A 337

Erratum to A . A N D R E O N I , R . C U B E D D U , S. D E S I L V E S T R I , G. J O R I , P . L A P O R T A , and E . R E D D I (Z. Naturforsch. 38c, 83 -89 (1983) 338

Con ten t s o f N u m b e r 5/6

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

The Stereochemistry of Silybin (In German) H . L O T T E R and H . W A G N E R 339

Identification of Anthocyanins and Intermediates of Anthocyanin Biosynthesis from P e t u n i a h y b r i d a Using High Performance Liquid Chromatography A . W . S C H R Ä M , L . M . V . J O N S S O N , and P . D E V L A M I N G 342

Formation of Benzophenanthridine Alkaloids by Suspension Cultures of E s c h s c h o l t z i a c a l i f o r n i c a J . B E R L I N , E . F O R C H E , V . W R A Y , J . H A M M E R , and W . H Ö S E L 346

Circulardichroism of C-Phycocyanin: Origin of Op-tical Activity in Denatured Biliproteins and E v i ­dence for an Intermediate during Unfolding H . L E H N E R and H . S C H E E R 353

Contents VI

Chromopeptides from Phytochrome and Phyco-cyanin. N M R Studies of the Pf r and P r Chromo-phore of Phytochrome and E, Z Isomeric Chro-mophores of Phycocyanin F . T H Ü M M L E R , W. R Ü D I G E R , E . C M I E L , and S. S C H N E I D E R 359

Prunasin Biosynthesis by Cell-Free Extracts from Black Cherry ( P r u n u s s e r o t i n a Ehrh.) Fruits and Leaves J . E . P O U L T O N and STJN-IN S H I N 369

Comparison of Purine Metabolism in Suspension Cultured Cells of Different Growth Phases and Stern Tissue of C a t h a r a n i h u s roseus F . H I R O S E and H . A S H I H A R A 375

Uptake and Conversion of Three Different 5-Ring Polycyclic Aromatic Hydrocarbons (PAHs) in Cell Suspension Cultures of Various Chenopo-diaceae-Species H . H A R M S 382

Geranyl Derivatives as Inhibitors of the Caroteno-genesis in Synechococcus PCC 6911 (Cyanobacteria) F . J Ü T T N E R and 0 . B O G E N S C H Ü T Z 387

Interconversion of Carotenoids and Quinones after Onset of Photosynthesis in Chloroplasts of Higher Plants K . H . G R U M B A C H 393

The Influence of the Glycerol-3-Phosphate Level in the Stroma Space on Lip id Synthesis of Intact Chloroplasts A . S A U E R and K . - P . H E I S E 399

Inability of Petite Mutants of Industrial Yeasts to Utilize Various Sugars, and a Comparison with the Abil i ty of the Parent Strains to Ferment the the Same Sugars Microaerophilically J . F . T. S P E N C E R , D . M . S P E N C E R , and R. M I L L E R 405

Studies on the Regulatory Role of tfraws-Cinnamic Acid on the Activity of the Phenylalanine Am-monia-Lyase (PAL) in Suspension Cultures of Daucus c a r o t a L . W. N O E and H . U . S E I T Z 408

Üfefo-Methylation of Flavonol Rings A(8-) and B(3'-) Is Catalysed by Two Distinct O-Methyl-transferases in Lotus c o r n i c u l a t u s M . J A Y , V . D E L U C A , and R . I B R A H I M 413

Characteristic Features of the Regulatory Functions of the D-Ribulose 1,5-Bisphosphate Carboxylase/ Oxygenase from Spinach J . V A T E R , T. G A U D S Z U N , B . L A N G E , N . E R D I N , and J . S A L N I K O W 4 1 8

A Cysteine Desulfhydrase Specific for D-Cysteine from the Green Alga C h l o r e l l a fusca A. S C H M I D T and I. E R D L E 4 2 8

Regulation of Nitrogenase Activi ty in Rhodopseudo-monas c a p s u l a t a A D 2 K . A L E F 4 3 6

Nitrate Reductase from a Mutant Strain of C h l a m y -domonas r e i n h a r d i i Incapable of Nitrate Assimila­tion E . F E R N A N D E Z and J . C A R D E N A S 4 3 9

A Membrane Bound Cysteine Oxydase from the Cyanobacterium Synechococcus 6301 A . S C H M I D T and E . K R Ä M E R 446

A Specific and Sensitive Radioimmunoassay for Human Choriogonadotropin K . M U R A L I D H A R , G. C H A U D H U R I , J . L I P P E S , and 0 . P . B A H L 4 5 1

Effect of Pressure on the Absorption Spectra of Phycobiliprotein and P o r p h y r i d i u m c r u e n t u m S. S. B R O D Y and L . S T E L Z I G 4 5 8

Bioalkylation of Nucleic Acids in Mice by Insecti-cides, I. Alkylation of Liver R N A and D N A by Chlorpyrifos I. Y . M O S T A F A , Y . M . A D A M , and S. M . A . D . Z A Y E D 461

Electromagnetic Modulation of Biological Processes: Bicarbonate Effect and Mechanistic Considera-tions in the Ca-Uptake by Embryonal Chick Tibia i n v i t r o G. C O L A C I C C O and A . A . P I L L A 4 6 5

Electromagnetic Modulation of Biological Processes: ATPase Function and D N A Production by Raji Cancer Cells i n v i t r o G. COLACICCO and A . A . P I L L A 4 6 8

The Influence of the Extracellular Concentration of Calcium, Magnesium and Sodium on the Repo-

Contents

larizing Phase of the Receptor Potential of the L i m u l u s Ventral Nerve Photoreceptor H . S T I E V E , M . B R U N S , and H . G A U B E 471

Notes

Adsorbent Culture — Method for the Enhanced Production of Secondary Substances in Plant Suspension Cultures (In German) B . K N O O P and R . B E I D E R B E C K 484

The Structure of a Ring-A-seco-rcor-Triterpenol from H o y a a u s t r a l i s Leaf Wax W . J . B A A S 487

Studies on the Distribution of Phytosterols in D i o s c o r e a Species Tubers P. G. K A D K A D E , C. L U J A N , and C. R O L Z 488

Quercetin-3-a-L-(2-0-a-L-rhamnopyranosido-arabopyranoside), a New Flavonolglycoside from Seeds of B r a s s i c a n i g r a (L.) Koch. (In German) H . G E I G E R , H . M A I E R , and K . R . M A R K H A M 490

Verpacrocin, a Polyene Pigment from Mycelial Cul­tures of V e r p a d i g i t a l i f o r m i s (Pers.) Fr . (Asco-mycetes) (In German) H . B E S L , A . B R E S I N S K Y , B . M E I X N E R , U . M O C E K , and W. S T E G L I C H 492

Inheritance of Sesquiterpenoid Phenolic Acid Esters (Guayulins) in F i Hybrids of P a r t h e n i u m (Asteraceae) H . M . B E H L , B . M A R C H A N D , and E . R O D R I G U E Z 494

Chemical Composition of the Secretion from the Anal Sacs of C i v e t t i c t i s c i v e t t a (Schreber, 1776) J . J A C O B and H . S C H L I E M A N N 497

The Possible Common Origin of t R N A and 5S r R N A M . U J H E L Y I 501

Rotation of Cells and Ion Exchange Beads in the MHz-Frequency Range G. K Ü P P E R S , B . W E N D T , and T L Z I M M E R M A N N 505

Temperature Regulation in Wood Ant Nests ( F o r m i c a p o l y c t e n a Förster) (In German) K . H O R S T M A N N 508

VII

Conten t s o f N u m b e r 7/8

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

Crystal Packing of Odd-Chain Saturated Tri­glycerides D. L . D O R S E T 511

New Cembranoids from Tobacco, I I A . - M . B Y L O V , U . B R U M M E R , W. H A S S , F . S E E ­H O F E R , V . H E E M A N N , and V . S I N N W E L L 515

3,7,1 l,15-Cembratetraen-6-ol, a New Cembranoid from Tobacco, I I I V . H E E M A N N , A . - M . B Y L O V , U . B R U M M E R , W. H A S S , and F . S E E H O F E R 517

Synthesis of Mono-, D i - and Trigalloyl-/?-D-glucose by ß-Glucogallin-Dependent Galloyltransferases from Oak Leaves G. G. GROSS 519

Carboxylic Acids in Wheat, Rye and Barley E . L O H A U S , I. B L O S , and W. R Ü D I G E R 524

Separation, Purification, and Characterization of Two Isoforms of Glutamine Synthetase from Chlamydomonas r e i n h a r d i i F . J . F L O R E N C I O and J . M . V E G A 531

Transformations of Phenazines in Pseudomonas aureofaciens (In German) A . R Ö M E R and E . L A N G E 539

Differential Regulation of Flavone Glycosylation during Ontogeny of Silene pratensis J . M . S T E Y N S , G. V A N N I G T E V E C H T , G. J . N I E ­M A N N , and J . v. B R E D E R O D E 544

Ontogeny and Biosynthesis of Isovitexin 7-O-Ga-lactoside in a Mutant of Silene pratensis Unable to Glycosylate this Compound in the Petals J . V A N B R E D E R O D E and J . M . S T E Y N S 549

Relationship between Flower Development, Antho-cyanin Accumulation and Activity of Enzymes Involved in Flavonoid Biosynthesis in M a t t h i o l a i n c a n a R . Br . B . D A N G E L M A Y R , G. STOTZ, R . S P R I B I L L E , and G. F O R K M A N N 551

Contents VIII

Variable Fluorescence and Fluorescence Spectra of Algae after Herbicide-Induced Pigment Bleach-ing N . V . K A R A P E T Y A N , R. STRASSER, and P . B Ö G E R 556

Investigations on the Synthesis and Biological Sig-nificance of Glutamic-l-semialdehyde as a Pre-cursor of the Chlorophylls (In German) H . - U . M E I S C H and R. M A U S 563

Nitrogen Fixation and Nitrate Respiration by A z o s p i r i l l u m b r a s i l e n s e H . B O T H E , G. B A R B O S A , and J . D Ö B E R E I N E R 571

Cyclic A M P Is Not Involved as a Second Messenger in the Response of Soybean to Infection by P h y t o p h t h o r a megasperma f. sp. g l y c i n e a M . G. H A H N and H . G R I S E B A C H 578

Biosynthesis of Cyanogenic Glycosides in Butter­flies and Moths: Incorporation of Valine and Iso-leucine into Linamarin and Lotaustralin by Zy-g a e n a and H e l i c o n i u s Species (Lepidoptera) V . W R A Y , R . H . D A V I S , and A . N A H R S T E D T 583

Caffeine-Induced Surface Blebbing and Büdding in the Acellular Slime Mold P h y s a r u m p o l y c e p h a l u m J . K U K U L I E S , W. S T O C K E M , and K . E . W O H L F A R T H - B O T T E R M A N N 589

Regulation of Light Energy Distribution between Photosynthetic Pigment Systems; a Possible Role of Leaf Anatomy G. H A R N I S C H F E G E R and G. Z E N K 600

Energy Parameters, Macromolecular Synthesis and Cell Cycle Progression of i n v i t r o Grown Ehrlich Ascites Tumor Cells after Inhibition of Oxydative A T P Synthesis by Oligomycin W . K R O L L , M . L Ö F F L E R , and F . S C H N E I D E R 604

Embryonic Hemoglobins in Mammals: The Primary Structures of f-, e- and #-Chains of the Pig (Sita scrofa d o m e s t i c a ) (In German) F . A . B I E B E R , H . A S C H A U E R , S. M . B E K T A S , and G. B R A U N I T Z E R 613

Studies on the Transport of Anions and Zwitterions of Acidic Amino Acids in Streptomyces h y d r o g e n a n s J . F R I T S C H and W . GROSS 617

Hydrogen Bond Indices and Tertiary Structure of Yeast t R N A p k e M . S. D E G I A M B I A G I , M . G I A M B I A G I , and D . M . S. E S Q U I V E L 621

Effect of 5'-Terminated (2'—5')-OIigoadenylates on Cap Degrading Activities in Rat Liver Nuclei W . M I C H E L S and E . S C H L I M M E 631

Decreased Production of RNA-Streptolysin S in Streptococci Devoid of Extracellular RNase Ac­tivity A . T A K E T O and Y . T A K E T O 635

Activation and Inhibition of Na/K-ATPase by Filipin-Cholesterol Complexation. A Correlative Biochemical and Ultrastructural Study on the Microsomal and Purified Enzyme of the Avian Salt Gland D . G A S S N E R and H . K O M N I C K 640

Incorporation of a Voltage Sensitive Pore from Guinea Pig Heart Mitochondria into Black Lipid Membranes and Characterization of Electrical Properties W . S C H R E I B M A Y E R , H . H A G A U E R , and H . A . T R I T T H A R T 664

Notes

Flavonoids from the Leaf Resin of H y m e n o c l e a s a l -s o l a T . & G . (Asteraceae) P . P R O K S C H , E . W O L L E N W E B E R , and E . R O D R I -G U E Z 668

New Papaveraceae Alkaloids Including an 7,8-Oxygenated Isoquinoline, the Postulated Precur-sor of the Cularine Alkaloids G. B L A S C H K E and G. S C R I B A 670

Conical Kinetochores with Single Microtubules in the Micronuclear Mitosis of the Ciliate Colpoda s t e i n i i A , K U C K and A . R U T H M A N N 672

Time-Shifted Mating Periods in Two Closely Related Corixid Species J . T H E I S S 675

Contents

Con ten t s o f N u m b e r 9 /10

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

Epicuticular Waxes from Leaves and Sterns of J o j o b a ( S i m m o n d s i a c h i n e n s i s [Link] Schneider) P . - G . G Ü L Z and C. E I C H 679

Chemistry and Morphology of Epicuticular Waxes from Various Organs of J o j o b a { S i m m o n d s i a c h i ­nensis [Link] Schneider) P . - G . G Ü L Z and K . H Ä N G S T 683

Isolation and Structural Determination of a New Methylated Triterpenoid from Rhizomes of I r i s versicolor L. W. K R I C K , F . -J . M A R N E R , and L. J A E N I C K E 689

Antifungal Phytoalexins in Phaseolus a u r e u s Roxb. M . J. O ' N E I L L , S. A. A D E S A N Y A , and M . F. R O ­BERTS 693

Aureol and Phaseol, Two New Coumestans from Phaseolus a u r e u s Rosb. M . J. O ' N E I L L 698

Synthesis and Biological Evaluation of 7a,7a,7a,8a,8a,8a-Hexafluororiboflavin and 7a,7a,7a,8a,8a,8a-Hexafluoro-FMN P. NIELSEN, A. B A C H E R , D. D A R L I N G , and M . C U S H M A N 701

3'-Hydroxylation of 4'-Methoxyisoflavones by F u s a ­r i u m o x y s p o r u m f. l y c o p e r s i c i K . M A C K E N B R O C K and W. B A R Z 708

Kinetin-Mediated Stimulation of Accumulation of Buckwheat Flavonoids in the Dark U. M A R G N A and T. VAINJÄRV 711

Uptake and Release of Abscisic Acid by Runner Bean Root Tip Segments W. H Ä R T U N G and B. D I E R I C H 719

Distribution and Variation of the Catalytic Activity of the Enzyme Responsible for Carthamin Synthe­sis in Safflower Seedlings K. SAITO, Y . T A K A H A S H I , and M . W A D A 724

Further Studies on Induction of Enzymes of Phyto-alexin Synthesis in Soybean and Cultured Soy-bean Cells J. L E U B E and H . G R I S E B A C H 730

Effects of Pyridazinone Herbicides during Chloro-plast Development in Detached Barley Leaves. I.

IX

Effects on Pigment Accumulation and Fluores­cence Properties G. L A S K A Y , E. L E H O C Z K I , I. M A R Ö T I , and L. SZA-L A Y 736

Effects of Pyridazinone Herbicides during Chloro-plast Development in Detached Barley Leaves. IL Effects on Lipid Content, Fatty Acid Composition and Ultrastructure of Chloroplasts G. L A S K A Y , T. F A R K A S , E. L E H O C Z K I , and K . G U L Y A 741

Chemistry of Bacteriochlorophyll b: Identification of Some (Photo)Oxidation Products R. STEINER, E. C M I E L , and H . SCHEER 748

Urobiliverdin, a New Bile Pigment Deriving from Uroporphyrin E. BENEDIKT and H.-P. K Ö S T 753

Conformational Study of Catecholamines in Solu­tion P. SOLMAJER, D. K O C J A N , and T. SOLMAJER 758

The Effects of Tumor Sera on Cell Shape and Photo-synthesis of E u g l e n a g r a c i l i s H. G. R U P P E L and B. B E N N I N G H O F F 763

Lipids and Enzymatic Activities in Vacuolar Mem-branes Isolated via Protoplasts from Oat Primary Leaves B. V E R H O E K , R. H A A S , K . W R A G E , M . LINSCHEID, and E. H E I N Z 770

A Study on Oxygen Evolution and on the S-State Distribution in Thylakoid Preparations of the F i l -amentous Blue-Green Alga O s c i l l a t o r i a c h a l y b e a K . P. B A D E R , P. T H I B A U L T , and G. H . S C H M I D 778

The Use of 0 2 -Evolving Subchloroplast Particles to Study Acceptor and Inhibitor Sites on the Re-ducing Side of Photosystem II W . S. C O H E N and J . R. B A R T O N 793

Electroimmunodiffusion - a Powerful Tool for Quantitative Determinations of Both Soluble and Membrane Bound Chloroplast ATPase, C F X

P. Roos and R. J . B E R Z B O R N 799

Analogue of Hippuran: Labelled Ruthenocenoyl-glycin. Synthesis, Organ Distribution and Clear­ance (In German) M . W E N Z E L und G. SCHACHSCHNEIDER 806

X Contents

Immobilization of E . c o l i Cell as an Antigen by Ra­diation Polymerization Method I. K A E T S U , M . K U M A K U R A , S. K I K U C H I , S. A D A C H I , and M . S U Z U K I 812

Exchange of the Structural Water of Collagen and Alcanol-Induced Molecular Rearrangement (In German) T H . NEMETSCHEK, E. K N Ö R Z E R , W. F O L K H A R D , W. G E E R K E N , K . J E L I N E K , C. K U H L E M A N N , E. M O S -LER, and H. N E M E T S C H E K - G A N S L E R 815

Preparation and Characterization of Actin from Liver B. J A B E R G 829

Transient Activation of the Ca 2 +-ATPase from Sar-coplasmic Reticulum during Thiol Modification by 5,5'-Dithiobis(2-nitrobenzoate) G. SWOBODA and W. H A S S E L B A C H 834

Phosphorylation of Ca-ATPase of Sarcoplasmic Re­ticulum with Different Substrates H. B A R R A B I N and L. DE M E I S 845

Composition of the Heterogeneous Sex Attracting Secretion of the Dung Beetle, K h e p e r l a m a r c k i B. V. B U R G E R , Z. M U N R O , M . R O T H , H . S. C. SPIES, V. T R U T E R , G. D. TRIBE, and R. M . C R E W E

848

Stimulation of Juvenile Hormone Biosynthesis i n v i t r o by Locust Allatotropin H.-J. F E R E N Z and I. D I E H L 856

Notes

Photosensitized Effects of Furocoumarins: the Pos-sible Role of Singlet Oxygen D. V E D A L D I , F. D A L L ' A C Q U A , A. G E N N A R O , and G. RODIGHIERO 866

Supplementary Data on the Sex Attractant System of P a n o l i s ßammea E. PRIESNER and M . S C H R O T H 870

Receptors for Di-Unsaturated Pheromone Anaio-gues in the Male Summerfruit Tortrix Moth E. PRIESNER 874

Effect of Ecdysterone on Food Intake of L o c u s t a m i -g r a t o r i a Hoppers P. J. R A O and H . R E M B O L D 878

Aminoacyl-tRNA Synthetases in Liver, Spleen and Small Intestine of Aged Leukemic and Aged Nor­mal Mice H. -J. G A B I U S , S. G A B I U S , G. G R A U P N E R , F. C R A -MER, and S. R E H M 881

A Non-Thermal Effect of Millimeter Wave Radiation on the Pulling of Giant Chromosomes C. K O S C H N I T Z K E , F. K R E M E R , L. SANTO, P. Q U I C K , and A. POGLITSCH 883

Determination of Measles Virus Protein Molecular Weights on High Percentage, Highly Cross-Linked SDS Polyacrylamide Gels M . J. C A R T E R and K . B A C Z K O 887

Flavonoids from the Leaf Resin of H y m e n o c l e a s a l -s o l a T. & G. (Asteraceae) P. P R O K S C H , E. W O L L E N W E B E R , and E. R O D R I -G U E Z 668

Interaction of Ascorbic Acid with Disulfides J. E. F L E M I N G , K . G . B E N S C H , J. SCHREIBER, and

C o n t e n t s o f N u m b e r 11/12

W. L O H M A N N 859 O r i g i n a l C o m m u n i c a t i o n s

ESR Investigations on the Modification of the Ascor­bic Acid-Erythrocyte Interaction by Ferrous Ions W. L O H M A N N , B. K I E F E R , D. H O L Z , D. SCHMIDT, and J. SCHREIBER 862

The Effect of Alcohol Oxidase on the Ascorbic Acid Erythrocyte Interaction: ESR Investigations W. L O H M A N N , J. SCHREIBER, D. H O L Z , and F. R. HOPKINS 864

SJ^'^'^'-Pentahydroxyflavanone in the Bracts of H e i i c h r y s u m b r a c t e a t u m G. F O R K M A N N 891

The Flavones of the European Species of S i l e n e Sec-tion Elisanthe O. M A S T E N B R O E K , J. J. K N O R R , Pv. K A M P S - H E I N S -BROEK, J. W. M A A S , J. M . STEYNS, and J. V A N B R E ­DERODE 894

Contents XI

Isolation and Identification of Isoflavanone Phyto-alexins from Leaflets of D i p h y s a r o b i n i o i d e s J . L. I N G H A M and S. T A H A R A 899

Wounding-Induced Increase of Quinolizidine Alka-loid Accumulation in Lupin Leaves M. W I N K 905

Site of Action of Growth Inhibitory Tryptophan Analogues in C a t h a r a n t h u s roseus Cell Suspen­sion Cultures F. SASSE, M . B U C H H O L Z , and J . B E R L I N 9 1 0

Selection of Cell Lines of C a t h a r a n t h u s roseus with Increased Tryptophan Decarboxylase Activity F. SASSE, M . B U C H H O L Z , and J . B E R L I N 916

Structure of Ascorbic Acid and Its Biological Func­tion: V. Transport of Ascorbate and Isoascorbate across Artificial Membranes as Studied by the Spin Label Technique W. L O H M A N N and J . W I N Z E N B U R G 923

On the Origin of the Non-Haemic Iron Transferrin ESR Signal: ESR Investigations on Histidine-Iron-Citric Acid Systems W. L O H M A N N , D. H O L Z , B. K I E F E R , and D. SCHMIDT 926

Enhancement Effect of Hydrophobie Vinyl Com­pounds in Enzymatic Hydrolysis of Waste Papers M . K U M A K U R A and I. K A E T S U 929

Purification and Characterization of a Dissimilatory Nitrite Reductase from the Phototrophic Bacteri-um R h o d o p s e u d o m o n a s p a l u s t r i s M . PREUSS and J . - H . K L E M M E 933

Isolation and Fast Purification of Neocarzinostatin by FPLC-Ion Exchange Chromatography D. D E N K L A U , W. K Ö H N L E I N , G. L Ü D E R S , and J . S T E L L M A C H 939

1 5N-Coprobiliverdin, a New Model Chromophore H.-P. K Ö S T , E. B E N E D I K T , E. C M I E L , and S. SCHNEIDER 943

Circular Dichroism of Chromopeptides from Phyco-eyanin C. S C H A R N A G L , E. K Ö S T - R E Y E S , S. SCHNEIDER, H -P. K Ö S T , and H . SCHEER 951

Cytochromes and Anaerobic Sulfide Oxidation in the Purple Sulfur Bacterium C h r o m a t i u m w a r m i n -8Ü

U . W E R M T E R and U . FISCHER 960

Molecular Properties of High Potential Iron Sulfur Protein of C h r o m a t i u m w a r m i n g i i U . W E R M T E R and U . FISCHER 968

The Phycobiliproteids in C y a n o p h o r a p a r a d o x a as Accessoric Pigments and Nitrogen Storage Pro­teins H. E. A. S C H E N K , J . H A N F , and M . N E U - M Ü L L E R

972

The Influence of Cycloheximide and Chlorampheni-col on the Biosynthesis of the Photosynthetic Pig­ments in C y a n o p h o r a p a r a d o x a . I. Photosynthetic Oxygen Evolution (In German) H . E. A. S C H E N K and M . N E U - M Ü L L E R 978

The Influence of Cycloheximide and Chlorampheni-col on the Biosynthesis of the Photosynthetic Pig­ments in C y a n o p h o r a p a r a d o x a . II. Carotenoids (In German) M . N E U - M Ü L L E R , H. E. A . S C H E N K , and H . S T R A N S K Y 984

The Influence of Cycloheximide and Chlorampheni-col on the Biosynthesis of the Photosynthetic Pig­ments in C y a n o p h o r a p a r a d o x a . III. Chlorophyll a and Phycochromoproteids (In German) M . N E U - M Ü L L E R and H . E. A. S C H E N K 990

Distribution of Chlorophylls, Carotenoids and Qui-nones in Chloroplasts of Higher Plants K . H. G R U M B A C H 996

Chlorophyll-Lipid-Interactions in Monomolecular Layers H. HEITHIER and H . M Ö H W A L D 1003

Sex Pheromone Components of the Gamma Moth, A u t o g r a p h a g a m m a (L.) (Lepidoptera: Noctuidae) E. D U N K E L B L U M and S. G O T H I L F 1011

A Conformational Transition of the Sarcoplasmic Reticulum Calcium Transport ATPase Induced by Vanadate W . H A S S E L B A C H , P. M E D D A , A. M I G A L A , and B. A G O S T I N I 1015

XII Contents

Enzymatic Removal of 0 6-Ethylguanine versus Sta-bility of 0 4-Ethylthymine in the D N A of Rat Tis-sues Exposed to the Carcinogen Ethylnitrosourea: Possible Interference of Guanine-O 6 Alkylation with 5-Cytosine Methylation in the D N A of Repli-cating Target Cells R. M Ü L L E R and M . F. R A J E W S K Y 1023

Rate of O H Radical Induced Strand Break Forma­tion in Single Stranded D N A under Anoxic Con-ditions. An Investigation in Aqueous Solutions Using Conductivity Methods E. B O T H E , G. A. QURESHI , and D. S C H U L T E - F R O H -

LINDE 1030

The Intensity Dependence of the Receptor Potential of the L i m u l u s Ventral Nerve Photoreceptor in Two Defined States of Light- and Dark Adapta­tion H. STIEVE, M . B R U N S , and H . G A U B E 1043

Incorporation of 1 5 N-Ammonia into Serotonin in Cotyledons of Maturing Walnuts W. GROSSE and F. A R T I G A S 1057

N-Terminal Sequence of a Porphobilinogen-Syntha-se (In German) B. L I N G N E R and T. K L E I N S C H M I D T 1059

Sequence Analysis of the Cloned C u c u m i s m e l o Highly Repetitive Satellite D N A A. B R E N N I C K E and V H E M L E B E N 1062

Variation in Cephalic Volatile Substances in Rela­tion to Worker Age and Behavior in the Stingless Bee, S c a p t o t r i g o n a p o s t i c a W. F R A N C K E , W. SCHRÖDER, E . E N G E L S , and W.

E N G E L S 1066

Antibodies against the a-Factor Pheromone of Sac-c h a r o m y c e s c e r e v i s i a U . T I L L M A N N and H . H A H N 1069

Notes

Identification of the Major Anthöcyanin of Carrot Cells in Tissue Culture as Cynidin 3-(Sinapoylxy-losylglucosylgalactoside) J . B . H A R B O R N E , A . M . M A Y E R , and N . B A R - N U N

1055

A New Cultural Method for T r i c h o p l a x adhaerens F. E. S C H U L Z E (In German) K . G. G R E L L 1072

Subject Index 1073

Authors Index 1095

Circulardichroism of C-Phycocyanin: Origin of Optical Activity in Denatured Biliproteins and Evidence for an Intermediate during Unfolding Harald Lehner

Institut für Organische Chemie, Universität Wien, Währinger Str. 38, A-1090 Wien 9

Hugo Scheer

Institut für Botanik, Universität München, Menzinger Str. 67, D-8000 München 19

Z. Naturforsch. 38 c, 353-358 (1983); received January 17, 1983

Phycocyanin, Phycoerythrin, Phytochrom, Circulardichroism, Denaturation The circulardichroism spectra (cd) of native and (partially) urea-denaturated C-phycocyanin

(PC) in the spectral ränge 700—210 nm are presented. The large ellipticities observed in the chromophoric region of native PC are retained in the fully denatured State (8 M urea). This is similar to the behavior of the red form of phytochrome (P,.), but in contrast to C-phycoerythrin (PE). These differences are rationalized in terms of epimeric equilibria between P- and M-helix shaped chromophores. Depending on the number and location of the chirality centers present in the tetrapyrrol moities a priori, the excess populations of the inherently chiral P- and M-helices differ, thus accounting for large (PC, Pr) or small (PE) ellipticities in the denatured pigments. Hence, the large optical activity observed for the former is generated by an excess population of the P-helix induced by the asymmetric C-2, C-3 and C-3'. In PE the additional chirality center at C-16 counteracts the influence of the others. The excess population of the inherently chiral species is therefore lowered, in agreement with the nearly vanishing cd reported for denatured PE.

The cd has also been studied at intermediate urea concentrations. Unfolding of PC with urea can be interpreted from these data as a stepwise process. Monitoring the urea induced unfolding of PC by cd at different wavelengths (A = 220, 345, and 610 nm); the "melting point" of the apoprotein (4-5 M urea) coincides with the extrema of the titration curves obtained in the chromophore region (345, 610 nm). These results give direct evidence for the existence of an intermediate species whose population reaches a maximum at 4 - 5 M urea.

Introduction

Phycobiliproteins are light harvesting pigments of certain algae (for some recent reviews, see e.g. refs. 1-4).

They contain chromophores like 1 covalently bound to the apoproteins. Whereas the denatured pigments have properties typical to free bile pig­ments of similar structure, e.g. 3, these properties are conspicously changed in the native pigments, in an adaptation to their function. From UV-Vis studies and complementary MO-calculations it has been concluded, that these changes are in part due to a conformational transition from a cyclic helical (e.g. 1 a) to a more extended conformation (e.g. 1 b,

Abbreviation: PC, C-phycocyanin; PE, C-phycoerythrin; P r , red absorbing form of phytochrome; cd, circulardi­chroism; UV-Vis, absorption spectroscopy in the ultra­violett and visible ränge. Reprint requests to Prof. Dr. H. Scheer. 0341-0382/83/0500-0353 $ 01.30/0

for leading references, see refs. 4), and that this process involves at least one intermediate State [5]. The denaturation of PE [6] and phytochrome P r and Pfr [7] has been studied in detail by a combinatory evaluation of absorption, fluorescence, and circular­dichroism spectroscopy. It could be shown that these techniques are complementary in exhibiting different sensitivities to the processes involved during denaturation. As an example in PE both the fluorescence and the cd-signals of the chromophores [4] decrease to almost zero upon complete un­folding of the protein [6]. On the other hand, the optical activity of phytochrome (P r , 2) is retained upon unfolding of the protein [7].

We here wish to report the corresponding cd spectra for the urea-induced denaturation of PC. The results obtained permit conclusions with respect to the origin of optical activity of bil i­proteins in general, especially in their denaturated states. Moreover, more direct evidence has been obtained for a stepwise unfolding of PC proposed earlier from UV-Vis data [5].

354 H. Lehner and H. Scheer • Circulardichroism of C-Phycocyanin

Experimental part

PC from S p i r u l i n a p l a t e n s i s was prepared by the method reported earlier [8], Urea was reagent grade (Merck, Darmstadt) or ultrapure (Schwarz & Mann, New York). In the spectroscopic studies, stock Solu­tions of PC (% 20 UM) in potassium phosphate buffer (0.05 M , pH 7) were mixed with this buffer, and with buffer containing urea (8 M) in the appro-priate ratios. UV-Vis and fluorescence spectra were recorded on a model D M R 2 2 (Zeiss, Oberkochen) spectrometer equipped with thermostated cell holder, at 20 °C. Cd-spectra were recorded on a dichrograph Mark III (Jobin-Yvon, France) using thermostated (20 ± 1 °C) cylindrical quartz cuvettes with variable path length (10-0.1 mm). The base lines were obtained by measuring the appropriate buffer-urea Solutions under the same conditions.

Results

The cd of native PC (Fig. 1) has a positive long-wavelength band peaking at 632 nm and a negative near-uv cd-band peaking at 342 nm, which are due to the chromophores. The bands below 280 nm arise primarily from the apoprotein. Both chromophore

bands are structured in a similar fashion as the absorption bands (Fig. 1), although there are small but distinct variations between the cd- and UV-Vis maxima {yide infrä).

PC contains three chromophores of identical molecular structure (1), but in different protein environments (see refs. 1-4). The fine structure of

30 25 v » 1 0 ° [ c m - ' ]

Fig. 1. Cd ( ) and UV-Vis absorption spectrum ( ) of PC (4 x 10"6 M) in the chromophoric region in phosphate buffer (0.05 M, pH 7) at room temperature. Molar extinction coefficients (e) and Jf-values are given for the monomer containing three chromophores.

H. Lehner and H. Scheer • Circulardichroism of C-Phycocyanin 355

the long-wavelength cd-band is roughly similar to that of the absorption band. In the latter, the major band corresponds to two chromophores, one each on the a- and /?-chain. The Shoulder at shorter wave-lengths which is resolve as a second band at low temperatures [9] arises from the second chromo­phore on the /?-chain. This assignment is likely from the spectra of the isolated subunits (Scheer et a L , unpublished). The similarity of the cd-band shape to this pattern indicates a similar differentiation. Upon denaturation, the long-wavelength cd-band (>W = 6T5nm) exerts a hypsochromic shift by

200 X[nm]-

250

30 25 * — vxlO-3 ( cm- ' ]

Fig. 2. Cd ( ) and UV-Vis absorption spectrum ( ) of PC (4xlO~ 6 M) in phosphate buffer (0.05 M, pH 7) containing urea (8 M) at room temperature. See Fig. 1 for molar extinction coefficients and Je-values.

30 25 vxlO- 'fcm-O

Fig. 3. Cd (- ) and UV-Vis absorption spectrum ( ) of PC (4X10" 6M) in aqueous urea (8 M) titrated with HCl to pH 1.5, at room temperature. See Fig. 1 for molar extinction coefficients and Je-values.

1x103-

2x103-

50 45 vx10- 3 [cm-l]

Fig. 4. Cd spectra of PC (4x 10~6M) in the peptide ab­sorption region at various urea concentrations (0 M, 2 M, 3 M, 4 M, 5 M, 6 M, and 8 M) in 0.05 M phosphate buffer at pH 7; room temperature.

17 nm, and the zle-value is decreased only by about 40% (Fig. 2). The changes in the negative band at 342 nm are less pronounced, there is even a slight increase of the ellipticities. Both bands loose their fine structure with increasing urea concentrations.

For a comparison, the cd of PC in 8 M urea at pH 1.5 is given in Fig. 3. The chromophhore is present in its protonated form under these condi-tions. In the UV-Vis spectrum, this leads to an increased absorption and a concomitant red-shift of the long-wavelength band [10], whereas the near-uv band remains almost unchanged. The red-shift is matched by the cd-band. The anisotropy factor of both bands is decreased by a factor of two.

The region of the peptide cd-bands (200-250 nm) shows a steady decrease with increasing urea con­centrations (Figs. 4, 5), with an inflection at about 4.5 M urea. The cd-titration curves at wavelengths ^ 300 nm (corresponding to chromophore absorp-tions) do not show similarly steady changes but rather irregularities which are most pronounced in the region between 4 and 5 M urea. Two such curves are shown in Fig. 5, and phenomenologically similar curves are obtained at any other wavelength cor­responding to chromophore rather than peptide absorptions.

356 H. Lehner and H. Scheer • Circulardichroism of C-Phycocyanin

X = 610(chromophor)

r t N

\ \

o \ ^

•,0°" \ I ; ' ' ' b

v " ° X= 3A5(chromophor)

Fig. 5. Titration curves of PC with urea at three wave-lengths: a: 220, b: 345 and c: 610 nm, monitored by cd.

Discussion

a . N o n c o i n c i d e n c e s between cd-a n d UV-Vis a b s o r p t i o n e x t r e m a

Whereas the cd fine structure in the native pig-ment can be interpreted like the electron absorption spectra by the three chromophores present in different environments (see refs. 1-4), the batho-chromic shift of the cd-spectra with respect to the electron absorption spectra in the chromophoric region needs further discussion.

Principally, it may arise from several origins: i) A dynamic equilibrium between different con-formers of each chromophore, with the populations absorbing more to the red exhibiting a stronger optical activity. ii) Each of the three chromophores is in a rather distinct environment, but the one ab­sorbing mostly to the red has a proportionally low absorption and high optical activity. This Situation is similar to (i), but represents a frozen rather than a

dynamic State, iii) At least two of the chromophores are close enough and appropriately oriented to each other to produce an exciton couplet with a positive band at the long-wavelength side.

Whereas several lines of evidence (see ref. 4) render the possibility (i) unlikely, both (ii) and (iii) are compatible with results assigning each chromo­phore type to a rather distinct and rigid site. Exciton coupling (e.g. iii) seems at least possible from the following arguments: In native PC the long-wavelength cd-maximum occurs at 632 nm, which is shifted by 306 cm"*1 (12 nm) to the red as compared to the long-wavelength absorption maxi-mum. A corresponding negative extremum would be expected around 608 nm. The pronounced dip in the cd-curve at 602 nm is indicative of this and may arise from a superposition of the negative part of the couplet with the positive band due to the "s"-chromophore. The opposite changes around 630 (negative) and 600 nm (positive) upon denaturation would also support this explanation. Excition Splittings have earlier been proposed for a crypto-phytan PC [11] and several allophycocyanins [12], and also discussed for PE [6], but only in the former case there exists more than circumstantial evidence.

b) O r i g i n of o p t i c a l a c t i v i t y i n n a t i v e a n d d e n a t u r e d b i l i p r o t e i n s

The spectral properties and the conformation of the prosthetic groups of native biliproteins are strongly influenced by the corresponding apopro-teins to which they are covalently bound. Thus, the chromophores exhibit an extended conformation which is chiral and rigid due to non covalent inter-actions with the apoprotein. On destroying the influence of the apoprotein by denaturation with urea, the conformation of the tetrapyrrol chromo­phores changes to a cyclic, helical arrangement (see ref. 4 for leading references). Hence, denatured biliprotein-chromophores predominantly adopt a cyclic helical conformation (2 a and la respectively). In open-chain tetrapyrrols lacking any chiral in­fluence the P- and M-helices are enantiomeric and thus equipopulated. Since the inversion barrier of the tetrapyrrol-helix is low (42 kJ • m o l - 1 in biliver-din [13]) and inversion fast at ambient,temperature, the P/M-helix ratio can be changed by exerting a diastereoisomeric relationship on it. Since such

H. Lehner and H. Scheer • Circulardichroism of C-Phycocyanin 357

helices represent inherently chiral chromophores large optical activity is to be expected even i f the excess population is small. An example for an external chiral influence represents the cd of achiral bile pigments complexed with the protein, serum albumin [14] or dissolved in chiral, optically active solvents [15-17].

In the denatured State of biliproteins the optical activity observed, is governed mainly by internal chiral influences, viz. of the chirality centers present in the chromophores. The PC chromophores possess chirality centers at C-2, C-3 and C-3 1 inducing a twist of the C-5 methine bridge, which is ener-getically favoured in one distinct direction. Hence, the large optical activity of even denatured PC may be rationalized in terms of an excess population of the P-helical arrangement being diastereoisomeric to the less populated M-helix. This interpretation is further corroborated by recent Ramachandran-type calculations [18, 19] indicating a twist of the C-5 methine bridge due to steric hindrance of the asym-metrically substituted ring A with rings B and D. Accordingly, the P-helical form would be favoured, in agreement with both the sign and the strong optical activity of denatured PC.

This interpretation likewise accounts for both the low optical activity observed for denatured PE (chromophore 4) [6] and the large Je-values reported for denatured red-absorbing phytochrome (P r , chro­mophore 2) [7]. The chromophores of P r (2) and PC (1) are constitutionally and configura-tionally closely related. The reason for the high optical activity in the denatured State of P r may then likewise be attributed to the influence of the chirality centers at C-2, C-3 and C-3 1, in generating an excess population of the P-type tetra-pyrrol-helix. In PE (4), the additional chirality center at C-16 leads to a Pertubation at the C-15 methine bridge as well. However, this influence counteracts the one excerted by C-2, C-3 and C-3 1. In a Situation similar to that in the i(=inactive)-urobilins with asymmetric C-4 and C-16 [20] these opposing effects in PE account for the extremly low optical activity observed for denatured PE [6].

For a more relevant comparison of PC with P r we have also measured a cd-spectrum of the urea-unfolded PC under acidic conditions (Fig. 3). The cd-spectrum of protonated PC (Fig. 3) possesses the same phenotype as that of protonated P r . It is positiv in the long wavelength band, negative in the

band around 360 nm. Even the zJ^-values are very similar: P r : 660 nm, Ae = + 2Q', 375 nm, A e = — 15; PC (values for one chromophore): 658 nm, Ae = + 101/3 = + 33; 350 nm, As = - 75/3 = - 25. In the native State, by contrast, the cd-spectra of phyto­chrome P r [7] and of PC (Fig. 1) are (expectedly) quite different indicating the varying influence of the two different proteins on the chromophores.

The close resemblance of the cd-spectra of the denatured PC (Fig. 2, 3) and phytochrome in the P r-form [7] with that of a model pigment, 2,3-di-hydrooctaethylbilindion (3), in chiral, optically active solvents [17] further corroborates the argu-ments made above. The cd-maxima of the latter at 610/348 nm [17] are only slightly shifted as com-pared to denatured PC at 615/358 nm (Fig. 2). Moreover, the similar ratios of Ae-values (0.7 for dihydrooctaethylbilindion 3 and 0.8 for PC in 8 M urea) indicate a similar pitch for the helix of the two chromophores.

c. Stepwise unfolding of P C d u r i n g d e n a t u r a t i o n

In an earlier study of PC unfolding [5] an inter­mediate during the denaturation process of PC with urea has been postulated to account for the Singular behavior of the UV-Vis absorption spectra. Fig. 5 nicely shows that the cd-titration curves yield more direct evidence for this assumption. The titration curve of the apoprotein (X = 220 nm, curve a in Fig. 5) taken from the data of Fig. 4 has an inflec-tion point between 4 M and 5 M urea. This point roughly represents the "melting point" of the apo­protein of PC. However, monitoring the chromo-phoric region by cd, e.g. at 380 nm and 610 nm (curves b and c in Fig. 5), curves are obtained whose maxima approximately coincide with the in-flection point of curve a for the peptide region (arrows). In a mathematical sense, curves b and c are derivatives of a. This means, that at least three species of PC are involved in the denaturation process: Between 0 M and 2 M urea the native species dominates. With increasing urea concentra-tion, the concentration of this species decreases and an intermediate, new species appears reaching a maximum at the "melting point" of the apoprotein (4 M - 5 M urea). Higher urea concentrations then favour the completely randomized, denatured PC and the concentration of the intermediate vanishes again at 8 M urea.

358 H. Lehner and H. Scheer • Circulardichroism of C-Phycocyanin

A c k n o w l e d g e m e n t s

H. L. thanks the "Hochschuljubiläumsstiftung der Stadt Wien" and the "Österreichischer Fonds zur

Förderung der wissenschaftlichen Forschung" (project 3033) for support, and H. S. acknowledges support of this work by the Deutsche Forschungs­gemeinschaft, Bonn.

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