ZEITSCHRIFT FÜR NATURFORSCHUNG - uni … · zeitschrift fÜr naturforschung section c biosciences...
Transcript of ZEITSCHRIFT FÜR NATURFORSCHUNG - uni … · zeitschrift fÜr naturforschung section c biosciences...
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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