Z E I T S C H R I F T FÜR N A T U R F O R S C H U N G Section C

A Journal of Biosciences

ISSN 0341-0382

Managing Editor:

H . Hausen, Tübingen

Council:

E. Bünning, Tübingen A. Butenandt, München M . Eigen, Göttingen

Editors:

A . Hager, Tübingen K. Hahlbrock. Köln W. Hasselbach, Heidelberg P. Karlson, Marburg F. Kaudewitz, München J. Klein, Tübingen J. St. Schell, Köln E. Wecker, Würzburg

Associate Editors:

N . Amrhein, Bochum B. A. Askonas, London W. Barz, Münster P. Böger, Konstanz G. Bornkamm, Freiburg D. Bückmann, Ulm K. G. Götz, Tübingen G. Gottschalk, Göttingen P. Gruss, Heidelberg G. Isenberg, Köln R. Jaenicke, Regensburg V. ter Meulen, Würzburg G. F. Meyer, Tübingen M . Rajewsky, Essen H . Schimassek, Heidelberg D. Schulte-Frohlinde,

Mühlheim/R. G. Schulz, Freiburg F. F. Seelig, Tübingen J. Seelig, Basel H . Simon, München W. Steglich, Bonn H . Stieve, Aachen J. Suko, Wien A . Trebst, Bochum G. Weissenböck, Köln G. Wiek, Innsbruck V. Zimmermann, Würzburg

Information for Contributors Contributions to Z. Naturforsch., Section C (biological sciences), may be (i) original papers, (ii) research notes not exceeding two pages in print and (iii) reports on current research of special interest. Two copies of each contribution should be sent to Dr. Helga Hausen, P.O. Box 2645, D-7400 Tübingen. (Section B (chemical sciences): Dr. Heide Voelter, P.O. Box 2645, D-7400 Tübingen; Section A (physical sciences): Mrs. Tamara Littmann, Lenzhalde 21, D-7082 Oberkochen). Contributors are encouraged to suggest possible reviewers of their papers. The manu­script should be type written using double spacing throughout. The title should be concise but informative. The names of the authors, with first names and initials, and of the institution where the work was carried out follow the title. A running title with not more than 60 characters should be indicated if the title is longer than this. In a footnote on the first page, please give an address for reprint requests. The title, a selfconsistant abstract, suitable for direct use by the abstracting journals (no references to the main text), and five keywords, all in English, must precede the main text of each contribution. The main text should meet the highest standards as to novelty of the material, organiza­tion and conciseness. A qualified colleague and, if the text is in a foreign language, a person who thoroughly knows it , should have been given the opportunity to check the paper before its submission for publication. Papers, reporting mainly spectroscopic, X-ray or other data of no general relevancy should not be submitted. References and footnotes should be numbered (e.g. [1]) and listed at the end of the paper (e.g. [1] A . Meyer, Z. Naturforsch. 30c, 633 (1976)). Tables with the appropriate captions and a list of the figure legends should follow at the end of the paper. Allowance should be made for the reduction in printing of the drawings (line thickness, lettering!). Original drawings larger than 21 x 30cm should be replaced by copies of reduced size, when the manuscript is submitted and only be sent in when the manu­script is accepted. On all illustrations, the figure number and the author's name must be written in pencil. The authors will receive page proofs. Changes in the text after acceptance of the paper and drawings not fit for direct reproduction cause delay and create extra costs which may be charged to the author. 50 reprints are free of charge.

Information for Subscribers

The subscription prices per year are for normal subscribers:

Section A Section B Section C D M 5 1 0 . - D M 6 1 0 . - D M 425.-

for authors and their institutions if they order directly from the publishers:

Section A Section B Section C D M 408. - D M 488.- D M 340. -

plus postage and handling

Section A Section B Section C D M 3 5 . - D M 3 5 . - D M 2 5 . -

Single copies and back-numbers are available. Subscriptions will remain standing for the following year unless cancellations are made by postage October 1st.

Informations for Advertisers The price for a small advertisement (breadth 43 mm, height 57 mm) in all three sec­tions of the journal is D M 60 . - . For larger advertisments please order the price list from the publishers.

V E R L A G DER ZEITSCHRIFT FÜR NATURFORSCHUNG, T Ü B I N G E N P.O. Box 2645, D-7400 Tübingen (Postscheck-Konto Stuttgart 8039-700).

I Bayerische ]

ZEITSCHRIFT FÜR NATURFORSCHUNG

SECTION C

A EUROPEAN JOURNAL OF

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 , M ü n c h e n M . E I G E N , Gö t t i ngen

N . A M R H E I N , Bochum B . A . A S K O N A S , London W . B A R Z , M ü n s t e r P . BÖGER, Konstanz G. B O R N K A M M , Freiburg D . B Ü C K M A N N , U l m

K . G. G Ö T Z , T ü b i n g e n G . GOTTSCHALK, G ö t t i n g e n P . GRUSS, Heidelberg

Edi tor ia l Board

A. H A G E R , T ü b i n g e n K . H A H L B R O C K , K ö l n 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 , M ü n c h e n J. K L E I N , T ü b i n g e n J. ST. S C H E L L , K ö l n E. W E C K E R , W ü r z b u r g

Advisory Edi tor ia l Board

G. ISENBERG, Köln R. J A E N I C K E , Regensburg V . TER M E U L E N , W ü r z b u r g G . F. M E Y E R , T ü b i n g e n M . R A J E W S K Y , Essen H . SCHIMASSEK, Heidelberg D . S C H U L T E - F R O H L I N D E , M ü h l h e i m / R .

G. S C H U L Z , Freiburg F. F. SEELIG, T ü b i n g e n

J. S E E L I G , Basel H . S I M O N , M ü n c h e n W . S T E G L I C H , Bonn H . STIEVE, Aachen J. S U K O , W i e n A . TREBST, Bochum G. W E I S S E N B Ö C K , Köln G . W I C K , Innsbruck V . Z I M M E R M A N N , W ü r z b u r g

E D I T E D I N C O L L A B O R A T I O N

W I T H T H E I N S T I T U T E S O F T H E M A X - P L A N C K - G E S E L L S C H A F T

Volume 42 c 1987

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

Anschrift des Verlages: Postfach 26 45, D-7400 Tübingen Satz und Druck: Allgäuer Zeitungsverlag GmbH, Kempten

Nachdruck — auch auszugsweise — nur mit schriftlicher Genehmigung des Verlages

Section a Physics, Physical Chemistry, Cosmic Physics

Section b Inorganic and Organic Chemistry

Volume 42c Zeitschrift für Naturforschung 1987

Contents

C o n t e n t s o f N u m b e r 1/2

Original Co mm imications

X-Ray Structure Analysis and Spectroscopic Data of the Ant ib io t ic 8-(Dichloroacetyl)-5-hydroxy-2,7-dimethyl-l ,4-naphthoquinone from the Fungus Mollisia sp. G. W E B E R , T . H Ü B N E R , A . G I E R E N , J. S O N N E N -

BICHLER, T . K O W A L S K I , and O. H O L D E N R I E D E R 1

Structure of Azadiracht in B H . R E M B O L D , H . FORSTER, and J. SONNENBICHLER 4

n-Alkylphenols f rom Schinus terebinthifolius R A D D I (Anacardiaceae) ( In German)

G. SKOPP, H . - J . OPFERKUCH, and G . SCHWENKER 7

Methylation of Guanine in vivo by the Organo-phosphorus Insecticide Methamidophos S. M . A . D . Z A Y E D and F. M . M A H D I 17

Cytogenetic Effects of the Insecticide Methamido­phos in Mouse Bone Marrow and Cultured Mouse Spleen Cells S. M . A M E R and M . A . SAYED 21

Molluscicidal Properties of Quinones ( In German) H . S C H I L D K N E C H T and J. L U B O S C H 31

Effects of Plant Bioregulators on the Production of I r ido id Der ived Terpenoids in Valeriana wallichii and Fedia cornucopiae Cell Suspension Cultures W . FÖRSTER and H . BECKER 33

Reactions of Substituted Arenediazonium Chlorides wi th Methylamine-Formaldehyde Premix Revisit­ed: Reactivity and Transformations of Methy lo l -amine Intermediates and Their Biological Signifi­cance G. F. K O L A R and M . SCHENDZIELORZ 41

Transformations of /rarcs-2-Hexenal by Botrytis cine­rea PERS. as Detoxification Mechanisms ( In German) I . U R B A S C H 64

Alkaloids in Stem Roots of Nicotiana tabacum and Spartium junceum Transformed by Agrobacterium rhizogenes M . W I N K and L . W I T T E 69

Flavonoids f rom the Leaf Resin of Snakeweed, Gutierrezia sarothrae D . H R A D E T Z K Y , ' E . W O L L E N WEBER, and J . N . R O I T -

M A N 73

The 14ß-Hydroxy la t ion in the Biosynthesis of Carde-nolides in Digitalis purpurea. The Role of 3 ß -Hydroxy-5ß-p regn-8 (14) -en -20-one M . E . D E L U C A , A . M . SELDES, and E . G . GROS 77

Biotransformation of Humulene by Fungi and Enan-tioselectivity of the Strains Used W . - R . A B R A H A M and B . STUMPF 79

Enzymatic Synthesis of 1,6-Digalloylglucose from ß-Glucogallin by ß-Glucogal l in : ß-Glucogal l in 6-0-Galloyltransferase from Oak Leaves S. W . S C H M I D T , K . D E N Z E L , G . S C H I L L I N G , and

G. G. GROSS 87

PAPS-Reductase from Escherichia coli: Characteri­zation of the Enzyme as Probe for Thioredoxins J. D . S C H W E N N and U . SCHRIEK 93

Protein Transport in Chloroplasts: A T P is Prere-quisit C. SCHINDLER, R. H R A C K Y , and J. S O L L 103

Lip id Influence on the Structure of the Light Har­vesting B 800-850 Proteins J. PESCHKE and H . M O H W A L D 109

Interaction in vitro of Non-Epithelial Intermediate Filament Proteins wi th Histones P. T R A U B , G . PERIDES, S. K Ü H N , and A . SCHER-

BARTH 47

Daffodil Chromoplast D N A : Comparison wi th Chlo-roplast D N A , Physical Map , and Gene Localiza­tion P. H A N S M A N N 118

I V Contents

Uptake of Protoporphyrin and Viole t Light Photo-destruction of Propionibacterium acnes T . B . M E L 0 123

Evolu t ion of E. coli t R N A I l e : Evidence of Derivat ion f rom Other t R N A s M . P. STAVES, D . P. B L O C H , and J. C. L A C E Y , Jr.

129

Intermolecular H-Abstract ion of T h i y l Radicals f rom Thiols and the Intramolecular Complexing of the T h i y l Radical wi th the Th io l Group in 1,4-Dithio-threi tol . A Pulse Radiolysis Study M . S H . A K H L A Q and C. VON SONNTAG 134

The Contents of the Dufour Gland of the A n t Har-pagoxenus sublaevis N y l . (Hymenoptera: Formi-cidae) D . G. O L L E T T , E . D . M O R G A N , A . B . A T T Y G A L L E ,

and J. P. J. B I L L E N 141

A GC/MS Study of the Propolis Phenolic Constitu­ents V . B A N K O V A , A . D Y U L G E R O V , S. POPOV, and

N . M A R E K O V 147

Investigation of the Screening Pigment System in the Compound Eye of the M o t h Agrotis segetum (fam. Noctuidae) by Visible Reflectometry T. N O R D T U G and T. B . M E L 0 152

Notes

Epicuticular Leaf Wax of Cistus albanicus, Cistaceae T . V O G T and P.-G. G Ü L Z 157

Isopulegol from L iqu id Cultures of the Fungus Cera-tocystis coerulescens (Ascomycotina) W . - G . K O C H and V . S I N N W E L L 159

Inh ib i t ion of cAMP-Phosphodiesterase by M o l y b -date B . O F E N L O C H - H Ä H N L E and K . E I S E L E 162

Polyenic Hydrocarbons as Sex Attractants for Geo-metrids and Amatids (Lepidoptera) Found by Field Screening in Hungary G . S z ö c s , M . T Ö T H , H . J. B E S T M A N N , O .

VOSTROWSKY, R. R. H E A T H , and J. H . T U M L I N S O N

165

Species and Sex Specificity in the Odour Composi­t ion of T w o Panurgine Bees (Hymenoptera , A n -drenidae) W . F R A N C K E , W . SCHRÖDER, A . K . B O R G - K A R L S -

SON, G . BERGSTRÖM, and J. T E N G Ö 169

C o n t e n t s o f N u m b e r 3

Original Commutations

Analysis of Single Channel Currents wi th a Micro­processor Based Device W . SCHREIBMAYER, E . H O F E R , P. W O L F , A .

LUEGER, and H . A . TRITTHART 173

Natural Waxes Investigated by Soft Ionization Mass Spectrometry H.-R . S C H U L T E N , K . E . M U R R A Y , and N . S I M M L E I T

178

Epicuticular Leaf Wax of Euphorbia dendroides L . . Euphorbiaceae P.-G. G Ü L Z , H . H E M M E R S , J. B O D D E N , and F.-J.

M A R N E R 191

Cell-Free Synthesis of the Alkaloids Ammodendr ine and Smipine M . W I N K and L . W I T T E 197

Comparison of the Solid State C P M A S and Solution Carbon-13-NMR Spectra of Humic Acids Ex­tracted from Composted Municipal Refuse R. F R Ü N D , F. J. G O N Z A L E Z - V I L A , H . - D . L Ü D E ­

M A N N , and F. M A R T I N 205

Glutamic Acid-l-semialdehyde, a Hypothetical In ­termediate in the Biosynthesis of 5-Amino-levulinic A c i d A . K A H and D . D Ö R N E M A N N 209

Interaction of Sodium, L i t h i u m , Caesium, and Potassium Ions wi th Ascorbyl Radicals P. W I E C Z O R E K , T. O G O N S K I , and Z . M A C H O Y 215

The Effect of Guanidinium Chloride on the Self-Association of Bovine Liver Glutamate Dehydro­genase: a Gel Fi l t rat ion Study A . M A Z Z I N I and R. F A V I L L A 217

Contents

Molecular Mechanics Investigation on Side-Chain Conformations of a 1 7 a - E t h y l - 1 7 ß - h y d r o x y Steroid wi th Regard to Receptor Binding M . B O H L 221

Characterization and A m i n o A c i d Composition of a Hypertrehalosaemic Neuropeptide from the Cor­pora cardiaca of the Cockroach, Nauphoeta cine­rea G. G Ä D E 225

The Rate of A T P Hydrolysis Catalyzed by Reconsti­tuted CFoFpLiposomes G. SCHMIDT and P. GRÄBER 231

Phosphate Accumulat ion by Muscle in vitro and the Influence of Vi t amin D 3 Metabolites T. B E L L I D O and R. B O L A N D 237

A Correlation between Detergent Tolerance and Cell Wal l Structure in Green Algae S. B I E D U N G M A I E R , G. W A N N E R , and A . SCHMIDT

245

Further Characterization of Chickpea Isoflavone 7-0-Glucoside-6"-0-malonate: malonylesterase: Evidence for a Highly Specific, Membrane-Bound Enzyme in Roots of Cicer arietinum L . W . H I N D E R E R , J. KÖSTER, and W . B A R Z 251

Chromophore Assignment in C-Phycocyanin from Mastigocladus laminosus S. S IEBZEHNRÜBL, R. FISCHER, and H . SCHEER 258

The Effect of Phosphinothricin (Glufosinate) on Photosynthesis. I . Inhibi t ion of Photosynthesis and Accumulation of Ammonia A . W I L D , H . SAUER, and W . R Ü H L E 263

The Effect of Phosphinothricin (Glufosinate) on Photosynthesis. I I . The Causes of Inhib i t ion of Photosynthesis H . SAUER, A . W I L D , and W . R Ü H L E 270

Sethoxydim-Uptake by Leaf Slices of Sethoxydim Resistant and Sensitive Grasses I . STRUVE, B . G O L L E , and U . L U T T G E 279

Light-Induced C a 2 + Influx into Spinach Protoplasts K . H E I M A N N , G. KREIMER, M . M E L K O N I A N , and E.

L A T Z K O 283

Acyclonucleoside Analogues Consisting of 5- and 5,6-Substituted Uracils and Different Acyclic

V

Chains: Inhibi tory Properties vs Purified E. coli Uridine Phosphorylase A . K . D R A B I K O W S K A , L . LISSOWSKA, M . D R A M I N -

S K I , A . Z G I T - W R O B L E W S K A , and D . SHUGAR 288

A Simple Analysis of Purine and Pyrimidine N u ­cleotides in Plant Cells by High-Performance L i ­quid Chromatography H . A S H I H A R A , K . M I T S U I , and T . U K A J I 297

Magnetotactic Bacteria from Freshwater M . O B E R H A C K , R. SÜSSMUTH, and H . F R A N K 300

C o n t e n t s o f N u m b e r 4

Original Communications

Stereochemistry and Mechanism of Reactions Cata­lyzed by Tyrosine Phenol-Lyase from Escherichia intermedia M . M . PALCIC, S.-J. S H E N , E . SCHLEICHER, H .

K U M A G A I , S. S A W A D A , H . Y A M A D A , and H . G .

FLOSS 307

Distant Precursors of Benzylisoquinoline Alka lo ids and Their Enzymatic Formation M . RUEFFER and M . H . Z E N K 319

Characterization of 2ß ( J R)-17 -0 -Ace ty l a jma lan : Acetylesterase — a Specific Enzyme Involved in the Biosynthesis of the Rauwolfia A l k a l o i d A j ma-line L . P O L Z , H . SCHÜBEL, and J. STÖCKIGT 333

Induction and Characterization of a N A D P H - D e -pendent Flavone Synthase from Cell Cultures o f Soybean G . K O C H S and H . GRISEBACH 343

Proposal for the Mechanism of Ac t ion of Urocanase. Inference from the Inhib i t ion by 2-Methyluroca-nate E. GERLINGER and J. R £ T E Y 349

Re-Investigation of the Protein Structure o f Co­enzyme B 1 2 -Dependent D i o l Dehydrase K . T A N I Z A W A , N . N A K A J I M A , T . T O R A Y A , H . T A N A -

K A , and K . SODA 353

VI Contents

Methanogenesis from Acetate by Methanosarcina barkeri: Catalysis of Acetate Formation from Methyl Iodide, C 0 2 , and H 2 by the Enzyme Sys­tem Involved K . L A U F E R , B . E I K M A N N S , U . FRIMMER, and R. K .

T H A U E R 360

Divergent Evolut ion of 5S r R N A Genes in Methano-coccus G. W I C H , L . S I B O L D , and A . B Ö C K 373

Characterization of Some Claviceps Strains Derived from Regenerated Protoplasts B. S C H U M A N N , W . M A I E R , and D . GRÖGER 381

Phenylalanine and Tyrosine Biosynthesis in Spore-forming Members of the Order Actinomycetales H . - K . H U N D , B . K E L L E R , and F. L I N G E N S 387

E. coli Maltodextr in Phosphorylase: Primary Struc­ture and Deletion Mapping of the C-Terminal Site D . P A L M , R. G O E R L , G. W E I D I N G E R , R. Z E I E R , B .

FISCHER, and R. S C H I N Z E L 394

Fermentation of D-Xylose to Ethanol by Bacillus macerans H.-J. SCHEPERS, ST. B R I N G E R - M E Y E R , and H . S A H M

401

Semicontinuous and Continuous Production of Citric Ac id wi th Immobil ized Cells of Aspergillus niger H . E I K M E I E R and H . J. R E H M 408

Microbial Hydroxylat ion of Cedrol and Cedrene W . - R . A B R A H A M , P. W A S H A U S E N , and K .

K I E S L I C H 414

6-Methylpurine, 6-Methyl-9-ß-D-ribofuranosylpu-rine, and 6-Hydroxymethyl-9-ß-D-ribofuranosyl-purine as Ant iv i ra l Metabolites of Collybia maculata (Basidiomycetes) K . L E O N H A R D T , T. A N K E , E . H I L L E N - M A S K E , and

W . STEGLICH 420

Enzymatic Synthesis of Riboflavin and F M N Specifi­cally Labeled wi th 1 3 C in the Xylene Ring H . SEDLMAIER, F. M Ü L L E R , P. J. K E L L E R , and A .

B A C H E R 425

A Vi tamin D 3 Steroid Hormone in the Calcinogenic Grass Trisetum flavescens W . A . R A M B E C K , H . WEISER, and H . Z U C K E R 430

Functional Group Recognition of Pheromone Mole ­cules by Sensory Cells of Antheraea polyphemus and Antheraea pernyi (Lepidoptera: Saturniidae) H . J. B E S T M A N N , W . C A I - H O N G , B . D Ö H L A , L I -

K E D O N G , and K . E . K A I S S L I N G 435

Building Blocks for Oligonucleotide Syntheses wi th Uni fo rmly Fragmentable ß - H a l o g e n a t e d Protect­ing Groups ( In German) P. L E M M E N , R. K A R L , I . U G I , N . B A L G O B I N , and J.

CHATTOPADHYAYA 442

Experiments on the Optical Resolution of Condur-amine Analogs by Enzymatic Transesterification in Organic Solvents ( In German) G. K R E S Z E and M . S A B U N I 446

Steric Course of the Rhodium-Catalyzed Decarbony-lation of Chiral 4 -Methy l - [ l - 3 H,2- 2 H 1 ]pen tana l H . O T S U K A and H . G. FLOSS 449

Synthesis of Immobil ized Peptide Fragments on Polystyrene-Polyoxyethylene for Af f in i ty Chro­matography ( In German) E. B A Y E R , H . H E L L S T E R N , and H . E C K S T E I N 455

Biopterin Synthesis in Mouse Spleen during Bone Marrow Transplantation Correlates wi th U n i m ­paired Hemopoietic Engraftment I . Z I E G L E R and ST. THIERFELDER 461

In vivo Screening of Glutathione Related Detoxifica­t ion Products in the Early State of Drug Devel­opment A . PROX, J. S C H M I D , J. N I C K L , and G. E N G E L ­

HARDT 465

Synthesis and Complexing Features of an Ar t i f i c i a l Receptor for Biogenic Amines ( In German) F. P. SCHMIDTCHEN 476

Metabolism of the Herbicide 2-(2,4-Dichloro-phenoxy)-propionic A c i d (Dichlorprop) in Barley (Hordeum vulgare) G. B Ä R E N W A L D , B . SCHNEIDER, and H . - R .

SCHÜTTE 486

Site Directed Antisera to the D-2 Polypeptide Sub-unit of Photosystem I I R. GEIGER, R. J. B E R Z B O R N , B . D E P K A , W . O E T T -

MEIER, and A . TREBST 491

Contents VII

C o n t e n t s o f N u m b e r 5

Original Communications

Epicuticular Wax Hydrocarbons of Ericaceae in Ger­many I . SALASOO 499

On the Essential Oils from Chrysanthemum balsa-mita L . ( I n German) H . STROBEL, K . K N O B L O C H , and E. Z I E G L E R 502

Epoxy-rra/25-isodihydrorhodophytin, a New Metab­oli t from Laurencia obtusa ( In German) S. I M R E , H . LOTTER, H . W A G N E R , and R. H .

THOMSON 507

Structure Elucidation of Kwakhur in , a New Preny-lated Isoflavone from Pueraria mirifica Roots S. T A H A R A , J . L . I N G H A M , and S. Z . D Z I E D Z I C 510

Biosynthesis of Vi texin and Isovitexin: Enzymatic Synthesis of the C-Glucosylflavones Vi texin and Isovitexin wi th an Enzyme Preparation from Fagopyrum esculentum M . Seedlings F . KERSCHER and G . F R A N Z 519

Effect of p H on Glycolate and Ammonia Excretion in L - M S O Treated Chlorella Cells Y . SHIRAIWA and G. H . S C H M I D 525

Derepression of Arylsulfatase Act iv i ty by Sulfate Starvation in Chlorella fusca I . NIEDERMEYER, S. B I E D L I N G M A I E R , and A .

SCHMIDT 530

Root Hai r Specific Proteins in Glycine max D . W E R N E R and A . B . W O L F F 537

Synthesis, Analysis and Characterization of the Coenzyme A Esters of o-Succinylbenzoic A c i d , an Intermediate in Vi tamin K 2 (Menaquinone) Bio­synthesis R . K O L K M A N N and E. LEISTNER 542

Chloride Avai labi l i ty Affects the Malate Content and its Control by the Circadian Clock in Pulvini of Phaseolus coccineus L . W . - E . M A Y E R , W . A . R Ü G E , N . STARRACH, and R .

H A M P P 553

Enzymatic Acylo in Condensation of Acyclic Alde ­hydes W.-R. A B R A H A M and B . STUMPF 559

Sn-Glycerol-3-phosphate is a Product of Starch Degradation in Isolated Chloroplasts from Chla-mydomonas reinhardii G. K L Ö C K and K . KREUZBERG 567

Substrate Flow from Photosynthetic Carbon Metabolism to Chloroplast Isoprenoid Synthesis in Spinach Evidence for a Plastidic Phosphoglyc-erate Mutase D . SCHULZE-SIEBERT, A . H E I N T Z E , and G .

SCHULTZ 570

Energy-Dependent Chlorophyl l Fluorescence Quenching in Chloroplasts Correlated wi th Quan­tum Yie ld of Photosynthesis G. H . K R A U S E and H . L A A S C H 581

Spontaneous Release of Malondialdehyde from Ultraviolet Light Exposed Liposomal Membranes S. A G A R W A L , A . G H O S H , and S. N . CHATTERJEE

585

Pyrimidine Homoribonucleosides: Synthesis, Solu­tion Conformation, and Some Biological Proper­ties P. LASSOTA, J . T . K U S M I E R E K , R. STOLARSKI, and

D . SHUGAR 589

Reactivation of Streptolysin S by Oligonucleotide A . T A K E T O and Y . T A K E T O 599

Ring Opening Reactions of Bioreactive Lactam Sys­tems ( In German) H . FRISTER and E. S C H L I M M E 603

Embryotoxici ty Induced by Alky la t ing Agents. Some Methodological Aspects of D N A Alky la t i on Studies in Murine Embryos Using Ethylmethane-sulfonate T. P L A T Z E K , G. BOCHERT, U . R A H M , and D .

NEUBERT 613

Tetraponerine-8, an Alka lo ida l Contact Poison in a Neoguinean Pseudomyrmecine A n t , Tetraponera sp. J . C. B R A E K M A N , D . D A L O Z E , J . M . PASTEELS, P.

VAN H E C K E , J . P. DECLERCQ, V . S I N N W E L L , and W .

FRANCKE 627

Contents X

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

Original Communications

ZZE-Configurat ion of Chromophore ß-153 in C-Phycocyanin from Mastigoclaclus laminosus G . SCHMIDT, S. S I E B Z E H N R Ü B L , R . FISCHER, W .

R Ü D I G E R , H . SCHEER, T. SCHIRMER, W . B O D E , and

R . H U B E R 845

a-Diceroptene: A New Dimer ic Structure for Iso-ceroptene C H . V I L A I N , A . H U B E R T , L . D U P O N T , K . R . M A R K -

H A M , and E . W O L L E N W E B E R 849

Stereochemistry of T w o Hydroxybiflavanonols from Garcinia cola Nuts J. SONNENBICHLER, I . M A D U B U N Y I , and H . SCHEER

855

"Epicuticular Waxes" from Exine Material of Pine Pollen C H . NIESTER, P . - G . G Ü L Z , and R . W I E R M A N N 858

Bryoflavone and Heterobryoflavone, Two New Iso-flavone-flavone Dimers f rom Bryum capillare H . GEIGER, W . STEIN, R . M U E S , and H . D . Z I N S -

MEISTER 863

Site of Lupanine and Sparteine Biosynthesis in Intact Plants and in vitro Organ Cultures M . W I N K 868

Occurrence of Amavadin in Mushrooms of the Genus Amanita ( In German) E . K O C H , H . K N E I F E L , and E . B A Y E R 873

Ascorbic A c i d and Glutathione Contents of Spruce Needles from Different Locations in Bavaria W . F. O S S W A L D , H . SENGER, and F. E . ELSTNER

879

Ace ty l -CoA: 4-Hydroxybutinylbithiophene O-Ace-tyltransferase Isoenzymes f rom Tagetes patula Seedlings G. M E T S C H U L A T and R . S Ü T F E L D 885

Uptake and Uti l izat ion of Sulfonic Acids in the Cyanobacterial Strains Anabaena variabilis and Plectonema 73110 S. B I E D L I N G M A I E R and A . S C H M I D T 891

Partial Characterization of an Enzyme from the Fun­gus Ascochyta rabiei for the Reductive Cleavage of Pterocarpan Phytoalexins to 2'-Hydroxyisofla-vans B. H Ö H L and W . B A R Z 897

Comparing Short-Term Effects of A m m o n i a and Methylamine on Nitrogenase Act iv i ty in Anabae­na variabilis ( A T C C 29413) S. R E I C H , H . A L M O N , and P. BÖGER 902

Reversible pH-Induced Dissociation of Glucose De­hydrogenase from Bacillus megaterium. I I . Kine­tics and Mechanism E. M A U R E R and G. PFLEIDERER 907

On the Possible Relation between Morphology and Precursors of the Crystallities in Calcified Tissues F. C. M . DRIESSENS, R. A . TERPSTRA, P. B E N N E -

M A , J . H . M . W Ö L T G E N S , and R. M . H . VERBEECK

916

In the Search for New Anticancer Drugs, X X I . Spin Labeled Nitrosoureas G. SOSNOVSKY, S. W . L I , and N . U . M . R A O 921

Transmission of Hormonal Imprin t ing in Tetrahyme-na Cultures by Intercellular Communication G. CSABA and P. KOVACS 932

M T D Calculations on Quantitative Structure-Activi­ty Relationships of Steroids Binding to the Proge­sterone Receptor M . B O H L , Z . S I M O N . A . V L A D , G. K A U F M A N N , and

K . PONSOLD 935

Synthesis of Heat Shock Proteins during A m i n o Ac id or Oxygen Limita t ion in Bacillussubtilis relA^ and relA ( In German) M . HECKER, A . RICHTER, A . SCHROETER. L . W Ü L ­

FEL, and F. M A C H 941

Sexual Pheromones and Related Egg Secretions in Laminariales (Phaeophyta) I . M A I E R , D . G. M Ü L L E R , G. G A S S M A N N , W . B O -

L A N D , and L . JAENICKE 948

Comparison of Dufour Gland Secretions of T w o Species of Leptothorax Ants (Hymenoptera: For-micidae) M . F. A L I , E . D . M O R G A N , A . B . A T T Y G A L L E , and

J . P. J . B I L L E N 955

Contents X I

Comparative Sex Pheromone Biosynthesis in the Obliquebanded Leafroller, Choristoneura rosa-ceana, and the Redbanded Leafroller, Argyro-taenia velutinana, Moths ST. P. FOSTER and W . L . ROELOFS 961

Effect of Hydroxylamine Derivatives on Photorespi­ration in the Tobacco Aurea Mutant Nicotiana tabacum Su/su G. H . S C H M I D , K . P. B A D E R , A . R A D U N Z , C. J .

V A N A S S C H E , N . R E I N I E R , and B . C O U R T I A D E 965

Estimation of the Extent of D N A Platination after Interaction of d s -DDP with D N A and Chromatin I . K U L A M O W I C Z and Z . W A L T E R 970

Reversed Light Reaction of the Screening Pigment in a Compound Eye Induced by Noradrenaline A . J U S E , G. H O G L U N D , and K . H A M D O R F 973

Light and Elect ion Microscopic Studies Regarding Cell Contractili ty and Cell Coupling in Light Sen­sitive Smooth Muscle Cells from the Isolated Frog Iris Sphincter K . V . W O L F 977

Fluorescence Moni tor ing of Membrane Potentials: the Spatio-Temporal Resolution in Isolated Neurons of Helix pomatia T H . K N Ö P F E L and P. FROMHERZ 986

Growth Kinetics of the G2-Phase of Ehrl ich Ascites Turnor Cells, Separated from Anaerobically Treated Asynchronous Cultures C. K R O L L . W . K R O L L , and F. SCHNEIDER 991

Demonstration of Statistically Significant Correla­tions between 8 and 12 kHz Atmosferics and Sud­den Deafness G. RUHENSTROTH-BAUER, K . M E E S , R. S A N D H A -

G E N , H . B A U M E R , and B . F I L I P I A K 999

Notes

y-Decalactone, an Odoriferous Compound from the Ma le Butterfly, Lethe marginalis Motschulsky N . H A Y A S H I , H . K A W A G U C H I , A . N I S H I , and H .

K O M A E 1001

The Phosphatidylinositol Species of Suspension Cul­tu red Plant Cells S. H E I M and K . G. W A G N E R 1003

A r e Small R N A s Associated wi th Crohn's Disease? R. P E C H A N , H . K U N E R T , and H . J . GROSS 1006

Inhibi t ion of c A M P Phosphodiesterase by Some Phototherapeutic Agents L . B O V A L I N I , P. L U S I N I , S. S I M O N I , D . V E D A L D I , L .

A N D R E A S S I , F. D A L L ' A C Q U A , and P. M A R T E L L I

1009

Report

Chimeric Genes — Their Contr ibut ion for Molecular Biology and Plant Breeding ( In German) H . - H . STEINBISS and J . S C H E L L 1011

C o n t e n t s o f N u m b e r 9/10

Original Co mm unications

Terpenoids from a Black Sea Bryozoan Conopeum sen rat urn P. H A D J I E V A , S. POPOV, B . B U D E V S K A , A . D Y U L -

GEROV, and S. A N D R E E V 1019

Thin Layer Chromatographic and Infra Red Spectral Evidence for the Presence of Phosphonolipids in Cicada oni M . C. M O S C H I D I S 1023

Adnexal Glands Chemistry of Messor ebeninus Forel (Formicidae: Myrmic inae) M . C O L L , A . H E F E T Z , and H . A . L L O Y D 1027

A Novel Caffeic A c i d Derivative and Other Con­stituents of Populus B u d Excretion and Propolis (Bee-Glue) E. W O L L E N W E B E R , Y . A S A K A W A , D . S C H I L L O , U .

L E H M A N N , and H . W E I G E L 1030

Polyunsaturated Pheromones: Semi-Synthesis of (Z ,Z) -6 ,9 -Alkad ienes and ( Z , Z , Z) -3 ,6 ,9 -Alka-trienes from Natural ly Occurring Fatty Acids H . K . M A N G O L D , H . B E C K E R , and E . SCHULTE

1035

N M R Spectra of Flavone Di-C-glycosides from Apometzgeria pubescens and the Detection of Rotational Isomerism in 8-C-Hexosylflavones K . R. M A R K H A M , R. M U E S , M . S T O L L , and H . D .

ZINSMEISTER 1039

X I I Contents

Trichothecene Mycotoxins from Fusarium culmorum Cultures N . C. P. B A L D W I N , B . W . BYCROFT, P. M .

D E W I C K , D . C. M A R S H , and J. G I L B E R T 1043

Metabolism of the Prenylated Pterocarpan Edunol by Aspergillus flavus S. T A H A R A and J. L . I N G H A M 1050

Fungal Metabolism of the Prenylated Isoflavone 2,3-Dehydrokievitone S. T A H A R A , E . M I S U M I , J. M I Z U T A N I , and J. L .

I N G H A M 1055

Novel Flavonoids from the Fern Notholaena sul­phured F. J. A R R I A G A - G I N E R , M . I I N U M A , T . T A N A K A , M .

M I Z U N O , C. SCHEELE, and E . W O L L E N W E B E R 1063

Synthesis of Piperoyl Coenzyme A Thioester U . SEMLER, G . SCHMIDTBERG, and G. G. GROSS

1070

Geraniol-10-hydroxylase Act iv i ty and Its Relation to Monoterpene Indole A l k a l o i d Accumulation in Cell Suspension Cultures of Catharanthus roseus 0 . SCHIEL, L . W I T T E , and J. B E R L I N 1075

A New Detection Procedure for Aminoacylase Ac­t ivi ty of Microorganisms Direct ly on Plate Culture wi th o-Phthalaldehyde ( In German) Y . Y A M A Z A K I , W . H U M M E L , and M . - R . K U L A

1082

Small Angle X-Ray Study on the Structure of Act ive and Inactive Ribulose-l,5-bisphosphate Carboxyl­ase-Oxygenase from Spinach. Evidence for a Con-figurational Change 1. P I L Z , E . S C H W A R Z , G . P. P A L , and W . SAENGER

1089

Quantitative Histochemical Analysis of Starch, Malate and K + , together wi th the Act iv i ty of Phospho-enolpyruvate Carboxylase along an Elongating Primary Leaf of Hordeum vulgare R. H A M P P , W . H . O U T L A W Jr., and H . Z I E G L E R

1092

Biotransformation of Citral by Botrytis cinerea P. B R U N E R I E , I . B E N D A , G. B O C K , and P. SCHREIER

1097

Comparison of Various Strategies Designed to Optimize Indole A l k a l o i d Accumulat ion of a Cell Suspension Culture of Catharanthus roseus J. B E R L I N , C H . M O L L E N S C H O T T , and F. D I C O S M O

1101

Amperometr ic Ti t ra t ion Largely Overestimates Chloride Concentrations in Chloroplast Extracts G. SCHROPPEL-MEIER and W . M . K A I S E R 1109

Extrachloroplastic Site of Synthesis of Three Chloro­plast Proteins in Maize (Zea mays) L . A . K L E C Z K O W S K I , C. A . Z E I H E R , and D . D .

R A N D A L L 1113

Organolead Toxici ty in Plants: Tr ie thyl Lead ( E t 3 P b + ) Acts as a Powerful Transmembrane C I " / O H ~ Exchanger Dissipating H + -Gradients at Nano-Molar Levels A . H A G E R , I . MOSER, and W . B E R T H O L D 1116

Effect of p H on the Slow Phase Components of Delayed Luminescence in Chloroplasts £ . H I D E G and S. DEMETER 1121

Chemokinesis and Necrotaxis of Human Granulo­cytes: the Important Cellular Organelles H . G R U L E R and A . DE BOISFLEURY C H E V A N C E 1126

In vitro T j and T 2 Relaxation Times of Coagulating Blood and Thrombuses ( In German) U . M . L A N D L E R , K . H E R G A N , E . JUSTICH, and H .

STERK 1135

Dependence of a Sleeping Parameter from the N-S or E-W Sleeping Direct ion G . R U H E N S T R O T H - B A U E R , E . R Ü T H E R , and T H .

REINERTSHOFER 1140

Notes

Automat ic Turgor Pressure Recording in Plant Cells K . - H . B Ü C H N E R , G . W E H N E R , W . V I R S I K , and U .

Z I M M E R M A N N 1143

Distinct Substrate Specificity of Dihydroflavonol 4-Reductase from Flowers of Petunia hybrida G . F O R K M A N N and B . R U H N A U 1146

Erra tum 1149

Contents

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

Contents of Nos 1-12 I I I - X I V

Original Communications

Identification of Betul in in Archaeological Tar ( In German) F. SAUTER. E . W . H . H A Y E K , W . M O C H E , and

U . JORDIS 1151

Chemical Composition and Morphology of Epi ­cuticular Waxes from Leaves of Solanum tube­rosum A . S E N 1153

Asymmetr ic Reduction of 4(5)-Oxocarboxylic Acids by Baker's Yeast ( In German) M . GESSNER, C. G Ü N T H E R , and A . M O S A N D L 1159

Partial Lack of N-Acety l Substitution of Glu­cosamine in the Peptidoglycan of the Budding Phototrophic Rhodomicrobium vannielii U . J . JÜRGENS, B . R I E T H , J . WECKESSER, C. S.

D o w , and W . A . K Ö N I G 1165

Accumulat ion of Phenolic Compounds and Phyto-alexins in Sliced and Elicitor-Treated Cotyledons of Cicer arietinum L . U . JAQUES, H . K E S S M A N N , and W . B A R Z 1171

Natural Inhibitors of Germination and Growth I V Compounds from Fruit and Seeds of Mounta in Ash (Sorbus aucuparia) U . OSTER, I . B L O S , and W . R Ü D I G E R 1179

The v versus v[I] Plot A . C. BORSTLAP 1185

Development of New Plate Tests for the Detection of Microb ia l Hydrolysis of Esters and Oxidations of 2-Hydroxycarboxylic Acids ( In German) Y . Y A M A Z A K I and M . - R . K U L A 1187

Enzymatic Synthesis of 4 '- and 3 ' ,4 ' -Hydroxylated Flavanones and Flavones wi th Flower Extracts of Sinningia cardinalis K . S T I C H and G . F O R K M A N N 1193

Purification and Properties of Chalcone Synthase f rom Cell Suspension Cultures of Soybean R. W E L L E and H . GRISEBACH 1200

X I I I

4-(2'-Carboxyphenyl)-4-oxobutyryl Coenzyme A Ester, an Intermediate in Vi tamin K 2 (Mena-quinone) Biosynthesis R. K O L K M A N N and E. LEISTNER 1207

Role of Pyrophosphate: Fructose-6-phosphate 1-Phosphotransferase in Glycolysis in Cultured Catharanthus roseus Cells H . A S H I H A R A and T . H O R I K O S I 1215

The Separation of T w o Different Enzymes Catalyz­ing the Formation of Hydroxycinnamic Ac id Glucosides and Esters P. A . B A U M K E R , M . JÜTTE, and R. W I E R M A N N 1223

Protein Sequence and Structure of N-Termina l A m i n o Acids of Subunit Delta of Spinach Photo-synthetic ATP-Synthase CF, R. J . B E R Z B O R N , W . F I N K E , J . O T T O , and H . E.

M E Y E R 1231

Isolation and Characterization of a Supramolecular Complex of Subunit I I I of the ATP-Synthase from Chloroplasts P. F R O M M E , E . J . B O E K E M A , and P. GRÄBER 1239

The Polyphasic Rise of Chlorophyl l Fluorescence upon Onset of Strong Continuous I l luminat ion: I . Saturation Characteristics and Partial Control by the Photosystem I I Acceptor Side C H . N E U B A U E R and U . SCHREIBER 1246

The Polyphasic Rise of Chlorophyll Fluorescence upon Onset of Strong Continuous I l luminat ion: I I . Partial Control by the Photosystem I I Donor Side and Possible Ways of Interpretation U . SCHREIBER and C H . N E U B A U E R 1255

Benzofuroxan as Electron Acceptor at Photosystem I B . L O T I N A - H E N N S E N , A . G A R C I A , M . A G U I L A R ,

and M . A L B O R E S 1265

C A R S Investigation of Changes in Chromophore Geometry of C-Phycocyanin from Mastigocladus laminosus Induced by Ti t ra t ion with p-Chloromer-curibenzenesulfonate S. SCHNEIDER, F. B A U M A N N , and U . K L Ü T E R 1269

Inhibi t ion by Sethoxydim of Pigment Accumulat ion and Fatty A c i d Biosynthesis in Chloroplasts of Avena Seedlings H . K . L I C H T E N T H A L E R , K . K O B E K , and K . I S H I I

1275

XIV Contents

Bioenergetics Studies of the Cyanobacterium Ana-baena variabilis S. SCHERER, H . S A D O W S K I , and P. BÖGER 1280

Radiochemical Methods for Studying Lipase-Cata-lyzed Interesterification of Lipids R. SCHUCH and K . D . M U K H E R J E E 1285

cAMP-Dependent Protein Kinase Act iv i ty in Yeast Mitochondria G . M Ü L L E R and W . B A N D I . O W 1291

Twisted Fibrils are a Structural Principle in the Assembly of Interstitial Collagens, Chordae Tendineae Included W . F O L K H A R D , D . C H R I S T M A N N , W . G E E R C K E N .

E. K N Ö R Z E R . M . H . J. K O C H , E. M O S L E R , H .

NEMETSCHEK-GANSLER, and T. NEMETSCHEK 1303

Lysine Decarboxylase from Hafnia alvei: Purifi­cation, Molecular Data and Preparation of Poly­clonal Antibodies H . BEIER, L . F. FECKER, and J. B E R L I N 1307

Recognition of H L A Class I I Molecules by Ant ipep-tide Antibodies Elicited by Synthetic Peptides Selected from Regions of H L A - D P Antigens A . CHERSI , R. A . H O U G H T E N , M . C. M O R G A N T I .

and E. M U R A T T I 1313

Active Cyanogenesis — in Zygaenids and Other Lepidoptera K . W I T T H O H N and C. M . N A U M A N N 1319

Efficacy of Sustained-Release Radioprotective Drugs in vivo J. S H A N I . S. B E N I T A , M . A B D U L R A Z I K , and

A . Y E R U S H A L M I 1323

MHC-Ant igens : Constituents of the Envelopes of Human and Simian Immunodeficiency Viruses H . G E L D E R B L O M . H . R E U P K E . T. W I N K E L .

R. K U N Z E , and G . P A U L I 1328

Evidences for Orcadian Rhythmicity in the per° Mutant of Drosophila melanogaster C H . H E L F R I C H and W . E N G E L M A N N 1335

Notes

Conformational Changes in Proteins Induced by Low Temperatures: an Infrared Study H . L . C A S A L , U . K Ö H L E R , H . H . M A N T S C H , F. M .

G O N I , and J. L . R. A R R O N D O 1339

Thermal Labil i ty of Membrane Proteins of Age Separated Erythrocytes as Studied by Electron Spin Resonance Spin Label Technique G. BARTOSZ, G. CHRIST, H . BOSSE. R. STEPHAN,

and H . GÄRTNER 1343

Lipids in the Gular Gland Secretion of the American All igator (Alligator mississippiensis) P. J . W E L D O N , A . S H A F A G A T I , and J . W . W H E E L E R

1345 Identification of the Sex Pheromone of Eggplant

Borer Leucinodes orbonalis Guenee (Lepidop­tera: Pyralidae) Z H U P I N G C H O U , K O N G F A N L E I , Y U S H E N G D I ,

Y u Y O N G Q I N G , J I N SHUPING, H U X I N H U A , and

Y u J I A N W E I 1347

(Z)-5-Dodecen-l-ol . Another Inhibi tor of Phero-monal Attract ion in Coleophora laricella E. PRIESNER 1349

(Z)-3-TetradecenyI Acetate as a Sex-Attractant Component in Gelechiinae and Anomologinae (Lepidoptera: Gelechiidae) E. PRIESNER 1352

Mammalian Pheromone Studies. V I . Compounds from the Preorbital Gland of the Blue Duiker , Cep halophus mon tico I a B. V . BURGER and P. J . PRETORIUS 1355

Structural Features and Biological Functions in Blue Copper Proteins Y . N I S H I D A 1358

Inhibi t ion of the Acety l -CoA Carboxylase of Barley Chloroplasts by Cycloxydim and Sethoxydim M . FOCKE and H . K . L ICHTENTHALER 1361

Formation of Large Thioredoxin / Accompanies Chloroplast Development in Scenedesmus obli-quus P. L A N G L O T Z and H . F O L L M A N N 1364

Cellular Spin Resonance of Yeast in a Frequency Range up to 140 M H z R. H O L Z E L and I . LAMPRECHT 1367

Melt ing Pressure. Volume and Stability of Blood at High Pressure ( In German) A . K L U G E and H . L E N T Z 1370

Subjekt Index

Authors Index

1373

1403

Chromophore Assignment in C-Phycocyanin from Mastigocladus laminosus S. S i ebzehnrüb l , R. Fischer, and H . Scheer

Botanisches Institut der Universität München, Menzinger Straße 47, D-8000 München 19, Bundesrepublik Deutschland

Z. Naturforsch. 42c, 258-262 (1987); received November 4, 1986

Photosynthesis, Cyanobacteria, Energy Transfer, Antenna Pigments, Cystein Modification

C-phycocyanin from the cyanobacterium, Mastigocladus laminosus, and its subunits have been treated with p-chloromercuribenzenesulfonate (PCMS). A single reactive site was found on the ß-subunit, and assigned to the single free cystein-ßl09. The concomitant spectral changes (absorp­tion, fluorescence, circular dichroism), together with the known close proximity of cys-ßl09 to chromophore ß82, allowed an unambiguous assignment of the three spectrally, biochemically and functionally different chromophores to specific binding sites on the two peptide chains (a84: 616-618, ß82: 622-624, ß 153: 598-600 nm).

Introduction

C-phycocyanins (PC), the light-harvesting pig­ments from cyanobacteria, are composed of two sub-units (a , ß) bearing one and two open-chain tetrapyr-rolic chromophores, respectively [1—5]. In spite of their common structures [ 1 , 2, 6, 7 ] , the three chromophores differ in their spectroscopic properties [8—10] and their reactivities [ 1 0 - 1 2 ] . These differ­ences, which arise from the different environments of the individual chromophores in the native chromoprotein, are essential for the fine tuning of their biological functions [1—5, 8, 10, 13].

The individual environments and conformations of the three chromophores, have recently been mapped in detail by sequenation [14—19] and X-ray crystal­lography [20, 21]. The data indicate a considerable degree of homology around the respective chromo­phores in PC's from different organisms. The single chromophore on the a-subunit is attached to Cys-84, and the chromophores on the ß-subuni t to Cys-82 and Cys-153. The two PC's studied by high-resolu­t ion X-ray crystallography [20, 21] (from Mastigoc­ladus laminosus and Agmenellum quadruplicatum), have furthermore almost identical chromophore and protein conformations and differ only in their aggre­gation state.

A correlation between the spectrally and structur­ally distinct chromophores has hitherto been lacking, but is very important in view of the recent progress in energy transfer studies (see e.g. [13]). Based on the

Reprint requests to Prof. Dr. H . Scheer. Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen 0341-0382/87/0300-0258 $ 01.30/0

Observation of a single binding site ( c y s - ß l 0 9 ) for mercurials [20, 21], we have now ti trated PC and its subunits from M. laminosus wi th the th io l reagent, PCMS, which led to the unambiguous assignment of the chromophores. This situation is more clear-cut than in other biliproteins, e.g. phycoerythrin [22] containing more than a single free cystein available for reaction wi th mercurials [ 1 - 5 ] .

Materials and Methods

M. laminosus was grown in Castenholz medium [23] in 300 1 batch cultures [24] and provided to us and stored deep frozen. PC and the subunits were prepared as described previously [6] . p-Chloro-mer-curi-benzenesulfonic acid (PCMS, A ld r i ch ) was a gift of T . Schirmer, all other chemicals came f rom Merck (Darmstadt) .

Absorpt ion spectra were recorded on a model 8451A spectrophotometer (Hewlett-Packard), fluo­rescence spectra on a model D M R 2 2 fluorimeter (Zeiss, Oberkochen) and circular dichroism spectra on a model V dichrograph (Jobin-Yvon, Unter­haching).

Titrations were carried out by adding aliquots of a stock solution of PCMS (2.26 I H M , in potasium phos­phate buffer, 100 ITIM, p H 7.0) to the chromoprotein solutions (1—3 \XM in the same buffer). The following extinction coefficients were used for the concentra­t ion determinations: PC trimer = 870,000 c m V 1

corresponding to 290,000 per protomer (aß)\ and 97,000 per chromophore); ß-subuni t = 164,000 cor­responding to 82,000 per chromophore; a-sub­unit = 122,000. They were determined by unfolding

S. Siebzehnrübl et al. • Chromophore Assignment in C-Phycocyanin from Mastigocladus laminosus 259

the proteins w i t h 8 M urea, on the basis of an extinc­t ion coefficient of 15,400 per chromophore in the denatured state [11].

Results

Integral PC

Treatment of PC tr imer wi th a threefold molar excess of PCMS (on a monomer basis for the pro­tein) results in a partial bleaching of the major ab­sorption (612 nm) (Fig. l a ) . I t is accompanied by a

minor blue-shift and the formation of a longer-wavelength shoulder. The integrated negative band (612 nm) in the difference spectrum, is about twice as large as the concomitant increase (655 nm) . There is also a distinct increase in the near-uv band. Ti t ra­t ion of PC gives a saturation of the reaction at 1 mol PCMS per mole PC (Fig. 2 a). The same general fea­tures are observed in PC tr imer containing l inker peptides ( X m a x = 629 n m ) , but here the negative peak in the difference spectrum is located at 632 nm (data not shown).

300 400 500 600 700 X (nm)

300 400 500 600 700 X (nm)

260 S. Siebzehnrübl etal • Chromophore Assignment in C-Phycocyanin from Mastigocladus laminosus

A A

0.08

- 0.04

0.00

-0.04

0.08

400 500 600 X ( n m )

I C Fig. 1. Treatment of integral PC-trimer ( A ) , its ß-subunit (B) and its a-subunit (C) with PCMS. Absorption (left scales) and absorption differ­ence spectra (right scales). 1 A : PC (4.8 nmol, spectrum a) treated with 13.6 nmol PCMS. Difference spectra re­corded after 30 (c), 60 (d) and 100 min (e), final absorp­tion spectrum (b) 180 min after addition. I B : Absorp­tion spectrum (a) of the ß-subunit (5.66 nmol) and final difference spectrum (b) after addition of PCMS (9.06 nmol, /=18min) . 1C: Absorption spectrum (a) of the a-subunit (6.9 nmol) and final differ­ence spectrum (b) after addi­tion of PCMS (9.1 nmol, / = 10 min).

660

-650

3 4 PCMS / Pigment

2B

Fig. 2. Titration of integral PC and its ß-subunit with PCMS. 2 A : Amplitude of the absorption difference signal (maximum-to-minimum). The absorptions before addi­tion of the reagent were 0.68 (—-), 0.62 ( ) and 0.66 (•••). Labels see inset. 2B: Relative fluorescence emis­sion intensities ( I F , left scale) and maxima (X m a x in nm, right scale). Labels see inset.

S. Siebzehnrübl et ai • Chromophore Assignment in C-Phycocyanin from Mastigocladus laminosus 261

The fluorescence of PC trimer is reduced to a min imum of 35% of the original value and saturates at « .8 mol PCMS, its wavelength is shifted by 10 nm to the red (Fig. 2 b ) . In the C D spectrum, the most notable feature is the development of a shoulder on the red-wing o f the long-wavelength band at 650 nm (data not shown, similar to the ß-subuni t shown in Fig. 3) .

Subunits

The spectral changes of the ß-subuni t are very similar to the ones observed in trimeric PC. The ab­sorption difference is again centered to the red of the absorption band. The amplitude of the difference spectrum (min imum to maximum) is even larger than PC in integral (Fig. l b ) , and the reaction saturates at « . 6 5 mol PCMS (Fig. 2). The fluorescence de­creases by 45%, and the amplitude of the red C D maximum by 7% w i t h a concomitant rise of a shoul­der around 650 nm and an increase of the near-uv band by 17% (Fig. 3) .

The a-subunit is essentially unreactive wi th PCMS (Fig. 1 c). There is only a minor feature in the difference spectrum wi th an intensity of 6% as com­pared to the ß - subun i t , which probably corresponds to a minor contamination wi th the latter ( « 5 % ac­cording to S D S - P A G E ) . The changes in the fluores­cence and the C D spectra are negligible as well (data not shown).

2 .00QE-4

Discussion

Besides the three cystein residues bound to the chromophores, PC contains only a single free cystein at position 109 on the ß-chain . I n the native chromoprotein, this cystein is very close ( ~ 4 A ) to the ß 8 2 chromophore, wi th an essentially protein-free hole between them, which is the single binding site for mercurials used for heavy-atom substitution [20, 21]. The distances to the other chromophores (a 84, ß 153) both on the same monomer and on the other monomers in the tr imer are considerably larger ( > 2 2 A ) . I t is, therefore, expected that the ß 8 2 chromophore is affected rather specifically upon binding of mercurials. A differential response of in ­dividual chromophores has earlier been reported for phycoerythrin [22], but could not be explored further due to lack of structural data at that t ime.

The saturation behavior in solution confirms the binding of only a single molecule of PCMS both in the PC protomer and its ß-subuni t . This is further corroborated by the lack of reaction wi th the a-sub­unit bearing no free cystein. A l l spectra can be rationalized by a single chromophore absorbing around 620 nm in the ß-subuni t and the t r imer, and around 630 nm in the linker-containing tr imer, re­spectively, being affected by the reaction of the free cys-109 wi th PCMS. T w o spectrally [10, 13], chemi­cally [10, 11] and photochemically [12] distinct chromophores have been identified on the ß-subuni t

en in cci ac Q

- 1 . 6 0 0 E -250 .0

Fig. 3. Circular dichroism spectra of the ß-subunit before (a) and after (b) reaction with PCMS. The spectra have been taken from the samples shown in Fig. 1B in absorption (traces a and b).

262 S. Siebzehnrübl et al. • Chromophore Assignment in C-Phycocyanin from Mastigocladus laminosus

of PC from M. laminosus. One of them ( ß l ) absorbs around 620 nm, is only weakly optically active and strongly fluorescent ( in the absence of any accep­tors), and the reversible photochemistry observed in partly denatured PC or its ß-subuni t resides on i t . The other one ( ß 2 ) absorbs below 600 nm, is strongly optically active and weakly fluorescent due to effi­cient energy transfer to ß 1 and (in integral PC) to the a-chromophore(s), and is photochemically inactive. I t is clear f rom the data, that the former chromo­phore ( ß l ) is the one close to the PCMS binding site and hence identical wi th ß 8 2 , whereas the latter ( ß 2 ) is identical w i th chromophore ß 153. I f judged from the absorption changes (increase in the near-uv, de­crease in the visible band), the ß 8 2 chromophore assumes a more helical conformation [11] after PCMS binding.

This assignment relates the photochemical reactiv­ity to the chromophore, ß 8 2 , which is least well de­fined in the X-ray structure [20, 21] and hence prob­ably also least rigidly bound. The data obtained wi th the linker-containing t r imer ( X m a x = 629 nm) also in ­dicate, that the ß 8 2 chromophore is the one shifted towards longer wavelengths (from « 6 2 2 to « 6 3 2 nm) . These linkers, which are substantial in the spectral modulation and organisation of bi l ipro-tein aggregates, are most l ikely located in the inner

[1] W. Rüdiger, Ber. Deutsch. Bot. Ges. 92, 413 (1979). [2] E . Gantt, Ann. Rev. Plant Physiol. 32, 327 (1981). [3] H. Scheer, in: Light Reaction Path of Photosynthesis

(F. K. Fong, ed.), pp. 7-45, Springer Verlag, Berlin 1983.

[4] A. N. Glazer, Ann. Rev. Biochemistry 52, 125 (1983). [5] W. Wehrmeyer, in: Proteins and nucleic Acids in Plant

Systematics (U. Jensen and D. E . Fairbrother, eds.), Springer Verlag, Berlin 1983.

[6] W. Kufer, O. Schmid, G. Schmidt, and H. Scheer, Z. Naturforsch. 41c, 437 (1986).

[7] J . C. Lagarias, A. N. Glazer, and H. Rapoport, J . Am. Chem. Soc. 101, 5030 (1979); see however: H. Rapoport, and A. N. Glazer, in: Optical properties and structure of Tetrapyrroles (G. Blauer and H. Sund, eds.), pp. 411-423, de Gruyter, Berlin, New York 1985.

[8] F. W. J . Teale and R. E . Dale, Biochem. J. 116, 161 (1970).

[9] J . Friedrich, H. Scheer, B. Zickendraht-Wendelstadt, and D. Haarer, J. Amer. Chem. Soc. 101, 1030 (1981).

[10] M. Mimuro, P. Füglistaller, R. Rümbeli, and H. Zuber, Biochim. Biophys. Acta 848, 155 (1986).

[11] W. Kufer and H. Scheer, Hoppe-Seyler's Z. Physiol. Chem. 360, 935 (1979).

[12] W. John, R. Fischer, S. Siebzehnrübl, and H. Scheer, in: Antennas and Reaction Centers of Photosynthetic Bacteria: Structure, Interaction and Dynamics (M. E . Michel-Beyerle, ed.), Springer Verlag, Berlin 1985.

hole of the doughnut-shaped t r i - and hexamers [4 , 5] . In PC, the ß 8 2 chromophore partly extends into this hole, which nicely fits wi th its spectral shift upon binding of l inker peptides.

Treatment wi th the mercurial , PCMS, thus allows a straightforward chromophore assignment in PC and its aggregates. I t should be useful as well w i th other biliproteins in view of the increased structural data available [1—5]. Interestingly, there are also large differences among the binding kinetics of PCMS to the different PC aggregates including phycobilisomes. This may be helpful in topological studies and is currently explored.

Acknowledgements

This work was supported by the Deutsche For­schungsgemeinschaft, Bonn . We are grateful for many stimulating discussions wi th and the provision of unpublished material by T . Schirmer and R. Huber (Mart insr ied) . The large-scale culture of M. laminosus by G . Reng at the Gesellschaft für biotechnologische Forschung (S töckhe im) is ac­knowledged.

Note added in proof: The amino acid residue numbering of the ß-subunit has been used differently in the literature [14, 20]. The one used corresponds to the original sequence data [14].

[13] K. Sauer, H. Scheer, and P. Sauer, Photochem. Photobiol., submitted; in part published in Proc. VII Int. Congr. Photosynthesis (J. Biggins, ed.), p. I. 1. 139, Martinus Nijhoff, Dordrecht.

[14] G. Frank, W. Sidler, H. Widmer, and H. Zuber, H. Hoppe-Seyler's Z. Physiol. Chem. 359, 1491 (1978).

[15] P. Freidenreich, G. S. Apell, and A. N. Glazer, J . Biol. Chem. 253, 212 (1978).

[16] G. D. Offner, A. S. Brown-Mason, M. M. Ehrhardt, and R. F. Troxler, J . Biol. Chem. 256, 2167 (1981).

[17] R. F . Troxler, M. M. Ehrhardt, A. S. Brown-Mason, and G. D. Offner, J. Biol. Chem. 256, 2176 (1981).

[18] T. J . Pilot and J. L . Fox, Proc. Natl. Acad. Sei. USA 81, 6983 (1984).

[19] R. Lorimier, D. A. de Bryant, R. D. Porter, W. Y . Liu. E . Jay, and S. E . Stevens jr., Proc. Natl. Acad. Sei. USA 81, 7946 (1984).

[20] T. Schirmer, W. Bode, R. Huber, W. Sidler. and H. Zuber, J. Mol. Biol. 184, 257 (1985).

[21] T. Schirmer, R. Huber, M. Schneider, W. Bode, M. Miller, and M. L . Hackert, J . Mol. Biol. 188, 651 (1986).

[22] J . Pecci, and E . Fujimori, Biochem. Biophys. Acta 154, 332 (1968).

[23] R. W. Castenholz, Schweizer Z. Hydrol. 35, 538 (1970).

[24] 300 1 mass cultures in the phototrophic fermenter at the Gesellschaft für Biotechnologische Forschung, 3301 Stöckheim, F R G .