ZEITSCHRIFT FÜR NATURFORSCHUNG

SECITON c

A EUROPEAN JOURNAL OF

BIOSCIENCES

C o u n c i l Edi tor ia l Board

E . B Ö N N I N G , T ü b i n g e n A . H A G E R , Tüb ingen

A . B U T E N A N D T , M ü n c h e n K . H A H L B R O C K , K ö l n

M . E I G E N , G ö t t i n g e 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. S T . 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

P. B Ö G E R , Konstanz H . S C H I M A S S E K , Heidelberg

D . B Ü C K M A N N , U l m D . S C H U L T E - F R O H L I N D E , M ü l h e i m / R .

K . G . G Ö T Z , T ü b i n g e n F. F. S E E L I G , T ü b i n g e n

G . G O T T S C H A L K , Gö t t i ngen J . S E E L I G , Basel

R. J A E N I C K E , Regensburg H . S I M O N , M ü n c h e n

G . F . M E Y E R , T ü b i n g e n W. S T E G L I C H , Bonn

M . R A J E W S K Y , Essen A . T R E B S T , Bochum

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

V O L U M E 39 c 1984

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 39c Zeitschrift für Naturforschung 1984

Contents

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

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

Nove l Ceramides from C y s t o b a c t e r f u s c u s (Myxobac-terales) (In German) H . E C K A U , D . D I L L , and H . B U D Z I K I E W I C Z 1

Pistallarin, a Characteristic Metabolite o f C l a v a r i a -d e l p h u s p i s t i l l a r i s and Several R a m a r i a Species (Basidiomycetes) (In German) W . S T E G L I C H , B . S T E F F A N , K . S T R O E C H , and M .

W O L F 10

New Dextrorotatory Peterocarpan Phytoalexins from Leaflets o f N i s s o l i a f r u t i c o s a J. L . I N G H A M and K . R. M A R K H A M 13

6-Hydroxyflavones and Other Flavonoids o f C r o c u s J. B. H A R B O R N E and C . A . W I L L I A M S 18

Conformation about the Glycosidic Bond and Sus-ceptibility to 5'-Nucleotidase o f 8-Substituted Analogues o f 5 ' - G M P P. L A S S O T A , R. S T O L A R S K I , and D . S H U G A R 55

Identification o f N ^ G l y c o l y l b i u r e t in the G a m m a Radiolysis o f Aerated Aqueous Solution o f Cyto-sine. Influence of the p H M . P O L V E R E L L I , J . U L R I C H , and R. T E O U L E 64

Purification and Partial Characterization o f the Soluble N A D H Dehydrogenase from the Photo-trophic Bacterium Rhodopseudomonas c a p s u l a t a T . O H S H I M A , M . O H S H I M A , and G . D R E W S 68

Causal Relationships among Metabolie Circadian Rhythms in L e m n a K . G O T O 7 3

Light Dependent A m m o n i u m Inhibit ion of Nitrate Assimilat ion in Rhodopseudomonas c a p s u l a t a A D 2 K . A L E F 85

Aliphat ic 1-Amino A c i d Decarboxylase from Ferns (Fil icopsida) T . H A R T M A N N , K . B A X , and R. S C H O L Z 2 4

Elaboration of the 6 ,7 ,8 Oxygenation Pattern in S im-ple Coumarins : Biosynthesis o f Puberul in in A g a -t h o s m a p u b e r u l a Fourc. S. A . B R O W N , D . E . A . R I V E T T , and H . J . T H O M P -

S O N 3 1

Biosynthesis o f the Bufadienolide R i n g o f Scil l irosid in S c i l l a m a r i t i m a L . R. G A L A G O V S K Y , A . M . P O R T O , G . B U R T O N ,

and E . G . G R O S 3 8

A n Adsorbent as Phytohormone Reservoir in a Plant C e l l Suspension Culture (In German) R. B E I D E R B E C K and B . K N O O P 4 5

Hormone Induced Changes in Carotenoid Compos i -tion in R i c i n u s C e l l Cultures. I. Identification o f Rhodoxanthin H . K A Y S E R and A . R. G E M M R I C H 5 0

Nickel-Dependent Uptake-Hydrogenase Activi ty in the Blue-Green A l g a A n a b a e n a v a r i a b i l i s H . A L M O N and P. B Ö G E R 9 0

Effect o f Electron Transfer Inhibitors and U n c o u -pl ing Agents on the Chlorophy l l Fluorescence Lifetime during Slow Fluorescence Decline in Bean Leaves and Intact Chloroplasts E. A . K O T O V A and M . D . I L ' I N A 9 3

The Influence of A m i n o A c i d Ligands and Vi ta -min C on the Reduction Potential o f Fe(III): Pola-rographic and Electron Spin Resonance Investiga-tions B. K I E F E R , H . S A P P E R , and W . L O H M A N N 1 0 2

Effects of N - A r y l - N ' ^ ' - D i a l k y l - l ^ - E t h a n e d i a m i n e s on A T P Format ion in Chloroplasts. Q S A R o f A m i n e Uncouplers G V A N D E N B E R G and N . B R A N D S E 1 0 7

Inhibit ion by Sethoxydim o f Chloroplast Biogenesis, Development and Replicat ion in Barley Seedlings H . K . L I C H T E N T H A L E R and D . M E I E R 1 1 5

IV Contents

Legume Root Response to Symbiotic Infection. E n -zymes o f the Peribacteroid Space R. B . M E L L O R , E . M Ö R S C H E L , and D . W E R N E R 1 2 3

C e l l Cycle Kinet ics and Metabol ism o f Ehr l i ch A s c i -tes Tumor Cells i n the Presence o f Chlorampheni -col as Inhibitor o f Mi tochondr ia l Protein Synthesis W . K R O L L and F . S C H N E I D E R 126

Immune Serum against Ant i~DNA-8-Methoxypsora -len Photoadduct Z . Z A R E B S K A , M . J A R Z A B E K - C H O R Z E L S K A , T.

C H O R Z E L S K I , and S. J A B L O N S K A 136

X - R a y Studies on Phosphol ipid Bilayers. III. Struc-ture and Morphology of L-a-Dilaurylphosphatidyl-ethanolamine ( D L P E ) M . S U W A L S K Y , C . G . S E G U E L , and F . N E I R A 141

Fusion o f Large Uni lamel la r Liposomes Conta in ing Hemocyanin with Planar Bilayer Membranes F. P A S Q U A L I , G . M E N E S T R I N A , and R. A N T O L I N I

147

E S R Investigations on Lyoph i l i zed B lood : Mixtures with Ascorbic A c i d H . N E U B A C H E R 174

Electron Microscopic Evidence for the Transmem-brane Displacement o f C a l c i u m ATPase D . J. S C A L E S and S. R . H I G H S M I T H 177

The Influence o f Cysteine on the Reaction o f d - G u -anosine with ds -Diammined ich lorop la t inum (II) I. K U L A M O W I C Z , R. O L I N S K I , and Z . W A L T E R 180

Insulin Bind ing Sites Induced in the Tetrahymena by Rat Liver Receptor Ant ibody G . C S A B A , P. K O V Ä C S , and A . I N C Z E F I - G O N D A 183

Intranuclear Crystals i n the Intestinal Ep i the l ium o f the Snail M a r i s a c o r n u a r i e t i s (Prosobranchia) R. G . L U T F Y and A . R U T H M A N N 186

Inheritance of mitochondrial D N A in O e n o t h e r a ber-t e r i a n a and O e n o t h e r a o d o r a t a Hybr ids A . B R E N N I C K E and B . S C H W E M M L E 191

The Tri ton X - 1 0 0 and H i g h Salt Resistant Residue o f Saccharomyces cerevisiae Nuclear Membranes, II. Isolation o f the Nuclear Membrane Insoluble Re-sidue from G 1 Arrested Cells and Immunological Comparison with the Corresponding Vertebrate Nuclear Fraction K . M A N N and D . M E C K E 1 5 6

Conductivity o f N o r m a l and Pathological H u m a n Erythrocytes (Homozygous ß -Tha lassemia ) at R a -diowave Frequencies C. B A L L A R I O , A . B O N I N C O N T R O , C . C A M E T T I , A .

R O S I , and L . S P O R T E L L I 1 6 0

S a l m o n e l l a / M i c i o s o m e Mutagenicity o f 1-Nitro pyre-ne-2-ol, a Nitropyrene Phenol Formed in the Pho-tolysis o f 1-Nitropyrene G . L Ö F R O T H , L . N I L S S O N , E . A G U R E L L , and A .

Y A S U H A R A 193

The Chromophore o f the Visua l Pigment in Some Insect Orders K . V O G T 1 9 6

The Life Span and Osmotic Fragi l i ty o f Erythrocytes in Mice Bearing Benzo(a) Pyrene - Induced F i -brosarcoma M . R . R A Y and J . R O Y C H O W D H U R Y 198

Notes

N e w C innamoyl Esters o f Quin ic A c i d from Meum a t h a m a n t i c u m D . B A R R O N , M . K A O U A D J I , and A . - M . M A R I O T T E

1 6 7

Product Specificity dur ing Incubation of Me thy l Linoleate with Soybean Lipoxygenase-I A . H A T A N A K A , T. K A J I W A R A , J . S E K I Y A , and M .

A S A N O 171

C o n t e n 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

Fie ld Desorption and Fast A t o m Bombardment Mass Spectrometry o f Spirostanol and Furostanol Saponins from P a r i s p o l y p h y l l a H . - R . S C H U L T E N , S. B . S I N G H , and R. S. T H A K U R

201

Contents

M i o t o x i n - A : A Nove l Macrocycl ic Trichothecene from the Brazi l ian Plant B a c c h a r i s c o r i d i f o l i a G . G . H A B E R M E H L , L . B U S A M , and J . S T E G E M A N N

212

Two Different Pathways Leading to Phenanthrenes and 9,10-Dihydrophenanthrenes of the Genus D i o s c o r e a K . - H . F R I T Z E M E I E R , H . K I N D L , and E . S C H L Ö S S E R

217

Glycoprotein Biosynthesis in P h y t o p h t h o r a mega-sperma f. sp. g l y c i n e a . A n i n v i t r o Study P. B A B C Z I N S K I 222

Catabol ism of (Ä) -Amygda l in and (Ä)-Vicianin by Partially Purified /3-Glycosidases from P r u n u s s e r o t i n a Ehrh . and D a v a l l i a t r i c h o m a n o i d e s G . K U R O K I , P. A . L I Z O T T E , and J . E . P O U L T O N 232

Volatile Sulfur Compounds Produced by Methionine Degrading Bacteria and the Relat ionship to C o n -crete Corros ion M . P O H L , E . B O C K , M . R I N K E N , M . A Y D I N , and

W. A . K Ö N I G 240

About the Similari ty between Polymaleic A c i d and Water Soluble H u m i c Fractions F. M A R T I N , F . J . G O N Z A L E Z - V I L A , and H . - D . L Ü -

D E M A N N 244

A Micromethod for Rap id Quantitative Determina-tion o f Phosphonate Phosphorus V . M . K A P O U L A S , S. K . M A S T R O N I C O L I S , I. C . N A K -

H E L , and H . J. S T A V R A K A K I S 249

Structure-Dependent Biological Activi ty o f Racemic 1-Substituted 2-0-Hexadecylglycero-3-phospho-cholines and Analogues S. K L U G E , H . -P . K E R T S C H E R , and G . O S T E R M A N N

252

Glutamate Dehydrogenase of Pisum s a t i v u m : Heat-Dependent Interconversion o f the Mul t ip le Forms A . E H M K E , H . - W . S C H E I D , and T. H A R T M A N N 257

Mechanisms o f Adriamycin-Dependent Oxygen Activat ion Catalyzed by N A D P H - C y t o c h r o m e c - (Ferredoxin)-Oxidoreductase E . P A U R , R. J . Y O U N G M A N , E . L E N G F E L D E R , and

E . F . E L S T N E R 261

V

Regulation of Acetyl-Coenzyme A Carboxylase and Acetyl-Coenzyme A Synthetase in Spinach C h l o -roplasts A . S A U E R and K . - P . H E I S E 268

Aurintr icarboxylic A c i d and Polynucleotides as Nove l Inhibitors o f Ribonucleotide Reductases H . B A U M A N N , R. H O F M A N N , M . L A M M E R S , G .

S C H I M P F F - W E I L A N D , and H . F O L L M A N N 276

Activat ion Volumes o f the Ca lc ium Dependent p a r a -Nitrophenyl Phosphate Hydrolysis o f the Sarco-plasmic Ret iculum Ca lc ium Transport Enzyme K . G . K Ö N I G and W . H A S S E L B A C H 282

Alterat ion of Acylphosphate Format ion of Cardiac Sarcoplasmic Ret iculum ATPase by Ca lmodu l in -Dependent Phosphorylation C . P I F L , B . P L A N K , G . H E L L M A N N , W . W Y S K O V S K Y ,

and J . S U K O 289

Isolation and Characterization o f Tributyl t in Resis-tant Mutants o f E s c h e r i c h i a c o l i A. P. S I N G H and K . S I N G H 293

Photoinactivation o f P r o p i o n i b a c t e r i u m acnes by Near-Ultraviolet Light B . K J E L D S T A D 300

Notes

Flavonoid Aglycones in the Leaf Resin of Some C i s t u s Species E. W O L L E N W E B E R and K . M A N N 303

Herpetol, a New Dimer ic L igno id from H e r p e t o s p e r -mum c a u d i g e r u m W a l l M . K A O U A D J I and J . F A V R E - B O N V I N 307

Structure Determination of 6-C-ß-D-glucopyrano-syl-8-C-a-L-arabinopyranosyltricetin from R a d u l a c o m p l a n a t a K . R. M A R K H A M and R. M U E S 309

A Convenient Synthesis of /?-Hydroxy-/3-[carboxy-methyl]-cinnamic A c i d (Sphagnum Acid ) (In Ger-man) D . W Ä C H T E R and H . R U D O L P H 311

VI Contents

y-Butyrolactone from the Black Stink Bug: Äethus i n d i c u s Westwood (Hemiptera: Pentatomidae) T . O. O L A G B E M I R O , M . N . K H A N , and A . M O H A M -

M E D 3 1 3

Identification o f a Major Pathway of Strand Break Formation in Poly U Induced by O H Radicals in Presence of Oxygen D . S C H U L T E - F R O H L I N D E and E . B O T H E 3 1 5

A T P Synthesis Dr iven by a Val inomycine Induced K + Diffusion Potential in Liposomes Bearing Chloroplast A T P Synthase M . D I N A N T and K . K A M I N S K I 3 2 0

Yeast-Like Endosymbionts i n an Ichneumonid Wasp J. M I D D E L D O R F and A . R U T H M A N N 3 2 2

Erratum to 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 , Z . Naturforsch. 38 c, 7 4 1 - 7 4 7

( 1 9 8 3 ) 3 2 6

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

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

Mode of Action of Herbicides in Photosynthesis

Preface J . J . S. V A N R E N S E N , A . T R E B S T , P. B Ö G E R 327

I. Herbicides and the Photosynthetic Electron Transport

Interference by Herbic idal Inhibitors o f Electron Transport with Phosphorylation and Permeability Properties o f Chloroplast Membranes D . E . M O R E L A N D and W. P. N O V I T Z K Y 329

Cyanoacrylate Inhibitors o f Photosynthetic Electron Transport. Nature o f the Interaction with the Re-ceptor Site J . P H I L L I P S and J. H U P P A T Z 335

Photosynthetic Electron Transport Inhibitors: Some Problems Related to an Accurate Determinat ion o f the Molecular Site o f Ac t ion C . J . V A N A S S C H E 338

A Nitroxide D i u r o n Ana log as a Probe for the Mode of Act ion o f Herbicides Y . S I D E R E R , N . L A V I N T M A N , C . G I L O N , and I.

O H A D 342

The Effects of 3-(3,4-Dichlorophenyl)- l , l -dimethyl-urea on the Photosynthetic Oxygen Complex M . - J . D E L R I E U 347

D C M U - I n d u c e d Fluorescence Changes and Photo-destruction o f Pigments Associated with an Inhibi-tion o f Photosystem I Cyc l ic Electron F low S. M . R I D L E Y and P. H O R T O N 351

Use of Chlorophy l l Fluorescence Induction Kinetics to Study Translocation and Detoxication of D C M U - T y p e Herbicides in Plant Leaves J. M . D U C R U E T , P. G A I L L A R D O N , and J. V I E N O T

354

Measurements o f Penetration and Detoxification o f PS II Herbicides i n Whole Leaves by a Fluorome-tric Method M . Voss, G . R E N G E R , P. G R Ä B E R , and C . K Ö T T E R

359

Studies on the Funct ional Mechanism o f System II Herbicides i n Isoiated Chloroplasts G . R E N G E R , R. H A G E M A N N , and W. F . J . V E R M A A S

362

Herb ic ide /Quinone Bind ing Interactions in Photo-system II W. F . J . V E R M A A S , G . R E N G E R , and C . J . A R N T Z E N

368

Interaction o f Photosystem II Herbidices with Bicar-bonate and Formate in Their Effects on Photo-synthetic Electron F low J . J . S. V A N R E N S E N 374

Comparison o f Bicarbonate Effects on the Variable Chlorophyl l a Fluorescence o f C 0 2 - D e p l e t e d and N o n - C 0 2 - D e p l e t e d Thylakoids in the Presence o f D i u r o n D . J . B L U B A U G H and G O V I N D J E E 378

The Effects of Bicarbonate Deplet ion and Formate Incubation on the Kinet ics o f Oxidat ion-Reduc-tion Reactions of the Photosystem II Quinone A c -ceptor Complex H . H . R O B I N S O N , J . J . E A T O N - R Y E , J . J . S. V A N

R E N S E N , and G O V I N D J E E 382

Contents

Formate as an Inhibitor of Photosynthetic Electron Flow J. F . H . S N E L , D . N A B E R , and J . J . S. V A N R E N S E N

386

Compar ison o f D i u r o n - and Phenol-Type Inhibitors: Addi t iona l Inhibitory Ac t ion at the Photosystem II Donor Site K . P F I S T E R and U . S C H R E I B E R 389

Herbicide and Plastoquinone Rind ing to Photosys-tem II W . O E T T M E I E R , H . - J . S O L L , and E . N E U M A N N 393

Redox-State Dependent Changes o f Inhibi tor-Bind-ing to the Photosystem II Acceptor Complex W . U R B A C H , H . L A A S C H , and U . S C H R E I B E R 397

Increased Bind ing of [ 1 4 C]Ioxyn i l in Unice l lu lar Green Algae under Anaerobiosis and by A d d i t i o n of Other Phenolic Herbicides or Uncouplers W , N E U M A N N , H . L A A S C H , and W . U R B A C H 402

Structure Act ivi ty Correlat ion o f Herbicides Affect-ing Plastoquinone Reduct ion by Photosystem II: Electron Density Distr ibution in Inhibitors and Plastoquinone Species A . T R E B S T , W . D O N N E R , and W . D R A B E R 405

The Molecular Basis of Triazine-Herbicide Resis-tance in Higher-Plant Chloroplasts J. H I R S C H B E R G , A . B L E E C K E R , D . J . K Y L E , L .

M C I N T O S H , and C . J . A R N T Z E N 412

Protein Sequence Homologies between Portions o f the L and M Subunits o f Reaction Centers o f R h o -dopseudomonas c a p s u l a t a and the Q B -P ro t e in o f Chloroplast Thylakoid Membranes: a Proposed Relat ion to Quinone-Binding Sites J . E . H E A R S T and K . S A U E R 421

Identification o f the Herbicide B ind ing Region of the Q B -P ro te in by Photoaffinity Labe l ing with Azidoatrazine P. K . W O L B E R and K . E . S T E I N B A C K 425

Herbicide-Binding Protein, B ind ing Sites and Elec-tron-Transport Act iv i ty : Quantitative Relations G . H E R R M A N N , A . T H I E L , and P. B Ö G E R 430

Isolation o f 32 -35 k D a Thylakoid Proteins from C h l a m y d o m o n a s r e i n h a r d i i N . P U C H E U and G . F . W I L D N E R 434

VII

Metribuzin-Resistant Mutants o f C h l a m y d o m o n a s r e i n h a r d i i N . P U C H E U , W . O E T T M E I E R , U . H E I S T E R K A M P , K .

M A S S O N , and G . F . W I L D N E R 4 3 7

Compar ison o f the Photosynthetic Capacity between Intact Leaves o f Triazine-Resistant and -Suscepti-ble Biotypes o f Six Weed Species J . L . P. V A N O O R S C H O T and P. H . V A N L E E U W E N

4 4 0

II. Bleaching Herbicides

Inhibit ion of Phytoene Desaturase - the Mode o f A c -t ion o f Certain Bleaching Herbicides G . S A N D M A N N , I. E . C L A R K E , P. M . B R A M L E Y , and

P. B Ö G E R 4 4 3

Compar ison of the Act ion o f Bleaching Herbicides J . F E I E R A B E N D 4 5 0

Herbicides which Interfere with the Biosynthesis o f Carotenoids and Their Effect on Pigment Exci ta-tion, Ch lo rophy l l Fluorescence and Pigment Composi t ion of the Thylakoid Membrane K . H . G R U M B A C H 4 5 5

Short Note on Dihydropyrones, a N e w Herbic idal Fami ly with Bleaching Properties J . V I A L and G . B O R R O D 4 5 9

Inhibi t ion o f Carotene Biosynthesis in C e l l Extracts o f Phycomyces blakesleeanus P . M . B R A M L E Y , I. E . C L A R K E , G . S A N D M A N N , and

P. B Ö G E R 4 6 0

Interaction of Herbicides with Pea Chloroplasts K . N . S I N G H , J . P R A K A S H , A . K . A G A R W A L , and G .

S. S I N G H A L 4 6 4

III. Peroxidations, Chloroplast Metabolism

Mult ip le Modes o f Ac t ion o f D ipheny l Ethers P. B Ö G E R 4 6 8

The Diphenyl-Ether Herbicide Oxyfluorfen: A Po-tent Inducer of L i p i d Peroxidation in Higher Plants K . J . K U N E R T 4 7 6

Photodynamic Damage to Isolated Chloroplasts: A Possible M o d e l for i n v i v o Effects o f Photosynthe-tic Inhibitor Herbicides M . P. P E R C I V A L and A . D . D O D G E 4 8 2

VIII Contents

Radical Format ion and Peroxidative Act ivi ty o f Phy-totoxic D ipheny l Ethers R. L A M B E R T , P. M . H . K R O N E C K , and P. B Ö G E R 4 8 6

Chloroplast Biogenesis, Its Inhibi t ion and Modif ika-tion by New Herbicide Compounds H . K . L I C H T E N T H A L E R 4 9 2

The Effect o f Phosphinothricin on the Assimila t ion of A m m o n i a in Plants A . W I L D and R. M A N D E R S C H E I D 5 0 0

Herbai Insecticides II [11. The Essential O i l from Leaves of Chrysanthemum b a l s a m i t a L . . Insectici-dal Act ivi ty and Composi t ion (In German) H . J . B E S T M A N N , B . C L A S S E N , U . K O B O L D , O.

V O S T R O W S K Y , F . K L I N G A U F , H . S T R O B E L , and K .

K N O B L O C H 5 4 3

Chemica l Defense o f Leguminosae. Are Qu ino l i z i -dine Alka lo ids Part o f the Ant imic rob ia l Defense System o f Lupins? M . W I N K 5 4 8

Physiological Changes i n M a t r i c a r i a i n o d o r a F o l -lowing Ioxyni l and Bromoxyn i l Treatment G . E . S A N D E R S , A . H . C O B B , and K . E . P A L L E T T

5 0 5

Increased Synthesis o f Photosystem II in T r i t i c u m v u l g a r e when G r o w n in the Presence o f B A S 1 3 - 3 3 8

S. B O S E , R. M . M A N N A N , and C . J . A R N T Z E N 5 1 0

C o n t e n t s o f N u m b e r 6

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

Hydroxycinnamoyl Esters of Ma l i c A c i d in Small Radish (Raphanus sativus L . var. s a t i v u s ) W. B R A N D L , K . H E R R M A N N , and L . G R O T J A H N 5 1 5

Epicuticular Waxes of Seed Coats from Species o f the Genus C i s t u s L . (Cistaceae) P. K R O L L M A N N , C . E I C H , and P . - G . G Ü L Z 5 2 1

Characterization o f Volati le Constituents from Pho-tomixotrophic C e l l Suspension Cultures o f R u t a g r a v e o l e n s F. D R A W E R T , R . G . B E R G E R , R . G O D E L M A N N , S.

C O L L I N , and W. B A R Z 5 2 5

The Structure o f Desmocarpin, a Pterocarpan Phyto-alexin from Desmodium g a n g e t i c u m J . L . I N G H A M and P. M . D E W I C K 5 3 1

Examinat ion of the Structure Activi ty Relationship of Antihepatotoxic Natura l Products (Si lybin-Antamanide) by X - R a y Analysis (In German) H . L O T T E R 5 3 5

Chemica l Defense of Lupins . Mollusc-Repellent Properties o f Quinol iz id ine Alkaloids M . W I N K 5 5 3

C o m m o n Identity o f U D P - G l u c o s e : Anthocyanidin 3-O-Glucosyltransferase and U D P - G l u c o s e : F l a -vonol 3-O-Glucosyltransferase in Flowers o f P e t u -n i a h y b r i d a L . M . V . J O N S S O N , M . E . G . A A R S M A N , J . B A S T I A -

A N N E T , W . E . D O N K E R - K O O P M A N , A . G . M .

G E R Ä T S , and A . W . S C H R Ä M 5 5 9

0 7 g and D 6 a : Two Flavone Glycosylat ing Genes in Silene, which are Only Expressed in Cotyledons and Rosette Leaves J . M . S T E Y N S , O. M A S T E N B R O E K , G . V A N N I G T E -

V E C H T , and J . V A N B R E D E R O D E 5 6 8

pH-Induced Inactivation of Glycero l Dehydrogenase from B a c i l l u s m e g a t e r i u m (In German) A . G A N Z H O R N , M . S C H A R S C H M I D T , and G . P F L E I -

D E R E R 5 7 5

Pheromone-Binding and Matr ix-Mediated Events in Sexual Induction o f V o l v o x c a r t e r i R. G I L L E S , C . G I L L E S , and L . J A E N I C K E 5 8 4

Cont ro l o f Fatty A c i d Incorporation into Chloroplast L ip ids i n v i t r o A . S A U E R and K . - P . H E I S E 5 9 3

Differences between Rat Splenic and Thymic Cells with Respect to the Effects o f E th id ium Bromide on the Unsheduled D N A Synthesis ( D N A Repa i r Synthesis) and the Nucleo id Sedimentation (In German) K . T E M P E L , A . G O E T T E , and I. S C H M E R O L D 6 0 0

Contents IX

Time-Resolved Polarized Fluorescence of C-Phyco-cyanin and Its Subunits from M a s t i g o c l a d u s l a m i -nosus P. H E F F E R L E , P. G E I S E L H A R T , T. M I N D L , S.

S C H N E I D E R , W . J O H N , and H . S C H E E R 606

Cyanoacrylate Inhibitors o f the H i l l Reaction. III. Stereochemical and Electronic Aspects of Inhibi -tor Bind ing J . L . H U P P A T Z and J . N . P H I L L I P S 617

Interaction of Photosynthetic and Respiratory Elec-tron Transport in Blue-Green Algae: Effect o f a Cytochrome c-553 Specific Ant ibody I. A L P E S , E . S T Ü R Z L , S. S C H E R E R , and P. B Ö G E R

623

Inhibi t ion o f Photosynthetic Reactions by Aureomy-cin J I - Y U Y E and U . H E B E R 627

Effect o f U V - B Radiat ion on Biomass Production, Pigmentation and Protein Content o f Mar ine Diatoms G . D Ö H L E R 634

A Granulopoiesis Inhibitor Partially Purified from Large-Scale Serum-Free Cultures o f Porcine L e u -kocytes M . K A S T N E R , H . R. M A U R E R , U . G E R L A C H , H .

R E N N E R , and J . H . W I S S L E R 639

Rabbit Antipeptide Antibodies against Restricted Domains of the Histocompatibil i ty Complex A . C H E R S I and R. A . H O U G H T E N 646

Purification of Soybean D N A - D e p e n d e n t R N A Polymerase I on a C o l u m n of Plasmid p H F K 206 Covalently Attached to Agarose K . G R O S S M A N N , H . F R I E D R I C H , and H . U . S E I T Z

652

Thermal Diffusion as a Mechanism for Biological Transport F. J . B O N N E R and L . O. S U N D E L Ö F 656

The Sensitivity Shift Due to Light Adapta t ion D e -pending on the Extracellular Ca l c ium Ion C o n -centration i n L i m u l u s Ventral Nerve Photorecep-tor H . S T I E V E , M . B R U N S , and H . G A U B E 662

Notes

H P L C Analysis o f Alka lo ids in Extracts of Callus Cultures o f C i n c h o n a Species R. V E R P O O R T E , T. M U L D E R - K R I E G E R , R. W I J N S M A ,

J . M . V E R Z U L , and A . B A E R H E I M S V E N D S E N 680

Abscisic A c i d in Phytopathogenic Fungi of the Genera B o t r y t i s , C e r a t o c y s t i s , F u s a r i u m , and R h i z o c t o n i a K . D Ö R F F L I N G , W . P E T E R S E N , E . S P R E C H E R , I.

U R B A S C H , and H . -P . H A N S S E N 683

Temperature Induced Spectral Changes o f Chloro-phy l l i n Micelles and Solution S. S. B R O D Y 685

Lysine-Enhanced Threonine-Inhibit ion o f Bacterial Aspartokinase: Concerted or Synergistic Feedback Inhibition? J . - H . K L E M M E 687

Lamoxirene. Structural P roof o f the Spermatozoid Releasing and Attracting Pheromone o f Lamina-riales F . - J . M A R N E R , B . M Ü L L E R , and L . J A E N I C K E 689

Packing Pattern o f D N A i n Bacteriophage T2 A . N . G H O S H , A . S E N , and N . N . D A S G U P T A 69?

C o n t e n 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

New Terphenylquinones from Mycel ia l Cultures o f P u n c t u l a r i a a t r o p u r p u r a s c e n s (Basidiomycetes) [1] (In German) H . A N K E , I. C A S S E R , R. H E R R M A N N , and W . S T E G -

L I C H 695

The Composi t ion of Terpene Hydrocarbons i n the Essential Oils from Leaves o f Four Cistus Species P . - G . G Ü L Z , U . K O B O L D , K . M I C H A E L I S , and O.

V O S T R O W S K Y 699

Chemosystematics o f Japanese H e t e r o t r o p a (Aristo-lochiaceae) N . H A Y A S H I , K . M A E S H I M A , T. M U R A K A M I , and H .

K O M A E 705

Contents X

Nove l Epicuticular Lea f Flavonoids from K a l m i a and G a u i t h e r i a (Ericaceae) E . W O L L E N W E B E R and G . K O H O R S T 710

Conversion o f Dihydroflavonols to Flavonols with Enzyme Extracts from Flower Buds o f M a t t h i o l a i n c a n a R. Br. R. S P R I B I L L E and G . F O R K M A N N 714

The Crystal Structure o f Anhydrous Xanthosine Displays Intramolecular 0 ( 2 ' ) H . . . 0 (3 ' ) Hydro-gen Bond B. L E S Y N G , C . M A R C K , and W . S A E N G E R 720

Tyrosine Oxidat ion by N 0 2 i n Aqueous Solution W . A . P R Ü T Z 725

Comparison o f Different Methods for the Determi-nation of Phenylalanine Hydroxylase Act iv i ty in Rat Liver and E u g l e n a g r a c i l i s R. M . F I N K and E . F . E L S T N E R 728

Studies on the Possible Mechanism of Inactivation o f Phenylalanine Hydroxylase by Destructive Oxy-gen Species R. M . F I N K and E . F . E L S T N E R 734

Two Dimensional Double-Quantum and C O S Y Spectra of Porcine Adenylate Kinase P. R Ö S C H and K . - H . G R O S S 738

O n the Reactivity o f Native Phytochrome P. E I L F E L D and W . R Ü D I G E R 742

Comparison of the Polypeptide Complement o f Dif -ferent Plastid Types and Mitochondr ia of N a r c i s -sus pseudonarcissus P. H A N S M A N N and P. S I T T E 758

Polycat ion-Cell Surface Interactions and Plasma Membrane Compartments in Mammals . Interfer-ence of Oligocat ion with Polycationic Condensa-tion S. A N T O H I and V . B R U M F E L D 767

Electron Microscopic Study o f the Po lymyxin Treated Goat Erythrocytes T K . M A N D A L and S. N . C H A T T E R J E E 776

Statistical Analysis o f Quantum B u m p Parameters in L i m u l u s Ventral Photoreceptors W . K E I P E R , J . S C H N A K E N B E R G , and H . S T I E V E 781

Mult ip le Periodicities in the Circadian System o f Unicel lu lar Algae M . H O F F M A N S - H O H N , W . M A R T I N , and K . B R I N K -

M A N N 791

System Analysis o f the Ci rcadian Rhy thm of E u g l e n a g r a c i l i s , II: Masking Effects and Mutua l Interac-tions o f Light and Temperature Responses T. K R E U E L S , R. J O E R R E S , W . M A R T I N , and K .

B R I N K M A N N 801

Mode of Discharge o f Haptocysts in E p h e l o t a gem-m i p a r a (Suctoria, Cil iata) (In German) G . B E N W I T Z 812

Uncoupl ing of Spinach Thylakoids by Gramine C . S. A N D R E O , E . G . O R E L L A N O , and H . M . N I E -

M E Y E R 746

The Radial Polar Pattern of Differentiation in T r i c h o -p l a x adhaerens F . E . Schulz (Placozoa) (In German) V. S C H W A R T Z 818

Effects o f Vi t amin A in the Presence o f Vitamins D 3 , E , K j on Red C e l l Membrane Structure R. C I C E R O , D . C A L L A R I , L . G U I D O N I , V . V I T I , M .

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

749

Hormone Induced Changes in C a r o t i n o i d Compos i -tum in R i c i n u s C e l l Cultures. II. Accumula t ion of Rhodoxanthin during Auxin-Cont ro l l ed Chromo-plast Differentiation A . R. G E M M R I C H and H . K A Y S E R 753

Notes

Exudate Flavanoids in H i e r a c e u m i n t y b a c e u m , an Alp ine Hawkweed (In German) E . W O L L E N W E B E R 833

Oxidized /?-Phenylenediamine Staining of Epoxy Resin Sections J . C . S T O C K E R T , R. A R M A S - P O R T E L A , O. D . C O L -

M A N , J . M . F E R R E R , and A . T A T O 835

Contents XI

The Distr ibution o f ß - C y a n o - L - a l a n i n e in Cyanoge-nic Lepidoptera (In German) K . W I T T H O H N and C . M . N A U M A N N 837

Specificity o f Synthetic Sex-Attractants i n Zygaena Moths E . P R I E S N E R , C . M . N E U M A N N , and J . S T E R T E N -

B R I N K 841

Analysis o f a Sex-Attractant System in the Noctu id M o t h R h y a c i a b a j a Schiff E . P R I E S N E R 845

The Pheromone Receptor System of Male E u l i a m i -n i s t r a n a L . , with Notes on Other Cnephasi in i Moths E. P R I E S N E R 849

Isolation and Respiratory Assay o f Ear thworm Body Wal l Mitochondria T V. R A O and U . C . B I S W A L 853

Neuroanatomical Evidence for Electroreception i n Lampreys B. F R I T Z S C H , M . - D . C . D E C A P R O N A , K . W Ä C H T -

L E R , and K . - H . K Ö R T J E 856

I n v i v o Metabolism of [4- 1 3 C]Phenacet in in an Isolated Perfused Rat Liver Measured by C o n -tinuous F low 1 3 C N M R Spectroscopy K . A L B E R T , G . K R U P P A , K . - P . Z E L L E R , E . B A Y E R ,

and F . H A R T M A N N 859

C o n t e n t s o f N u m b e r 9 / 1 0

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

Inorganic Bromide in Higher Fungi T. S T O V E 863

Characterization of M i c r o c y s t i s Strains by A l k y l Sul-fides and ß-Cycloci t ral F. J Ü T T N E R 867

Phthalide Derivatives from Meum a t h a m a n t i c u m Jacq. M . K A O U A D J I , A . M . M A R I O T T E , and H . R E U T E -

N A U E R 872

A l k a l o i d Sequestration by P a p a v e r s o m n i f e r u m Latex B. C . H O M E Y E R and M . F. R O B E R T S 876

Degradation o f the Isöflavone Biochanin A-7 -O-g lu -coside-6"0-malonate and Phenylacetic Acids by F u s a r i u m j a van i c um D . S C H L I E P E R , D . K O M O S S A , and W. B A R Z 882

Induced and Constitutive Isoflavonoids in Phaseolus mungo L . Leguminosae S. A . A D E S A N Y A , M . J. O ' N E I L L , and M . F . R O -

B E R T S 888

Cytochromes o f the Purple Sulfur Bacterium E c t o -t h i o r h o d o s p i r a s h a p o s h n i k o v i i W. H . K U S C H E and H . G . T R Ü P E R 894

Metabolie Activity of Hydroxycinnamic A c i d Glucose Esters i n Ce l l Suspension Cultures of C h e n o p o d i u m r u b r u m D . S T R A C K , M . B O K E R N , J . B E R L I N , and S. S I E G 902

Purification by Affinity Chromatography of Glu ta -thione Reductase ( E C 1.6.4.2) from E s c h e r i c h i a c o l i and Characterization of such Enzyme A . M . M A T A , M . C . P I N T O , and

J . L Ö P E Z - B A R E A 908

Occurrence of Mercaptopyruvate Sulfotransferase Act iv i ty in Photosynthetic Organisms A . S C H M I D T 916

Occurrence of the Bacter iochlorophyll-Binding Poly-peptides B870a and B 8 0 0 - B 8 5 0 a in the Mutant Strains Y5 and A l a + o f Rhodopseudomonas capsu-l a t a , which are Defective in Formation of the Light-Harvesting Complexes B870 and B800-850 , Respectively M . H . T A D R O S , R. D I E R S T E I N , and G . D R E W S 922

Functional and Regulatory Properties o f H + Pumps at the Tonoplast and Plasma Membranes o f Zea mays Coleoptiles A . H A G E R and W. B I B E R 927

D e novo Synthesis and Levels o f Cytochrome c and a Bil iprotein during Pupal -Adul t Development o f a Butterfly, P i e r i s b r a s s i c a e H . K A Y S E R 938

Development-Specific Incorporation o f [ 1 4 C ] 5 - A m i -nolevulinate and [ 3 H]Leucine into Cytochrome c and Bil iprotein in the Butterfly, P i e r i s b r a s s i c a e . Correlat ion with the Ecdysteroid Titer in the Pupa H . K A Y S E R and U . K R U L L - S A V A G E 948

Contents XII

Distribution of the H l Histone Subfractions in Syrian Hamster Chromat in Fractions H . M O D R Z E J E W S K A , G . G A t A Z K A , J . S Z E M R A J , and

H . P A N U S Z 958

Phospholipids and Glycerides Composi t ion during Spheroplasts Format ion of M y c o b a c t e r i u m s r n e g -m a t i s A T C C 14468 M . V . V . S. M U R T Y and T. A . V E N K I T A S U B R A M A N I -

A N 962

Surface Pressure Hysteresis o f M i x e d L i p i d / P r o t e i n Monolayers: Applicat ions to the Alveolar D y n a -mics R. M U T A F C H I E V A , I. P A N A I O T O V , and D . S. D I M I -

T R O V 965

Cel l Fusion by Simulated Atmospheric Discharges: Further Support for the Hypothesis o f Involve-ment o f Electrofusion in Evolu t ion G . K Ü P P E R S , K . - J . D I E D E R I C H , and U . Z I M M E R -

M A N N 973

Octopamine Modulates the Sensitivity of L i m u l u s Ventral Photoreceptor H . S T I E V E and E . A N D R £ 981

Influence of the Membrane Potential on the Intra-cellular Light Induced Ca 2 + -Concentra t ion Change o f the L i m u l u s Ventral Photoreceptor Moni tored by Arsenazo III under Voltage C l a m p Condit ions I. IvENS and H . S T I E V E 986

The Cel lular Substrate: A Very Important Require-ment for Baculovirus i n v i t r o Replicat ion H . G . MlLTENBURGER, W. L . NASER, J . P. HARVEY, J . H U B E R , and A . M . H U G E R 993

Notes

Production of C 6 - W o u n d Gases by Plants and the Effect on Some Phytopathogenic Fungi (In Ger-man) I. U R B A S C H 1003

Binding of the Fluorescent Dye 8-Anil inonaphtha-lene 1-Sufonic A c i d to the Native and Pressure Dissociated ß2-Dimer o f Tryptophan Synthase from E s c h e r i c h i a c o l i T H . S E I F E R T , P. B A R T H O L M E S , and R. J A E N I C K E

1008

Determination o f 4-Pregnene-3-ones in Thymus Tissue Samples by H i g h Performance L i q u i d Chromatography J . R E I S C H and J . N O R R E N B R O C K 1012

Effects o f V i t amin D 3 on i n v i v o Label l ing o f Ch ick Skeletal Muscle Proteins with [ 3 H]Leucine A . R. D E B O L A N D and R. L . B O L A N D 1015

A New Isolation Procedure for Acy lamino A c i d Amidohydrolase. Kinet ics o f the C o 2 + , M n 2 + , N i 2 + and C d 2 + Enzyme (In German) I. G I L L E S , H . - G . L Ö F F L E R and F . S C H N E I D E R 1017

Dose Rate Dependence of Radiat ion Induced I g G Membrane Receptor Alterat ion [1] F. O J E D A , D . M O R A G A , M . I. G U A R D A , and

H . F O L C H 1021

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

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

A N e w Cembranoid from Tobacco, I V V S I N N W E L L , V H E E M A N N , A . - M . B Y L O V , W .

H A S S , C . K A H R E , and F . S E E H O F E R 1023

Constituents o f A g a r i c u s x a n t h o d e r m u s Gene vier: The First Natural ly Endogenous A z o C o m p o u n d and Toxic Phenolic Metabolites M . G I L L and R. J . S T R A U C H 1027

Accumulat ion o f Volati le Flavour Compounds i n L i q u i d Cultures o f K l u y v e r o m y c e s l a c t i s Strains H.-P . H A N S S E N , E . S P R E C H E R , and A . K L I N G E N -

B E R G 1030

Dopamine Accumula t ion in P a p a v e r s o m n i f e r u m L a -tex B. C . H O M E Y E R and M . F. R O B E R T S 1034

Differential Synthesis o f Alka l ine Phosphatase in R h i z o b i u m Species Isolated from the Tropics A . P. S I N G H and J . B . S I N G H 1038

Preparation o f an Affinity Chromatographie System for the Separation o f A D P Bind ing Proteins E . B I E B E R , C . W O E N C K H A U S , and H . P A U L I 1042

A Simple Method to Prepare Affinity Resins on C e l -lulose Basis K . E I S E L E , F . D I A S C O S T A , C . P A S C U A L , and B .

O F E N L O C H - H Ä H N L E 1048

Contents XIII

Isolation of the Adducts o f Plat inum Complexes and Nucleic A c i d Bases on the Dowex 50 W C o l u m n R. O L I N S K I and Z . W A L T E R 1052

Isolation o f the Products Result ing from the Reac-tion o f eis and Irans Diaminedichloropla t inum [II] with D N A and Chromat in on the Dowex 50 W C o l u m n R. O L I N S K I and Z . W A L T E R 1057

Structural Studies on the Galactan from the A l b u -min G l a n d of A c h a t i n a f u l i c a O . H O L S T , H . M A Y E R , R. O . O K O T O R E , and W . A .

K Ö N I G 1063

Flash Photolysis o f Liposomes Conta in ing Chloro-phyl l and Zeaxanthin, as a Funct ion o f Tempera-ture ( 2 ° - 3 4 ° C ) S. S. B R O D Y 1108

Properties o f Membrane Fractions Prepared by Chromatophore-Liposome Fusion A . F . G A R C I A and G . D R E W S 1112

Light Induced Changes in the Conformation o f Spinach Thylakoid Membranes as Moni tored by 9 0 ° and 180° Scattering Changes: A Compara-tive Investigation S. J . C O U G H L A N and U . S C H R E I B E R 1120

Incorporation of Radiolabeled Tyrosine, N - A c e t y l -dopamine, N-/3-Alanyldopamine, and the A r y l -phorin Manduc in into the Sclerotized Cuticle o f Tobacco H o r n w o r m ( M a n d u c a sexta) Pupae L . G R Ü N and M . G . P E T E R 1066

H i g h Energy Radiation-Induced Cross l inking o f Histone Octamer Complexes K . - J . D E E G , L . K A T S I K A S , and W . S C H N A B E L 1075

Red-Light Effects Sensitized by Methylene Blue on Nitrate Reductase from Spinach { S p i n a c i a o l e r a -cea L.) Leaves S. G . M A U R I N O , M . A . V A R G A S , C . E C H E V A R R I A , P.

J . A J P A R I C I O , and J . M . M A L D O N A D O 1079

Fluorescence -ODMR of Reaction Centers of Rho-dopseudomonas v i r i d i s A . A N G E R H O F E R , J . U . V O N S C H Ü T Z , and H . C .

W O L F 1085

Flash Pattern o f Oxygen Evolu t ion i n Greening Etioplasts of Oat F. F R A N C K and G . H . S C H M I D 1091

Studies on the 0 2 Evolu t ion under Flash Light I l lu -mination in Preparations o f A n a c y s t i s n i d u l a n s E. K . P I S T O R I U S and G . H . S C H M I D 1097

Pressure Induced Shifts in Spectral Properties o f P ig -ment-Protein Complexes and Photosynthetic Or -ganisms S. S. B R O D Y and K . H E R E M A N S 1104

Effects o f Membrane-Act ing Drugs and Aerobiosis on Production o f Streptolysin S and Nuclease in Hemolyt ic Streptococci A . T A K E T O and Y . T A K E T O 1 1 2 8

Haemolytic Properties o f Cereal 5-«-Alk(en)yl resor-cinols A . K O Z U B E K 1 1 3 2

Dependence on Membrane Lip ids o f the Effect o f Vanadate on C a l c i u m and A T P Bind ing to Sarco-plasmic Ret iculum ATPase P. M E D D A and W . H A S S E L B A C H 1 1 3 7

Metabolie Effects o f Direct Current Stimulation on Cultured Vascular Smooth Muscle Cells H . H E I N L E , G . S I G G , A . R E I C H , and K . - U . T H I E D E -

M A N N 1141

Juvenile Hormone III as a Natura l L igand for Photo-affinity Label l ing o f J H - B i n d i n g Proteins H.-P . S T U P P and M . G . P E T E R 1 1 4 5

Titer o f Juvenile Hormone III in D r o s o p h i l a hydei during Metamorphosis Determined by G C - M S -M I S U . B Ü H R L E N , H . E M M E R I C H , and H . R E M B O L D 1 1 5 0

Feeding and M o l t Inhibi t ion by Azadirachtins A , B , and 7-Acetyl-azadirachtin A in Rhodnius p r o l i x u s Nymphs E. S. G A R C I A , P. D E A Z A M B U J A , H . F O R S T E R , and

H . R E M B O L D 1 1 5 5

Contents XIV

A High Frequency Mutat ion Starts Sexual Repro-duction in V o l v o x c a r t e r i B . W E I S S H A A R , R. G I L L E S , R. M O K A , and L .

J A E N I C K E 1159

Effect o f Extracellular A l k a l i Meta l Salts on the Elec-tric Parameters o f H u m a n Erythrocytes in N o r m a l and Pathological Condit ions (Homozygous ß -Tha -lassemia) C . B A L L A R I O , A . B O N I N C O N T R O , C . C A M E T T I , A .

R O S I , and L . S P O R T E L L I 1163

Impaired Diffusion Coupling-Source o f Arrhy thmia in C e l l Systems I. S C H R E I B E R , M . K U B I Ö E K , and M . M A R E K 1170

Notes

Arboxeniol ide-1, a New, Natural ly Occurr ing Xen io -lide Diterpenoid from the Gorgonian P a r a g o r g i a a r b o r e a o f the Crozet Is. (s. Indian Ocean) M . D ' A M B R O S I O , A . G U E R R I E R O , and F. P I E T R A

1180

Flavonoids o f E r i c a m e r i a l a r i c i f o l i a (Asteraceae) W . D . C L A R K and E . W O L L E N W E B E R 1184

Oxygen Supply of Roots by Gas Transport in Alde r -Trees W . G R O S S E and P. S C H R Ö D E R 1186

Inhibitors o f Calmodul in-Dependent Phosphoryla-tion Simultaneously Inhibit Ca lc ium Uptake and Calcium-Dependent ATPase Activi ty in Skeletal

Muscle Sarcoplasmic Reticulum and Transiently Induce C a l c i u m Release W. H A S S E L B A C H and A . M I G A L A 1189

Identification and F ie ld Evaluat ion of a Sex Phero-mone o f the European Pine Moth E. P R I E S N E R , H . B O G E N S C H Ü T Z , R. A L B E R T , D . W .

R E E D , and M . D . C H I S H O L M 1192

Stimulation o f Bra in Synaptosome - Associated Adenylate Cyclase by Ac id i c Phospholipids S . T S A K I R I S 1196

Stimulation o f Immunoreactivity against Endoge-nous Retroviruses and Protection against Leuke-mia of Older A K R Mice by Treatment with A n t i -bodies against Retroviral Surface Components. Role o f p l 5 ( E ) Ant ibody (In German) H . S C H W A R Z , H . - J . T ^ I I E L , K . J . W E I N H O L D , D . P.

B O L O G N E S I , and W . S C H Ä F E R 1199

Comment on: C . Koschnitzke, F . Kremer , L . Santo, P. Quick and A . Poglitsch, A Non-Thermal Effect o f Mi l l imeter Wave Radiat ion on the Puffing of Giant Chromosomes (Z. Naturforsch. 38 c, 883-886, 1983) A . H . F R U C H T 1203

Answer to the Comment of A . H . Frucht

F . K R E M E R 1204

Subject Index 1205

Authors Index 1231

Time-Resolved Polarized Fluorescence of C-Phycocyanin and Its Subunits from Mastigocladus laminosus* P. Hefferle, P. Geiselhart, T. M i n d l , and S. Schneider Institut für Physikalische und Theoretische Chemie der Technischen Universität München, D-8046 Garching, Bundesrepublik Deutschland

and W. John and H . Scheer Botanisches Institut der Universität, Menzinger Str. 67, D-8000 München 19, Bundesrepublik Deutschland

Z. Naturforsch. 39c, 606-616 (1984); received February 16, 1984

Photosynthesis, Biliproteins, Picosecond Spectroscopy, Energy Transfer, Protein Denaturation The influence of aggregation and temperature on the excited State kinetics of C-phycocyanin

from M a s t i q o c l a d u s l a m i n o s u s has been studied. Polarized fluorescence decay curves have been recorded using a synchronously pumped dye laser in conjunction with a synchroscan streak camera. The experimental data for all samples can be fit satisfactorily assuming a biexponential decay law. Fluorescence depolarization times have been interpreted in terms of energy transfer among the different chromophores. The influence of temperature is only moderate on the intra-molecular relaxation, but pronounced on the rates of energy transfer. Both are dependent on the size of the aggregate. The biexponential decay of the a-subunit containing only one chromo-phore, indicates the presence of different subsets of chromophores in these samples. The results are discussed in terms of variations of the chromophore arrangements upon temperature induced changes in the protein conformation.

Introduction

Phycobiliproteins are light harvesting pigments in certain algae. They contain 2 - 3 Polypeptide sub-units each bearing up to 4 covalently bound linear tetrapyrrolic chromophores [1 -3] . U n l i k e most other photosynthetic pigments, the phycob i l i -proteins are not integral membrane proteins and readily water soluble. The phycobil iproteins from blue-green and red algae are aggregated i n v i v o into microscopic particles, the phycobilisomes, which act as light harvesting and energy transfering units mainly to photosystem II. They also have a tendency for aggregation i n v i t r o . Here, much smaller and less complex structures are formed, which are believed to represent the phycobilisome bu i ld ing blocs [4, 5]. Earlier static fluorescence studies (see [6-8]) o f the readily accessible biliproteins have prompted a series of investigations by picosecond time-resolved spectroscopy using different excitation and detec-tion conditions [ 9 - 1 9 ] . The complex aggregation has, on the other hand, led to considerable technical and interpretational problems [11, 14, 16]. In order

* In part presented at the 6th Int. Congr. Photosynthesis, Brüssels 1983, Proceedings II. 2. 81.

Reprint requests to Prof. Dr. H. Scheer.

0341-0382/84/0600-0606 $ 01.30/0

to elucidate the influence of aggregation and other environmental factors (e.g. temperature) on the primary photophysical processes, we have begun systematic studies using polarized picosecond time-resolved emission spectroscopy [15, 16]. A s a conti -nuation of earlier work on higher aggregates i n -cluding integral phycobilisomes from M a s t i g o c l a d u s ( M . ) l a m i n o s u s [15], we here wish to report the results obtained with a series of increasingly complex aggregates of one of its bi l iproteins, C-phycocyanin (PC). This alga was chosen, because the primary structure of its biliproteins [20-22] as well as preliminary X-ray results of its P C [23] are known. The comparison with similar studies [16] on P C from a different alga, S p i r u l i n a (S.) p l a t e n s i s may also shed some light on the different properties of the two functionally similar pigments derived from a thermophilic and mesophilic organism, respectively.

Materials and Methods

B i o c h e m i s t r y

Cells of M . l a m i n o s u s were grown photoauto-trophically in Castenholz' medium [24] at 4 0 - 4 5 ° in 101 cultures. They were either used fresh for the measurement with whole cells, or stored frozen for

P. Hefferle et a l . • Time-Resolved Polarized Fluorescence and Its Subunits 607

the isolation of P C . The latter was isolated as described for S. platensis [16]. The tr imeric aggre r gates (5 2 o, w =5.6) were dissociated into the mono-mers (S 2 0,w = 2.8) with N a S C N ( I M ) . Subunits were obtained by preparative isoelectric focusing on Sephadex G75 gels (Pharmacia, Uppsala) and renatured without delay on Biogel P 2 desalting columns (Biorad, M ü n c h e n ) . The isoelectric points were at 6.3 (a-) and 5.1 (/?-subunit). The subunits were concentrated with aquacide (Calbiochem) to an optical density of ^ 0 . 5 c m " 1 at the red max i -mum. Both subunits were ^ 9 5 % pure from the respective other subunit and from colorless peptides i f judged from S D S - P A G E . The absorption and fluorescence spectra of the subunits are shown in Fig . 2. Analytical S D S Polyacrylamide gel electro-phoresis was performed according to Laemmli [25]. Analytical ultracentrifugation was done as described earlier [16] on a model E (Beckman, M ü n c h e n ) ultracentrifuge. The S-values were corrected for density of the Solutions. Standard correction factors for viscosity and partial volume of the protein were taken from the literature [26]. The viscosity correc-tion for N a C l was used for K S C N . The S 2o,w coefficients were then extrapolated to t = 0. Station-ary fluorescence was measured with a model D M R 2 2 (Zeiss, Oberkochen) photometer equiped

with a Single monochromator on the excitation side and a double monochromator on the emission side. Standard bandwidths were 33 and 15 nm, re-spectively, at 600 nm. Sedimentation and fluo-rescence measurements were done with aliquots o f the same preparations. A l l buffer Compounds and other chemicals used were reagent grade. Stationary fluorescence measurements were performed as described in the previous paper [16].

Time-resolvedfluorescence measurements a n d d a t a analysis

The experimental setup is s imilar to the one used in the previous Communications [15, 16]. The ex-citation pulses are derived from a dye laser (rhodamine 6 G , tuned to 600 nm) synchronously pumped by an acousto-optically mode-locked Argon ion laser. The pulse intensity at the sample is 10 1 3 photons • pu l se - 1 • c m - 2 . The fluorescence is monitored at 9 0 ° and passes through a filter (Kodak Wratten N o . 23, 620 nm cutoff) and a polarizing film directly into the entrance slit of the streak camera. The streaked image is monitored and digitized ( P A R model 1025 optical mul t i -channel analyzer ( O M A ) ) , and the data are trans-ferred to a minicomputer.

PL0TTER 0ATA

COMPUTING SYSTEM

A -

TO SHG-C0RRELATI0N

BS

FROM SYNCHRONOUSLY/'BS PUMPED M0DEL0CKED CW DYE LASER

0ELAY

- D -PINDIODE

.BS

BS MICHELSON INTERFEROMETER

RECORDER

TRIGGER

A/2

ND

OMA

< z >

PR,

STREAK CAMERA

SLIT

SAMPLE

7

Fig. 1. Schematic of the experimental setup for the fluorescence measurements. L = lenses; PR = fixed polarizer, f = emission filter, BS = beam Splitters, X/2 = adjustable polarizer, N D - neutral density filter. The Michelson inter-ferometer is used for calibration of time scale.

608 P. Hefferle et a l • Time-Resolved Polarized Fluorescence and Its Subunits

300 400 500 600 700 800 X/nm

Fig. 2. Absorption (—), fluorescence excitation (- - - - -) and emission spectra ( ) of the a (a) and /?-subunit (b) of PC from M . l a m i n o s u s . Al l spectra are normalized with respect to the red absorption maximum. Experimental conditions for the a-(ß)-subunit: A = 0.52 (0.48) at 617 (602) nm, emission at 650 nm for the excitation spectra, excitation at 600 (590) nm for the emission spectra. The skewing of the excitation as compared to the absorption bands is due to the experimental conditions.

The expressions / (/) = 7| (/) + 2 1 ± ( t ) and D ( t ) = / | (/) - 7 ± (/) were calculated from the decay curves with the analyzer being parallel (7ji (/)) and perpendicular (7 ± ( / ) ) to the adjustable polar izat ion of the exciting laser beam. 7 ( 0 corresponds to the decay of the excited State population, the "differ-ence function" D ( t ) to the product o f the former with the correlation function o f the absorption and emission dipoles [27, 28]. D ( t ) can be evaluated by means of a convolution (in contrast to the fluo-rescence anisotropy R ( t ) ) both i f the ind iv idua l functions are convoluted by the slower instrument response function (see e.g. [28]) and i f the recorded fluorescence is a superposition of fluorescence from two (or more) emitting species (see e.g. the appen-dix in [16]). The latter condition is prevalent in most biological samples.

In the case of fluorescence depolarization by orientational relaxation of the photoselected excited molecules, the correlation function is (multi-)ex-ponential and the analytically simple function D ( t ) can be evaluated to give the orientational relaxation time(s) T O R [29]. In the case of depolarization by energy transfer, the functional dependence of the

correlation function on energy transfer parameters is not yet solved. We have here assumed that the correlation function can be approximated by a (multi-)exponential and thereby derived a formal set of parameters T D E P which give a rough measure of the energy transfer kinetics.

Almost all decay curves can be fitted on the basis of a biexponential:

F ( t ) = A 0 + j E ( t - O - [AX exp ( - / V i , )

+ A 2 exp (- /7T 2 ) ]df'

where E ( t ) represents the excitation profile as recorded by the streak camera and AQ a constant background. The five parameters, A 0 , A X , A 2 I T\ and T 2 are determined using a non-linear least Squares routine based on the algorithm of M a r -quardt [30] and Berington [31]. The precision of the fit parameters depends not only on the signal-to-noise ratio (S /N) , but also on their relative magni-tudes [32]. The noise distribution is not well defined in a combined streak camera-optical multichannel detector System, in contrast to e.g. Single photon timing methods [13]. We have, therefore, performed simulations in order to establish some criteria for the realiability limits of the computed parameters. Two examples with S / N ratios of 2 and 5% are shown in F ig . 3 (see figure legend for details), which correspond roughly to the Situation en-countered with the /?-subunit (Figs. 4 a and 4 b). The deviation of the amplitude ratios ( A ] / A 2 ) is comparably small in the cases studied. The time constants can, however, deviate by up to 20% from the "true" value ( S = 1) for an S / N = 5%. This is in particular true for the long-lived component due to the limited time window ( 2 - 3 ns) of the streak camera System. If judged from these reliability tests, the absolute values of the calculated parameters are only approximate, but their variations with temper-ature and aggregation State should reliably reflect the trends.

Results and Discussion

T e m p e r a t u r e dependence o f t h e f l u o r e s c e n c e

The fluorescence intensity of all samples de-creased markedly with increasing temperature. This decrease is demonstrated in Fig . 3 for the a- and /?-subunits (normalized to equal excitation and detection conditions). The integrated fluo-

P. Hefferle et cd. • Time-Resolved Polarized Fluorescence and Its Subunits 609

N 26-

N 18-

16-

14-

12-

10-

N 14-

12-

10-

8-

0.92 a)

—\— 1,0 1.08 0,84 0.92 1.0 1.08 1,16

b)

2-

0.95 1.0 1.05

C) Fig. 3. Error analysis for the derived decay times. Noise from a random number generator corresponding to 2% (dashed lines) and 5% intensity (solid lines) with respect to the maximum amplitude has been superimposed on a theoretical decay curve (convolution of an excitation function with a biexponential, r?= 100 ps, T^= 1500ps, R ° = 5 ) . The procedure has been repeated 30 times, and the fit parameters were then determined by means of a program based on a Marquardt algorithm. The distribution of deviations (in relative units with respect to the "true" values, e.g. ö] = T]/iß]y

610 P. Hefferle et a l . • Time-Resolved Polarized Fluorescence and Its Subunits

msTisociADus ummnsus - ALPHA SUBUNIT OF PC

EXCITATION: 600 NM OBSERVATION:>620 NM

TEMPERATURE: 18,36,51 *C

. ' . ' V ' • • ' . A . ' ^ V . ' v *

1000 2000 3000 TIME ( Ps)

MAST06QCLADUS LAMINOSUS - BETA SUBUNIT 0F PC

EXCITATION: 600 NM 0BSERVATION:^620 NM

TEMPERATURE: 18,36,51 'C

1000 TIME

3000 ( P s )

Fig. 4. Isotropie decay /(/) of the chromophore fluorescence in the a-(top) and /?-subunits (bottom) of PC from M . l a m i n o s u s . Curves are normalized to equal excitation at 600 nm, maxi-mum intensity decreases with increasing temper-ature. All fluorescence with X ^ 620 nm has been recorded.

integral P C consisting of two subunits, and the dissociation of the former could be the process leading to this intermediate. The results obtained here for the a-subunit indicate, that another type o f intermediate must be invoked, e.g. one in wh ich the peptide chain has changed its conformation. The two-state model would predict a monotonous decrease of the fluorescence yield with temperature, and temperature independent decay constants. Since both the yield a n d the rates change, the data presented in this paper must be taken as further evidence for an intermediate State being present during the unfolding. As shown in F i g . 4, this is also true for the more complex aggregates.

A g g r e g a t i o n State a n d f l u o r e s c e n c e A similar set of data as shown in F i g . 4 has been

collected for the monomeric (aß) and tr imeric P C

(a/?)3 and for the whole algae. The data are sum-marized in Fig . 5 for both the isotropic (/(/)) and the anisotropic decay (£>(/), see experimental part for the definition of these funetions). T o emphasize the different kinetics with increasing temperatures, the curves have here been normalized to the max i -mum amplitude except in those cases, where an identification of the - indiv idual curves would be impossible due to an extensive overlap of data points. The results are in the following discussed in the order of increasing size of the aggregates.

a - s u b u n i t

The a-subunit of P C contains only a Single chromophore, and the decay should, therefore, be monoexponential, i f all chromophores were kept i n the same conformation by noncovalent interactions

P. Hefferle et cd. • Time-Resolved Polarized Fluorescence and Its Subunits

with the apoprotein. The decay curves can nonethe-less only be fitted by biexponentials (F ig . 5). A s imi-lar Observation has been made earlier for the isotropic fluorescence of the a-subunit of P C from S. p l a t e n s i s [16] and A n a b a e n a v a r i a b i l i s [19]. A n aggregation of the a-subunit is unlikely from (i) the low concentration, (ii) from the fact that N a S C N is present [16] and (i i i) the ultracentrifuge measurements ( S 2 0.w = 2.35). One must then assume, that there are two species present wi th different chromophore-protein arrangements and hence fluorescence lifetimes. The " long-l ivecl" species has a lifetime in the ränge of the integral biliproteins, whereas that of the "shor t - l ived" species is unusual in the sense that lifetimes of this intermediate ränge have only occasionally been reported for integral phycobiliproteins [16, 19].

In contrast to earlier work [16], it has now been possible to obtain also information of the depolar-ization with an acceptable S / N (Fig . 5). The dif-ference function D ( t ) of the a-subunit can again be fit only by a biexponential, with shorter decay times r than the respective T ' of the isotropic decay. Under the assumptions discussed above, the depolar-ization times r d e p can be obtained separately for both components from the relation

1/T'.

The slow depolarization components are subject to a large possible error (values in parenthesis in Table II) and wil l , therefore, not be discussed. The depolar-ization time of %1500ps for the short-lived com-ponent at lower temperatures (18 and 36 ° C ) can be reconciled with the torsional motion of a loosely bound chromophore. The chromophores of P C from M . l a m i n o s u s are covalently attached to the peptide chain by a Single thioether bond [38], but they are believed to be rigidly bound to the latter by additional strong non-covalent interactions (see Ref. [1]). The comparably rapid depolarization of the fast decaying fluorescence component would then indicate, that these interactions are weakened in the

Table II. Fluorescence depolarization times r d e p (in ps) in dependence on temperature and State of aggregation (for error analysis see text; values in parenthesis cor-respond to the slow decaying component).

d̂ep [PS] T[°C] d̂ep [PS]

18 36 51

a-subunit 1575 1355 573 (10778) (3306) -

/?-subunit 403 162 145 (2838) (2701) (773)

monomer 580 561 649

- (7165) (4400) trimer 70 242 160 - - (3520)

algae 0 0 0

"short-lived" species. This "loosened bol t" model would be supported by the decreased depolarizat ion time of 570 ps at higher temperature (51 ° C ) , where an even higher mobil i ty of the less tighly coupled chromophore is expected. This is also in accordance with the decreased isotropic lifetime of the "short-l ived" species, while that o f the " long- l ived" one is fairly insensitive to temperature.

There are two explanations for these results: The first is, that the fast decaying species is an experi-mental artefact due to irreversible denaturation. The isolation of the a-subunit involves a complete unfolding of the peptide chain over several hours, which may cause such problems. The second ex-planation suggested by Sauer (personal communica-tion, 1983) is based on the finding (contrary to our data) of a similar decay constant in the /?-subunit and in integral P C from A n a b a e n a v a r i a b i l i s [19]. It suggests, that the native biliproteins might be heterogeneous per se. The two possibilities are presently difficult to distinguish, but the micro-heterogeneity of a biopolymer is an intr iguing and potentially far-reaching idea. It may be supported by the fact, that biexponential decays have been observed for a-subunits of PC ' s from different

Fig. 5. Influence of temperature and aggregation on the isotropic (/(/), left side) and anisotropic decay (£>(/), right side) of the chromophore fluorescence in PC from M . l a m i n o s u s . Top: a-subunit, second row: /?-subunit, third row: monomeric PC, fourth row: trimeric PC and bottom: whole algae. No anisotropic fluorescence was detectable in the latter. Excitation wavelength was generally 600 nm, and all fluorescence with X ̂ 620 nm has been recorded. A l l curves are normalized with respect to peak intensity, except for few to allow a better identification of the individual curves. The original data are given by the points, and the fit parameters for the solid curve are given in the insets in the order T , , T 2 (in ps), amplitude ratios A ] / A 2 (in %). The data were collected at 18 °C (A), 36 °C (B), and 52 °C (C), in the figures the amplitudes at longer decay times decrease in that order (see Jabels Fig. 5 a) except for the anisotropic decay of the trimer (notice labels).

Isotropic Anisotropie

'5Ö0' ' 1000 1S00 2000 Ups)

P. Hefferle et cd. • Time-Resolved Polarized Fluorescence and Its Subunits 613

species and prepared by somewhat different proce-dures. It may also relate to the photochromic properties of phycobil iproteins under mi ld ly denaturing conditions including low p H [39], moderate concentrations of urea [40] or monomer formation by chaotropic salts inc lud ing N a S C N [41], or of isolated subunits o f bi l iproteins [42].

ß-Subunit

This subunit contains two chromophores, whose absorption maxima are about 20 nm or 550 c m " 1

apart. Both chromophores are about equally well excited with the chosen wavelength, v/z. 600 nm. The stationary emission spectrum o f the /?-subunit is at ambient temperature s imi la r to that of the monomer, which indicates an efficient energy transfer from the high-energy (sensitizing = "s" in the nomenclature of Teale and Dale [6]) to the low energy (fluorescing = "f") chromophore.

The postulated efficient energy transfer is sup-ported by the kinetic data. A satisfactory fit of all decay curves is again obtained with a biexponential fit (Fig. 5 c, d), although addi t ional long-l ived, low-amplitude components cannot be excluded in all cases. A t increased temperatures, the decay times of both components are reduced, but less pro-nounced than in the a-subunit. The depolar izat ion times r d e p (see above) of the fast decaying com-ponent are much faster than those o f the a-subunit and decrease with increasing temperature from T D E P % 400 to 150 ps (Table II). They are too fast for an orientational depolarizat ion, but are rather assigned to a depolarizat ion by energy transfer. A physical interpretation of r d e p as the energy transfer time is, however, ambiguous. There is no dissipative continuum, and back transfer can, there-fore, not be excluded, in which case T D E P would be only an effective energy transfer t ime. Processes o f this type have been discussed in Chlorophyl l an-tennas, which have s imi lar energy differences as isolated P C [43].

The longer l ived component in the fluorescence decay is considerably shorter than that o f the a-subunit and that o f al l other isolated P C ' s studied here (Fig. 5). This could indicate a part ial un-coupling of the chromophore as compared to the integral P C , because the free chromophores have lifetimes ^ 100 ps, whereas those o f native chromo-phores are « 1500 ps. There are two explanations to account for such a change: The first is again an

artefact due to the preparation, which involves the same denaturation-renaturation sequence as de-scribed above for the a-subunit. The second is a rearrangement of the peptide chain in the absence of the a-subunit. The absorption spectra o f the two subunits add up to that of the monomer (as ob-served earlier for other biliproteins, see e.g. [8]), but it is also known that the absorption spectra are far less sensitive than the fluorescence towards changes in the State o f the protein (see [1] for a discussion). A partial uncoupling of the chromophore is in -dicated by a small but distinct heterogeneity of the cw fluorescence (F ig . 2 b). It is also supported by the pronounced temperature sensitivity of the life-time. The reduction in the effective depolarization time would then indicate that the energy back transfer is more strongly reduced than the forward process.

M o n o m e r a n d t r i m e r

The isotropic fluorescence decay curves (/(/)) of the monomer (aß) and the trimer (a/?) 3 are similar to each other and to those of the /?-subunit. They can again be fit with biexponentials, with TX in the ränge of 200-500ps and r 2 in the ränge of 1600 — 2500ps (Fig. 4 e - h ) . The slow component is as-signed to the decay o f the " f " chromophore(s) in their native State, and the fast one is probably associated — as in the /?-subunit — with energy transfer. This interpretation is supported by the depolarization times. They are much shorter in the trimer bearing 9 chromophores than in the mono-mer bearing only three chromophores (Table II), i . e . they decrease with the number of possible ac-ceptors. A similar dependence on the aggregation has been observed earlier for the static fluorescence depolarisation [6—8]. It increases with an increasing number of chromophores, which can all act as acceptors. It should be pointed out in this context that the fluorescence anisotropy in the kinetic ex-periments never extrapolates to 0.4, e.g. the theo-retical max imum in a randomly oriented System. It has been estimated to %0.2 from the deconvoluted Limits of / ( / ) and D ( t ) extrapolated to / = 0, a value which is similar to results from other laboratories [51]. One explanation is the non-statistical orienta-tion of the chromophores in biliproteins, an as-sumption which is also supported by the non-vanishing anisotropy i n steady-state experiments or

614 P. Hefferle et a l . • Time-Resolved Polarized Fluorescence and Its Subunits

500 1000 1500 2000 tlps)

a)

b) Fig. 6. Isotropic (a) and anisotropic decay (b) of the chromophore fluorescence of PC at the same temperature (36 °C), but in different aggregation states. The decay of whole algae is also shown in (a). A l l spectra are normal-ized with respect to peak intensity.

at long times after excitation in kinetic experiments [ 6 - 8 , 50]. A n alternative explanation is a third component in the fluorescence decay which is faster than the time-resolution of our equipment. G i l b r o et a l . [50] have recently found two short-lived components in phycobilisomes from Synechococcus 6301 with T ^ I O and 9 0 ps, respectively. The latter is in the ränge observed by us for the fast

component of isolated P C . The presence of an addi -tional % 10 ps component (which is not contained in our biexponential fit) would also lead to a de-creased l imit ing value of the anisotropy for t = 0.

It should be pointed out that the aggregation of biliproteins is at present not yet fully understood. Ultracentrifugal measurements of our P C prepara-tions from M . l a m i n o s u s (this work) and S. p l a t e n s i s [16] gave the trimer (aß), as the predominant aggregate, with little to no hexamers (aß)6 detec-able. This is at variance with a large body o f earlier work showing the hexamer as the predominant species [4], but similar findings have occasionally been reported by others, too [see e.g. 44, 45]. There is growing interest i n the function of the generally colorless linker peptides present in phycobil isomes and - in varying amounts - i n preparations of isolated phycobiliproteins as well [46-49] . In particular have two of them been invoked in the aggregation of P C from Synechococcus 6301 [49].

The differences in aggregation could then be due to different amounts of the l inker peptides in different preparations. The samples studied by us contained only traces of these peptides i f judged from S D S - P A G E after staining with Coomassie blue, and the failure to observe aggregates higher than trimers may be l inked to this fact. Ear l ier work involving two of us [15] on P C from the same organism, M . l a m i n o s u s , but isolated by the con-trolled dissociation of phycobilisomes and subse-quent ultracentrifugation had indeed produced both trimers (as well as hexamers) with significantly different fluorescence decay times. In part icular was at ambient temperatures the shorter component of /( /) more pronounced and its lifetime was about half of the values given here, and the difference function D ( t ) could be fit satisfactorily wi th a Single short-lived exponential. F o r the present preparation a second component is needed with a l ifetime in the ränge of the isotropic decay (r « 2600 ps). Since the residual fluorescence polarization in bil iproteins is increased with a decreasing number of coupled chromophores [6-8] and also order-dependent, these differences in decay pattern probably reflect differences in aggregation and/or non-covalent chromophore-protein interactions in the two prep-arations. This point adds yet another hitherto neglected parameter in the sample characterization (besides the measuring technique, data analysis and species related differences) which renders the

P. Hefferle et a l . • Time-Resolved Polarized Fluorescence and Its Subunits 615

comparison of data in a generalized description rather difficult.

In spite of these problems, it is evident that the set of data obtained here for M . l a m i n o s u s shows distinct differences as compared to the data ob-tained earlier [16] under rather s imilar isolation and measuring conditions for P C from S p i r u l i n a p l a t e n s i s . The depolarization times are rather different for P C derived from the two organisms. In both cases, the fast components o f the monomer and trimer have similar isotropic, but different anisotropic decay times. The latter is « 600 ps in monomeric P C from M . l a m i n o s u s , and shorter and more sensitive to increased temperatures in the trimer. The Situation is opposite to that in P C from S. p l a t e n s i s , where this decay constant is rather sensitive in the monomer (300-760 ps), but con-stant ( « 9 0 ps) in the trimer. This difference in temperature dependence of the energy transfer characterized by the depolarization time, may be related to the fact that M . l a m i n o s u s is thermophil ic , although there seems to be no obvious ecological advantage in the observed behavior of P C from M , l a m i n o s u s . It is furthermore not yet clear how the excited State kinetics of small b i l iprote in aggregates relate to those of integral phycobilisomes.

Whole Algae

The fluorescence of whole algae is completely depolarized at our time resolution; therefore, only the isotrpic fluorescence decay has been analyzed. The recorded fluorescence is leakage fluorescence from different members of the energy transfer chain, which are to the most part indirectly excited. A t ambient temperatures, the integral fluorescence is dominated by a short lived component ( T « 120ps). The less intense long-lived component is also com-parably short (T « 600 ps), and disappears at 52 ° C . Since all fluorescence with X ^ 620 nm is recorded, it includes leakage not only from P C but also from its acceptor pigments, e.g. al lophycocyanin and Chlorophyll. A distinction is possible by a spectral analysis of the emission [15, 18], which was beyond the scope of this project. A tentative assignment is, however, possible from comparison with data from the literature. The isotropic fluorescence of whole phycobilisomes from another alga, Synechococcus 6301 has recently been shown [50] to have two short-lived components ( ^ 1 0 and 90 ps). The first com-

ponent is beyond our time resolution, but the second one could correspond to the 120 ps com-ponent in M . l a m i n o s u s (differences are expected from the differences in the phycobil isome composi-tion and Organization in the two organisms). The short-lived component is thus assigned to indirectly excited P C , quenched by transfer to al lophyco-cyanin. The longer l ived component in M . l a m i n o s u s would then arise from allophycocyanin and/or chlorphyll a , whose lifetime is determined by the energy transfer to the reaction centers. If this tentative assignment were correct, the decrease in amplitude of the longer-lived component at higher temperatures could indicate an increased rate of radiationless processes in the acceptor pigments (internal conversion, photochemistry) leading to similar short lifetimes of both P C and al lophyco-cyanin. M . l a m i n o s u s is generally grown at « 50 ° C , so that photosynthesis and in particular the energy transfer between the two pigments is still efficient at this temperature. Otherwise, the long-lived allo-phycocyanin emission should be detectable as in the case of integral phycobilisomes [15].

Concluding Remarks

The data presented demonstrate a strong in -fluence of chromophore-protein and chromophore-chromophore interactions on the photophysics of the chromophores in P C . This interaction is m o d i -fied by changes in the quaternary structure, but also in the protein conformations, as shown for the a-sub-unit. Only two parameters, e.g. the State of aggrega-tion and temperature have been investigated here, in addition to the species dependent differences. The major biochemical problem is the functional relationship of isolated pigments to the i n s i t u antenna System, since the State of the former depends on the isolation procedures. The major problem in deriving parameters for the photo-physical processes is the presence of more than one emitting species (in all but the a-subunit), and the large ränge of decay times. The main advantage of the repetitive streak-camera, v i z . the intrinsic high sensitivity, is in part lost by the necessity to record simultaneously very long and very short decay times. This has in addit ion significant consequences on the accuracy of the derived parameters. Since chromophore-protein interactions are a key for the understanding of chromo-protein structure and

616 P. Hefferle (?/#/. • Time-Resolved Polarized Fluorescence and Its Subunits

function, further studies using addit ional pico-second-time resolved techniques on the same Sys-tem are in preparation.

Acknovvledgements

We thank Prof. F . Doerr and Prof. W . R ü d i g e r for stimulating discussions and continuing support.

Financial support from the Deutsche Forschungsge-meinschaft, Bonn, is gratefully acknowledged. W e thank Dr. W. Hensel, M ü n c h e n for the ultracentri-fuge measurements and Dr . W . Nies in the labo-ratory of Prof. W. Wehrmeyer for a culture of M a s t i g o c l a d u s l a m i n o s u s .

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