ZEITSCHRIFT FÜR
NATURFORSCHUNG
SECTION C
Α EUROPEAN JOURNAL O F
BIOSCIENCES
Editorial Board
Council
Ε. B ü N N i N G , T ü b i n g e n
A. H A G E R , T ü b i n g e n
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 , Köln
M. E I G E N , Göttingen
W. H A S S E L B A C H , Heidelberg
P. K A R L S O N , M a r b u r g
F. K A U D E W I T Z ,
München
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 E d i t o r i a l B o a r d
G. ISENBERG, Köln
J. S E E L I G , Basel
Β. A . ASKONAS, London
R . J A E N I C K E , Regensburg
H . SIMON, M ü n c h e n
W . BARZ, Münster
V. TER M E U L E N , Würzburg
W. STEGLICH, Bonn
P. B Ö G E R ,
G. F. M E Y E R , Tübingen
H . S T I E V E , Aachen
M . R A J E W S K Y , Essen
J. S U K O , W i e n
N. AMRHEIN,
Bochum
Konstanz
G. BORNKAMM,
D.
Freiburg
BÜCKMANN, U l m
H . SCHIMASSEK,
Heidelberg
A . TREBST, Bochum
K. G . G Ö T Z , Tübingen
D. SCHULTE-FROHLINDE,
G. GOTTSCHALK,
G. S C H U L Z , Freiburg
G . W I C K , Innsbruck
F. F . S E E L I G , T ü b i n g e n
V. ZIMMERMANN, W ü r z b u r g
P. G R U S S ,
Göttingen
Heidelberg
Mühlheim/R.
G. WEISSENBÖCK,
Köln
EDITED I N C O L L A B O R A T I O N
W I T H T H E INSTITUTES OF T H E M A X - P L A N C K - G E S E L L S C H A F T
1986
V o l u m e 41 c
VERLAG D E RZEITSCHRIFT FÜR NATURFORSCHUNG
TÜBINGEN
1986
Zeitschrift für Naturforschung
Volume 41c
Contents
Contents of Number
Origin.il
1/2
Communications
Identification of (/?)-Vicianin in Davallia
ma no ides Blume
tricho-
P. A . L I Z O T T E and J . E . P O U L T O N
Use of I m m u n o t i t r a t i o n to Demonstrate Phytochrome-Mediated Synthesis de novo o f Chalcone
Synthase and Phenylalanine A m m o n i a Lyase i n
Mustard Seedling Cotyledons
R. B R Ö D E N F E L D T and H . M Ö H R
61
5
Differential Regulation of T w o Genes C o n t r o l l i n g
the Biosynthesis o f Isovitexin 7-O-Galactoside i n
Silene Plants
J. M . S T E Y N S and J. v. B R E D E R O D E
Tyrosine Biosynthesis i n Sorghum bicolor: Isolation
and Regulatory Properties of Arogenate Dehydrogenase
J. A . C O N N E L L Y and Ε . E . C O N N
69
9
Biosynthetic Capacity of Stachys Seedlings for Verbascoside and Related Caffeoyl Derivatives
C. A N D A R Y and R . K . I B R A H I M
Tyrosine Biosynthesis in Sorghum bicolor: Charac­
teristics of Prephenate Aminotransferase
D . L . S I E H L , J. A . C O N N E L L Y , and Ε . E . C O N N
79
18
Flavonoids and Terpenoids from the Exudates of
Isolation and Separation o f E p i d e r m a l and Mesophyll Protoplasts from Rye Primary Leaves - Tissue-Specific Characteristics of Secondary Phenolic
Product A c c u m u l a t i o n
M . S C H U L Z and G . W E I S S E N B Ö C K
W . G R A W E and D . S T R A C K
28
Phytoalexin
N . A . A L - D O U R I and P. M . D E W I C K
WOLLENWEBER,
H R A D E T Z K Y , F.
I . SCHOBER,
P.
J . ARRIAGA-GINER,
DOSTAL,
and
D.
G. Y A T -
SKIEVYCH
87
K . R. D A V I S , A . G . D A R V I L L , P. A L B E R S H E I M ,
A. D E L L
Μ . H E B E L , G. B R A N D N E R , Η . K . H O C H K E P P E L ,
D. G. B R A U N
and
94
Glycosphingolipid Analysis of H u m a n M y e l o i d L e u ­
B . K N I E P and P. F . M Ü H L R A D T
100
Properties o f V i n o r i n e Synthase — the Rauwolfia
Enzyme I n v o l v e d i n the F o r m a t i o n of the A j m a line Skeleton
A . P F I T Z N E R , L . P O L Z , and J. S T Ö C K I G T
103
and
39
I n h i b i t i o n of Phenylalanine Ammonia-Lyase in vitro
and in vivo by ( l - A m i n o - 2 - p h e n y l e t h y l ) p h o s phonic A c i d , the Phosphonic Analogue of Phenylalanine
B . L A B E R , H . - H . K I L T Z , and N . A M R H E I N
Transformation-Related Cellular Protein p53: I n ­
creased Level i n U n t r a n s f o r m e d Rat Cells Follow­
ing Treatment w i t h the T u m o r p r o m o t e r , Tetradecanoylphorbol-Acetate
kemias ( I n G e r m a n )
34
Host-Pathogen Interactions. X X X . Characterization
of Elicitors of Phytoalexin A c c u m u l a t i o n i n Soybean Released from Soybean Cell Walls by E n d o polygalacturonic A c i d Lyase
49
Flavin Nucleotide-Dependent 3 - H y d r o x y l a t i o n of
4-Hydroxyphenylpropanoid Carboxylic Acids by
Particulate Preparations from Potato Tubers
J. M . B O N I W E L L and V . S. B U T T
E.
22
Partial Purification and Some Properties of 1-Sinapoylglucose: Choline Sinapoyltransferase ("Sinapine Synthase") from Seeds o f Raphanus sativus
L . and Sinapis alba L .
Biosynthesis of the Furanoacetylene
Wyerone in Vicia faba
Some Baccharis Species
56
H i g h Performance L i q u i d Chromatography Coupled
w i t h Radioactivity D e t e c t i o n : A Powerful T o o l for
D e t e r m i n i n g D r u g M e t a b o l i t e Profiles i n B i o l o g i cal Fluids
K . - O . V O L L M E R , W . K L E M I S C H , and A . v.
HODEN-
BERG
115
Partial Purification and Characterization of S-Aden o s y l - L - M e t h i o n i n e : Norreticuline N - M e t h y l t r a n s ferases from Berberis Cell Suspension Cultures
C H I - K I T W A T , P. S T E F F E N S , and Μ . H . Z E N K
126
IV
Contents
Biosynthesis of 4-Formyl-4-imidazoline-2-on, the
Heterocyclic Base o f N i k k o m y c i n X
R . - M . S C H M I D T , H . P A P E , and M . J U N A C K
135
Metabolism of the Plant G r o w t h Regulator (E)[ H]2-Ethylhex-2-enoic A c i d i n Hordeum vulgare
3
Β . SCHNEIDER, H.-R.
S C H Ü T T E , and A . P R E I S S
141
the W h i t e - R o t Fungus, Phanerochaete
rium
chrysospo-
M . A R J M A N D and H . S A N D E R M A N N JR.
206
Further Studies o n the Biosynthesis o f Granaticin
XIAN-GUO
CHANG,
HE,
J . C.
CHIU-CHIN
VEDERAS, A.
CHANG,
G.
CHING-JER
MCINNES,
J.
W A L T E R , and H . G . F L O S S
Metabolism and Degradation of Nicotinic A c i d i n
Parsley (Petroselinum hortense) Cell Suspension
Cultures and Seedlings
L . S C H W E N E N , D . K O M O S S A , and W . B A R Z
148
Basal Blotches and Blotchlessness of Poppy Flowers:
A Chemogenetic Characterization ( I n G e r m a n )
H . BÖHM
158
Chemical Investigations o f T r o p i c a l M e d i c i n a l
Plants, X X I . L o n g Chain A l k y l Esters o f Ferulic
and p-Coumaric A c i d from Bauhinia manca
H.
ACHENBACH,
Μ.
S T Ö C K E R , and
M.
A. CON-
STENLA
164
M i n i m a l T i m e Requirement for Lasting E l i c i t o r Effects i n C u l t u r e d Parsley Cells
H . S T R A S S E R and U . M A T E R N
paradoxal
222
Elicitor-Stimulated Furanocoumarin Biosynthesis i n
Cultured Parsley-Cells: S - A d e n o s y l - L - M e t h i o n i n e : B e r g a p t o l and S - A d e n o s y l - L - M e t h i o n i n e :
X a n t h o t o x o l O-Methyltransferases
K . D . H A U F F E , K . H A H L B R O C K , and D . S C H E E L
228
The Topology of the Plastoquinone and Herbicide
B i n d i n g Peptides o f Photosystem I I i n the T h y l a k o i d Membrane
A. TREBST
Cyanophora
A.
215
240
Fatty Acids and Fatty A c i d
Synthesis in vitro
H . K L E I N I G , P.
B E Y E R , C.
SCHUBERT, B. LIEDVO-
G E L , and F . L Ü T K E - B R I N K H A U S
169
Degradation of N A D ( H ) by Endogenous
of Yeasts and Clostridia
H.-J.
Enzymes
S C H U E T Z and H . S I M O N
Contents of Number 3
172
Original
Communications
Genetic and Biochemical Studies on the Conversion
of Dihydroflavonols to Flavonols i n Flowers of
Petunia hybrida
G. FORKMANN,
ST. A . BROWN
P.
D E V L A M I N G , R.
SPRIBILLE,
W I E R I N G , and A . W . S C H R A M
179
from Ruta graveolens Cell Suspension
247
H.
Biosynthesis of A c r i d o n e A l k a l o i d s . A Cell-free System
Biosynthesis of D a p h n e t i n i n Daphne mezereum L .
Monogalloylhamamelose from Hamamelis
(In German)
G . S C H I L L I N G and A . K E L L E R
virginiana
253
CulActivation of Streptolysin S in vitro by Oligonu-
tures
A . B A U M E R T , G . S C H N E I D E R , and D . G R Ö G E R
187
cleotides
A . T A K E T O and Y . T A K E T O
258
Phytoalexin Production by Isolated Soybean Protoplasts
H . M I E T H , V . S P E T H , and J. E B E L
193
Composition of Lipopolysaccharides from V a r i o u s
Strains o f Rhodomicrobium
vannielii
O.
H O L S T , J.
Dow
WECKESSER, B.
RIETH,
and
C.
Optimization of Conditions for Accurate Phosphonate and T o t a l Phosphorus Assay on L i p i d Samples, i n Conjunction w i t h T h i n - L a y e r Chromatography
V . M . K A P O U L A S and G . T H . T S A N G A R I S
263
S.
202
Plant Biochemistry of Xenobiotics. Mineralization of
Chloroaniline/Lignin Metabolites from Wheat by
Effect o f the C 0 - C o n c e n t r a t i o n during G r o w t h on
the Oxygen E v o l u t i o n Pattern under Flash L i g h t
in Chlorella
2
Y . SHIRAIWA and G . H . S C H M I D
269
V
Contents
A Large Chloroplast T h i o r e d o x i n / F o u n d i n G r e e n
Algae
P. L A N G L O T Z , W . W A G N E R , and H . F O L L M A N N
Chlorophyll Photobleaching
Complexes
R.
C A R P E N T I E R , R.
in
275
Pigment-Protein
Influence of Temperature on the Transport of Ascorbate across Artificial Membranes as Studied by
the Spin L a b e l Technique
W . L O H M A N N , P. z. T I A N , and D . H O L Z
M . L E B L A N C , and
G.
284
Diurnal Changes of Fructose-6-phosphate,2-kinase
and Fructose-2,6-bis-phosphatase
Activities i n
Spinach Leaves
SÖLING, H .
348
BELLE-
MARE
M . STITT, G. M I E S K E S , H . - D .
Notes
GROSSE,
and H . W . H E L D T
291
The Influence of Spin L a b e l on the Transport o f A s corbate across A r t i f i c i a l Membranes
W . L O H M A N N , P. z. T I A N , and D . H O L Z
351
Effect of Magnetic Field on Ascorbic A c i d Oxidase
Activity, I
V.
S.
GHOLE,
P.
S.
DAMLE,
and
W.
THIEMANN
Molecular Mechanics Investigation on C o n f o r m a tional Flexibility of 14ß Steroids i n Drug-Receptor
Interactions
M . B O H L and M . W U N D E R W A L D
297
E . K A M B E R and L . K O P E I K I N A - T S I B O U K I D O U
301
355
l8
Oxygen I n c o r p o r a t i o n i n Cleavage of 0 - L a b e l e d
13-Hydroperoxylinoleyl A l c o h o l into 12-Hydroxy(3 Z)-dodecenal in Tea Chloroplasts
A.
Alterations i n the Activities of R a b b i t E r y t h r o c y t e
Membrane-Bound Enzymes Induced by Cholesterol Enrichment and D e p l e t i o n Procedures
HATANAKA,
T.
KAJIWARA,
J . S E K I Y A , and
TOYOTA
2+
M . V A R S Ä N Y I , G . B E H L E , and M . S C H Ä F E R
310
Relative Hypertrehalosaemic Activities o f N a t u r a l l y
Occurring Neuropeptides f r o m the A K H / R P C H
Family
G. G Ä D E
315
Broadband, Non-Thermal Millimeter-Wave
ence o n Giant Chromosomes
CHR.
KOSCHNITZKE,
F.
KREMER,
L.
New Pulvinic A c i d Derivatives from
Species (Boletales) ( I n German)
Pulveroboletus
363
Volatiles from the Defensive Secretions of T w o
Rove Beetle Species (Coleoptera: Staphylinidae)
K . D E T T N E R and G . S C H W I N G E R
366
Isolation o f Beef B r a i n Phosphonolipids by T h i n
Layer Chromatography: T h e i r Identification and
Silicic A c i d C o l u m n Chromatographic Separation
M . C. M O S C H I D I S
369
Influ-
SANTO,
P O G L I T S C H , and L . G E N Z E L
A.
Contents of Number 4
321
Original
The A n a l Sac Secretion of Viverrids from the Genus
Genetta
J . J A C O B and H . S C H L I E M A N N
325
Communications
Storage o f Quinolizidine A l k a l o i d s i n E p i d e r m a l Tissues
M. WINK
Some Remarks A b o u t Laser-Induced Mass Spectrometry of Bacteria
J . A L B R E C H T , E . W . S C H M I D , and R . S U S S M U T H
337
Transepithelial Cytophagy by Trichoplax adhaerens
F. E . Schulze (Placozoa) Feeding on Yeast
343
375
Indole A l k a l o i d s from Ochrosia elliptica Plant Cell
Suspension Cultures
K . - H . P A W E L K A and J . S T Ö C K I G T
H . WENDEROTH
H.
359
R. M A R U M O T O , C. K I L P E R T , and W . S T E G L I C H
Stimulation of Phosphatidylinositol Phosphorylation
in the Sarcoplasmic Reticular C a - T r a n s p o r t
ATPase by Vanadate
H.-P.
381
M a j o r Indole A l k a l o i d s Pruduced i n Cell Suspension
Cultures o f Rhazya stricta Decaisne
K . - H . P A W E L K A and J . S T Ö C K I G T
385
Contents
VI
D e t e r m i n a t i o n o f Hyoscyamine and Scopolamine i n
Datura
innoxia
Plants
by
High
Characterization
Performance
of a
Defective M u t a n t
of
L i q u i d Chromatography
J. M . W E R T Z , P. S M I T A M A N A , and S. S A R K A R
K . - H . P L A N K and K . G . W A G N E R
the
Dahlemense Strain o f Tobacco Mosaic V i r u s
477
391
Electronmicroscopical
Evidence for the Presence o f N e u t r a l Glycerylether
Contrast
by
Palladium
Chloride
Derivatives in Pollen Lipids o f Pine Tree Pinus
halepensis
Ν.
K . ANDRIKOPOULOS,
A . SIAFAKA-KAPADAI,
Y A N O V I T S - A R G Y R I A D I S , and C. A .
A Chemical Investigation o f Pueraria mirifica Roots
J. L . I N G H A M , S . T A H A R A , and S R . Z . D Z I E D Z I C
and
Glucosyltransferases
483
DEMOPOULOS
396
Characterization
J. M . F E R R E R , A . T A T O , and J. C. S T O C K E R T
N.
Properties
of
Isolated from
Notes
Cyanidin 3-Gentiobioside f r o m Primary Leaves o f
403
Different
Suspension-
C u l t u r e d Cells o f Daucus carota
E . I N G O L D and H . U . S E I T Z
409
Rye (Secale cereale L . )
E . B U S C H , D . S T R A C K , and G . W E I S S E N B Ö C K
:
485
H - N M R Studies o n the Effect o f Spin L a b e l on
Lipids
W . L O H M A N N and B . K I E F E R
487
T h e M a c r o m o l e c u l a r Structure o f Collagen i n TenConversion Rate o f Ozone w i t h V o l a t i l e Terpenes i n
the Surface Region o f Conifer Needles
(In German)
MOSLER,
HELLE,
H . Russi
421
Impact o f U V - B R a d i a t i o n on Photosynthetic A s ­
similation o f
don Fibres o f Dermatosparactic A n i m a l s
E.
14
C-Bicarbonate and Inorganic
1 5
N-
Compounds by Cyanobacteria
Ε.
W.
FOLKHARD,
KNÖRZER,
Μ.
W.
H . J.
GEERCKEN,
O.
KOCH, C H . M .
LAPIFJRE, H . N E M E T S C H E K - G A N S L E R , B . N U S G E N S ,
and T H . N E M E T S C H E K
Quasisynergism
489
as E v o l u t i o n a r y Advance to I n ­
crease Repellency o f Beetle Defensive Secretions
G . D Ö H L E R , I . B I E R M A N N , and J. Z I N K
426
K . D E T T N E R and R . G R Ü M M E R
493
Pentachlorophenol Inhibits Photosynthetic E l e c t r o n
Flow
and
Quenches C h l o r o p h y l l
Fluorescence
after P r e i l l u m i n a t i o n
C o n t e n t s o f N u m b e r 5/6
C H . N I E H R S and J. A H L E R S
433
The D i a z o Reaction o f B i l i r u b i n s and Phycorubins:
Original
Communications
A Quantitative Study
W.
KUFER,
O.
SCHMID,
G.
SCHMIDT,
and
SCHEER
H.
437
Pyoverdine Type Siderophores from
Pseudomonas
aeruginosa ( I n G e r m a n )
G . B R I S K O T , K . T A R A Z , and H . B U D Z I K I E W I C Z
497
O r i e n t a t i o n Measurements o n O r d e r e d M u l t i b i l a y ers
of
Phospholipids
and
Sphingolipids
from
Synthetic and N a t u r a l O r i g i n by A T R Fourier
A N e w Biflavone and Further Flavonoids from the
Moss Hylocomium
Transform Infrared Spectroscopy
K . B R A N D E N B U R G and U . S E Y D E L
453
Z O G , and H . G E I G E R
4-O-ß-D-Glucosides
The Dynamics o f B o n e M i n e r a l i n Some V e r t e brates.
507
o f Hydroxybenzoic and
Hy-
droxycinnamic Acids — T h e i r Synthesis and D e termination i n B e r r y F r u i t and Vegetable
F. C. M . D R I E S S E N S and R . Μ . H . V E R B E E C K
468
A Serum-Free in vitro C u l t u r e System for Crayfish
Organs
G . G E L L I S S E N , M . T R A U B , and K . - D .
splendens
R. B E C K E R , R . M U E S , H . D . Z I N S M E I S T E R , F . H E R -
B . S C H U S T E R , M . W I N T E R , and K . H E R R M A N N
Chemistry and M o r p h o l o g y o f Epicuticular Waxes
from Leaves o f Five Euphorbia
SPINDLER472
511
H . H E M M E R S , P.-G.
Species
G Ü L Z , and K . H Ä N G S T
521
VII
Contents
^-Acetyl-S-indolylmethylglucosinolate in Seedlings
of Tovaria pendula Ruiz et Pav.
H . S C H R A U D O L F and R. B Ä U E R L E
526
Effects o f Adenosine-3':5'-monophosphate ( c A M P )
on the A c t i v i t y o f Soluble Protein Kinases i n
Maize (Zea mays) Coleoptile Homogenates
B. JANISTYN
Changes i n Fructose-2,6-bisphosphate L e v e l during
the G r o w t h of Suspension C u l t u r e d Cells of
Caiharanthus roseus
H . ASHIHARA
529
Biosynthesis of Pyoluteorin: A M i x e d PolyketideTricarboxylic A c i d Cycle O r i g i n Demonstrated by
[ 1 , 2 - C ] Acetate I n c o r p o r a t i o n
!3
579
Effects of Pyridazinone Herbicides during C h l o r o plast Development i n Detached Barley Leaves.
I I I . Effects of S A N 6706 on Photosynthetic A c t i v i ­
ty and C h l o r o p h y l l - P r o t e i n Complexes
G.
LASKAY, Ε . LEHOCZKI, A.
L.
DOBI,
and
SZALAY
L.
585
2
D . A . C U P P E L S , C. R. H O W E L L , R. D .
STIPANOVIC,
A . S T O E S S L , and J . B . S T O T H E R S
532
The Expression o f the Isovitexin 7-O-Xylosylating
Gene gX in Silene pratensis and S. dioica is Restricted to the Petals
J . S T E Y N S and J . v. B R E D E R O D E
537
Ergosterol as a Biochemical Indicator of Fungal I n fection i n Spruce and Fir Needles from Different
Sources
W . F. O S S W A L D , W . H O L L , and E . F. E L S T N E R
542
Plant Defense Substances X X I X . Isolation, Characterization and Synthesis of Turgorines from Gleditsia triacanthos L . ( I n German)
H . S C H I L D K N E C H T , R.
M U L E Y , G.
M.
KRESBACH,
P. K U N Z E L M A N N , and D . K R A U S S
547
Preparation of Pheromones by Simple Procedures
Η . Κ . M A N G O L D and H . B E C K E R
555
Increased Lipoxygenase A c t i v i t y is I n v o l v e d i n the
Hypersensitive Response o f Wheat Leaf Cells I n ­
fected w i t h A v i r u l e n t Rust Fungi or Treated w i t h
Fungal Elicitor
C.
A.
OCAMPO,
B.
MOERSCHBACHER,
and
GRAMBOW
H.
J.
559
Stimulation of Photorespiration by the Carbonic
Anhydrase I n h i b i t o r Ethoxyzolamide i n Chlorella
vulgaris
Y . S H I R A I W A and G . H . S C H M I D
564
The Photosynthetic Apparatus o f
Ectothiorhodospira halochloris. 2. Accessibility of the M e m ­
brane Polypeptides to Partial Proteolysis and A n ­
tenna Polypeptide Assignments to Specific Chromophores
R . S T E I N E R , A . A N G E R H O F E R , and H . S C H E E R
571
Photocontrol of Chloroplast Lipids in Fern Gametophytes
S T . K R A I S S and A . R. G E M M R I C H
591
Changes i n the Stoichiometry of Photosystem I I
Components as an A d a p t i v e Response to H i g h Light and L o w - L i g h t Conditions during G r o w t h
A.
WILD,
M.
HÖPFNER,
W.
RÜHLE,
and
RICHTER
M.
597
The A c t i v a t i o n of the Cytochrome P-450 Dependent
Monooxygenase System by L i g h t
D . M Ü L L E R - E N O C H and H . G R U L E R
604
Specificity of Rabbit A n t i b o d i e s Elicited by Related
Synthetic Peptides
A.
C H E R S I , R.
A.
HOUGHTEN,
Z I T O , and D . C E N T I S
F.
CHILLEMI,
R.
613
Structure Investigations o f Agonists of the N a t u r a l
Neurotransmitter
Acetylcholine, I V . X-Ray
Structure Analyses of T r i m e t h y l p e n t y l a m m o nium-chloride
and ( 4 - A c e t o x y b u t y l ) t r i m e t h y l ammonium-iodide
A . G I E R E N and M . K O K K I N I D I S
618
Structure Investigations of Agonists of the N a t u r a l
Neurotransmitter A c e t y l c h o l i n e , V . Structure-Activity Correlations for Cholinergic Stimulants D e rived from Crystal Structures of T h e i r Halides
A . G I E R E N and M . K O K K I N I D I S
627
Structure Investigations o f Agonists o f the N a t u r a l
Neurotransmitter
Acetylcholine, V I . X - R a y
Structure Analysis of T r i m e t h y l [ 2 - ( p r o p i o n y l o x y ) ethyl] ammonium-iodide
(O-Propionylcholineiodide)
A . G I E R E N and M . K O K K I N I D I S
641
Is the Calcium Pump I n v o l v e d i n Calcium Release?
M . U N G E H E U E R , A . M I G A L A , and W . H A S S E L B A C H
647
VIII
Contents
Selective A b o l i t i o n o f Sarcoplasmic R e t i c u l u m Vesi­
cles' Calcium Releasing Mechanisms
of Pyridine-2,6-di(monothiocarboxylic A c i d ) from
Pyridine-2,6-dicarboxylic A c i d ( I n G e r m a n )
W. HASSELBACH, Μ. UNGEHEUER, A . MIGALA,
and
U . H I L D E B R A N D , Κ . T A R A Z , and H . B U D Z I K I E W I C Z
K . RITTER
652
691
Photoreceptor
A Furanoheliangolide in Helianthus debilis; I m p l i c a ­
Recovers after L i g h t A d a p t a t i o n i n
tions for a Chemotaxonomy o f the Genus Helian­
The Sensitivity o f the V e n t r a l Nerve
of Limulus
T w o Phases o f D a r k A d a p t a t i o n
thus
I . C L A S S E N - L I N K E and H . S T I E V E
657
O . S P R I N G , V . K L E M T , K . A L B E R T , and A . H A G E R
695
UDP-Glucose: A n t h o c y a n i d i n / F l a v o n o l 3 - 0 - G l u c o Notes
syltransferase in Enzyme Preparation f r o m F l o w e r
A n t i p e p t i d e A n t i b o d i e s : D o They Distinguish H L A -
incana R. B r .
Extracts o f Genetically D e f i n e d Lines o f Matthiola
Alloantigens?
A . C H E R S I , R.
C
M . T E U S C H , G . F O R K M A N N , and W . S E Y F F E R T
A . HOUGHTEN,
D. ZELASCHI,
CENCIARELLI
668
E r r a t u m to A . P F I T Z N E R , L . P O L Z , and
J . STÖCKIGT,
Ζ . Naturforsch. 41c, 1 0 3 - 1 1 4 (1986)
699
and
671
Cyanidin 3-Oxalylglucoside i n Orchids
D.
STRACK, Ε . B U S C H ,
V . WRAY,
L.
GROTJAHN,
and E . K L E I N
707
Characterization o f G l u t a m i n e Synthetase o f Roots,
Etiolated
Cotyledons
and
Green
Leaves
from
Sinapis alba ( L . )
Contents
Original
of Number
7/8
R. M A N D E R S C H E I D and A . W I L D
Communications
712
A r e Polyphosphoinositides I n v o l v e d in Signal Trans­
duction o f E l i c i t o r - I n d u c e d Phytoalexin Synthesis
Esters of Benzyl A l c o h o l and 2-Phenyl-ethanol-l i n
Epicuticular Waxes from Jojoba Leaves
P.-G.
G Ü L Z and F . - J . M A R N E R
673
in C u l t u r e d Plant Cells?
H . STRASSER, C H . H O F F M A N N , H . G R I S E B A C H ,
and
U . MATERN
717
Colletruncoic A c i d M e t h y l Ester, a U n i q u e M e r o t e r penoid from Colletotrichum
Herbal Insecticides I I I . Pyrethrin I in the Essential
truncatum
A . S T O E S S L and J. B . S T O T H E R S
677
Oil
of
Chrysanthemum
balsamita
L.
( I n German)
Complex Flavonoids from Pityrogramma F r o n d E x u dates: Synthesis
o f T w o Flavones
with
C—C-
H . J. B E S T M A N N , Β . C L A S S E N , U . K O B O L D , Ο .
Vo-
S T R O W S K Y , and F. K L I N G A U F
725
L i n k e d D i h y d r o c i n n a m o y l Substituents
M.
IINUMA, K . H A M A D A , M .
M I Z U N O , F.
and E . W O L L E N W E B E R
ASAI,
681
Interference o f Dimethazone w i t h F o r m a t i o n o f Ter­
penoid Compounds
G . S A N D M A N N and P. B Ö G E R
729
Occurrence of 2 - ( 2 - H y d r o x y - 4 , 7 - d i m e t h o x y - 2 H - l , 4 benzoxazin-3-one)-ß-D-glucopyranoside
cum aestivum Leaves and
in
Its Conversion
Tritiinto
6-Methoxy-benzoxazolinone
H . J . GRAMBOW,
Species-Specific Differences i n A c e t y l Coenzyme A
Synthesis o f Chloroplasts
H . - J . T R E E D E , B . R I E N S , and K . - P . H E I S E
J . LUCKGE, A. KLAUSENER,
E. M Ü L L E R
684
N i t r o g e n and Sulfur Starvation o f the
t e r i u m Synechococcus
6-(Hydroxythio)carbonylpyridine-2-carboxylic
and
Pyridine-2-carboxylic
733
and
Acid
Acid-6-monothiocar-
boxylic A c i d as Intermediates i n the Biosynthesis
Cyanobac-
6301. A n U l t r a s t r u c t u r a l ,
M o r p h o m e t r i c a l , and Biochemical C o m p a r i s o n
G. W A N N E R , G. H E N K E L M A N N ,
H . -P. K O S T
A . SCHMIDT,
and
741
Contents
Conjugated E n a m i n o Compounds, a N e w M o l e c u l a r
Probe for the Mechanism of Photosynthetic Elec­
tron Transport
T . A S A M I , N . T A K A H A S H I , and S. Y O S H I D A
IX
Contents of Number 9/10
Original
Communications
751
2-Hydroxy-4-methoxy-5-methyl
Acyclo
Nucleosides
Conformation
and
and
Nucleotides:
Other
Synthesis,
Properties,
and
Be­
haviour i n Some Enzyme Systems, o f 2',3'-Seco
Purine Nucleosides, Nucleotides and 3':5'-Cyclic
an A l
3 +
Pyridine N - O x i d e ,
Complexing Metabolite from
Pseudo­
monas cepacia ( I n G e r m a n )
ST.
WINKLER,
W.
OCKELS,
H.
BUDZIKIEWICZ,
H . K O R T H , and G . P U L V E R E R
807
Phosphates, Analogues of c A M P and c G M P
R . S T O L A R S K I , Z . K A Z I M I E R C Z U K , P. L A S S O T A ,
and
D . SHUGAR
758
Azadirachtin, a Chemical Probe for the Study o f
M o u l t i n g Processes i n Rhodnius
Characterization o f V o l a t i l e Constituents
from
H e t e r o t r o p h i c Cell Suspension Cultures o f Ruta
graveolens
M.
prolixus
E . S. G A R C I A , M . U H L , and H . R E M B O L D
771
Comparative Enzymatic Degradation o f Η 1 Subfractions f r o m Syrian Hamster Tissues
E . H R A B E C , A . P L U C I E N N I C Z A K , and H . P A N U S Z
JORDAN,
C. H . R O L F S , W . B A R Z , R . G . B E R -
G E R , H . K O L L M A N N S B E R G E R , and F. D R A W E R T
809
Identification and Biosynthesis o f Glucosylated and
Sulfated Flavonols in Flaveria bidentis
L . V A R I N , D . B A R R O N , and R. I B R A H I M
813
776
Polymerization o f A c t i n i n the Absence and Pres­
ence o f Cytochalasin B : Problems o f D e t e r m i n i n g
Substrate-Dependent Arylsulfatase A c t i v i t y i n the
Cyanobacterium Plectonema 7 3 1 1 0
S. M Ü L L E R and A . S C H M I D T
"Critical Concentration"
B . F U S S M A N N and P. D A N C K E R
820
781
Volatile Fragrance Compounds
Gloeophyllum
Proliferation Kinetics and Metabolic Features of in
vitro G r o w n E h r l i c h Ascites T u m o r Cells i n the
Presence o f Exogenous Pyruvate
W . K R O L L , S T . P O S T I U S , and F. S C H N E I D E R
787
H.-P.
from
the
Fungus
odoratum (Basidiomycotina)
HANSSEN,
V.
SINNWELL,
and
W.-R.
ABRAHAM
825
A n E l i c i t o r of the Hypersensitive Lignification Response in Wheat Leaves Isolated from the Rust
Triplet-Selective Chemistry: a Possible
Cause o f
Purification and Characterization
Biological M i c r o w a v e Sensitivity
F. K E I L M A N N
Fungus Puccinia graminis f. sp. tritici. I . Partial
795
B. MOERSCHBACHER, Κ . H . K O G E L , U . N O L L ,
and
H. J . REISENER
830
A n E l i c i t o r of the Hypersensitive Lignification ReNotes
sponse in Wheat Leaves .Isolated from the Rust
Fungus Puccinia graminis f. sp. tritici. I I . Induc-
Interaction between Spin Labels and D P P C Vesicles
W . L O H M A N N , B . K I E F E R , and W . S C H M E H L
799
Aspects o f Mycobacterial Response to Beryllate Ions
MACCORDICK
MOERSCHBACHER,
B.
HECK,
Κ.
H.
KOGEL,
839
802
Effect o f Tuftsin on the Phagocytotic A c t i v i t y o f the
U n i c e l l u l a r Tetrahymena. Does Primary Interac­
t i o n Develop I m p r i n t i n g ?
G . C S A B A , V . L A S Z L O , and P. K o v Ä c s
B.
Ο . O B S T , and Η . J . R E I S E N E R
in vitro
H.J.
tion of Enzymes Correlated w i t h the Biosynthesis
of L i g n i n
805
Metabolic Conversions o f Trichothecene M y c o toxins:
Biotransformation of 3-Acetyldeoxynivalenol into Fusarenon-X
N.
C.
P.
BALDWIN,
B.
D E W I C K , and J . G I L B E R T
W.
BYCROFT,
P.
M.
845
Contents
Χ
Biochemical Properties and Crystal Structure o f
Ethylmethylglyoxal Bis(guanylhydrazone) Sulfate
— an Extremely Powerful N o v e l I n h i b i t o r o f A d e nosylmethionine Decarboxylase
H. E L O , I . MUTIKAINEN, L.
(/?)Mandelonitrile and Prunasin, the Sources o f
Hydrogen Cyanide i n A l l Stages o f Paropsis atomaria (Coleoptera: Chysomelidae)
A . N A H R S T E D T and R. H . D A V I S
928
ALHONEN-HONGISTO,
R. L A I N E , J . J Ä N N E , and P. L U M M E
851
Protein Phosphorylation - D e p h o s p h o r y l a t i o n in
the Cytosol o f Pea M e s o p h y l l Cells
R. H R A C K Y and J. S O L L
Latitude Dependent Circadian Rhythms o f Carabid
Beetles
G . L E Y K and W . M A R T I N
Diethyldithiocarbamate, a N e w Photosystem I Electron D o n o r of M e h l e r - T y p e H i l l Reactions
B . L . U P H A M and Κ . K . H A T Z I O S
861
Notes
New Flavonoids from the Exudate
bigelovii (Asteraceae)
F.
Photodestruction o f Endogenous Porphyrins in Rela­
tion to Cellular Inactivation o f
935
856
Propionibacterium
acnes
Τ . B . M E L 0 and G . R E I S ^ T E R
867
The Photosynthetic Apparatus of
Ectothiorhodospira halochloris. 3 . Effect of Proteolytic Diges­
tion on the Photoactivity
R. S T E I N E R , Β . K A L U M E N O S , and H . S C H E E R
873
Phenolic Herbicides: C o r r e l a t i o n between L i p o philicity and Increased I n h i b i t o r y Sensitivity of
Thylakoids from H i g h e r Plant Mutants
J. D U R N E R , A . T H I E L , and P. B Ö G E R
881
J.
ARRIAGA-GINER,
E.
of
Baccharis
WOLLENWEBER,
946
The C-Glycosylflavone Pattern of Passiflora
nata L .
949
Antiproliferative A c t i v i t y of Derivatives o f transBis(salicylaldoximato)copper(II) in vitro. Some
in vivo Properties of the Parent C o m p o u n d
H . E L O and P. L U M M E
951
Effect of T u m o u r Regression on Serum and Tissue
Copper Concentration in Mice Bearing Induced
Fibrosarcoma
P.
K.
Biosynthesis and T u r n o v e r o f Cell W a l l G l y c o p r o teins during the Vegetative Cell Cycle of Chlamydomonas reinhardii
885
Nucleic A c i d - B i n d i n g Activities of the Intermediate
Filament Subunit Proteins Desmin and G l i a l
Fibrillary A c i d i c Protein
C. E . V O R G I A S and P. T R A U B
897
CHAKRAVARTY,
A.
GHOSH,
and
J.
R. B R U S T
910
Specific Binding of Calcium to Soluble C h r o m a t i n
R. B R U S T
Contents of Nos 1 - 1 2
Original
U . - A . H I R T H and R. L A W A C Z E C K
III - XII
Communications
Volatiles from L i q u i d Cultures
cochleatus (Basidiomycotina)
of
Lentinellus
H A N S S E N and W . - R . A B R A H A M
959
Biosynthesis o f Flavor Compounds by M i c r o o r g a n isms. 6. Odorous Constituents of Polyporus durus
(Basidiomycetes)
R.
G . B E R G E R , K . N E U H Ä U S E R , and
F.
DRAWERT
963
917
O n the Direct Observation of Water-Fluxes i n Tissues and Leaves
923
R.
956
Contents of Number 1 1 / 1 2
H.-P.
Soluble and Insoluble Rat L i v e r C h r o m a t i n is Different i n Structure and Protein Composition
incar-
H . G E I G E R and K . R. M A R K H A M
CHOWDHURY
J . VOIGT
and
D. HRADETZKY
Rhizomnium magnifolium and R.
pseudopunctatum,
the First Mosses to Y i e l d Flavone Glucuronides
R.
M U E S , G. L E I D I N G E R , V . L A U C K , H . D .
M E I S T E R , T . K O P O N E N , and K . R. M A R K H A M
ZINS971
XI
Contents
Flavonoid Constituents of Rhamnus lycioides L .
Μ . Ρ A Y Ä , S. M Ä N E Z , and A . V I L L A R
976
Adsorbent Culture o f Tobacco Cell Suspensions w i t h
Different Adsorbents
R. M A I S C H , Β . K N O O P , and R . B E I D E R B E C K
Green Algae (Scenedesmus obliquus)
Thioredoxins o f Regular Size
P. L A N G L O T Z , W . W A G N E R , and H . F O L L M A N N
979
A Simple and R a p i d M e t h o d for Isolation of 124 k D a
Oat Phytochrome
R. G R I M M and W . R Ü D I G E R
988
Investigation o f the Peptide Chain of 124 k D a Phytochrome: Localization of Proteolytic Fragments
and Epitopes for M o n o c l o n a l A n t i b o d i e s
R.
G R I M M , F. L O T T S P E I C H , H . A . W .
and W . R Ü D I G E R
SCHNEIDER,
993
Changes in Levels o f Cellular Constituents i n Sus­
pension Culture o f Catharanthus roseus Associ­
ated w i t h Inorganic Phosphate D e p l e t i o n
T . U K A J I and H . A S H I H A R A
lides from Pigment M u t a n t C-2 A ' of
Scenedesmus
obliquus
1001
Phosphoenolpyruvate
Carboxylase
from
Maize
Leaves. Studies Using ß - M e t h y l a t e d Phosphoenolpyruvate Analogues as Inhibitors and Substrates
I . U L R I C H and W . U L R I C H
H . P. K O C H and M . J. C Z E J K A
J. N . H E R A K and
K . HIRUMA
G.
1011
On the Role of Magnesium i n the Reaction of the
Pyruvate Kinase from Salmonella typhimurium
C. G A R C I A - O L A L L A and
A . GARRIDO-PERTIERRA
1018
Content and Metabolism of Indole-3-acetic A c i d
( I A A ) in Healthy and Rust-Infected Wheat L e a f
Segments
G . W I E S E and H . J. G R A M B O W
1023
The Production of Pyrenocines A and Β by a N o v e l
A Iternana species
Β . T A L and D . J. R O B E S O N
1032
G. B E H R E N S
1062
Alkylene-bis-isothiocyanates: N o v e l Insect G r o w t h
Purification of the Chloroplast Pyruvate D e h y d r o genase Complex from Spinach and Maize Mesophyll
HEISE
1057
Formation and Structure o f Radicals from D-Ribose
and 2-Deoxy-D-ribose by Reactions w i t h SOJ Rad­
icals i n A q u e o u s Solution. A n in-situ Electron
Spin Resonance Study
Regulators
H . -J. T R E E D E and K . - P .
1052
Evidence for the Intercalation o f T h a l i d o m i d e i n t o
D N A : Clue to the M o l e c u l a r Mechanism of Tha­
lidomide Teratogenicity?
1004
D . H . G O N Z A L E Z and C. S. A N D R E O
1045
Flow Cytometric D N A - A n a l y s i s of Plant Protoplasts
Stained w i t h D A P I
Isolation and Characterization of 3 Protochlorophyl-
K . K O T Z A B A S I S and H . S E N G E R
1040
Contain Three
C
9
MATOLCSY, I . UJVÄRY, L. M . RIDDIFORD,
and
1069
A l i p h a t i c Aldehydes: Possible Sex Pheromone
from M a l e T r o p i c a l West African Shield B u g ,
Sphaerocoris
annulus
A . J. E .
G O U G H , D . E.
GAMES, B. W.
STADDON,
D . W . K N I G H T , and T . O . O L A G B E M I R O
1073
I n d i v i d u a l V a r i a t i o n i n the Sex Pheromone Components o f the False C o d l i n g M o t h , Cryptophlebia
leucotreta (Lepidoptera: Tortriciade)
A . B . A T T Y G A L L E , J. S C H W A R Z , Ο .
VOSTROWSKY,
and H . J. B E S T M A N N
1077
Screening and Use of Sex Attractants i n M o n i t o r i n g
of G e o m e t r i d M o t h s in Bulgaria
M.
A.
S U B C H E V , J.
and H . J .
A.
GANEV,
O.
VOSTROWSKY,
BESTMANN
1082
Relative Potencies o f Antagonists of the L u t e i n i z i n g
H o r m o n e Releasing H o r m o n e w i t h L y s and A r g
and Substitutions in Positions 3, 5, 6, 7 and 8
8
Sensitivity of a Phototrophic Bacterium to the H e r b i ­
cide Sulfometuron M e t h y l , an I n h i b i t o r of
Branched Chain A m i n o A c i d Biosynthesis
I . S C H N E I D E R and J . - H .
KLEMME
1037
8
K . F O L K E R S , C. B O W E R S , P . - F . L . T A N G , M . K O B O T A , X . S H A O - B O , W . B E N D E R , and
L. YIN-ZENG
1087
XII
Contents
Effect of E x t e r n a l Calcium Concentration o n the I n ­
tensity Dependence of L i g h t - I n d u c e d M e m b r a n e
Current and Voltage Signals i n T w o D e f i n e d
States o f A d a p t a t i o n i n the Photo-Receptor o f
Limulus
H . S T I E V E , H . G A U B E , and J . K L O M F A S S
1092
Effects o f L o w Frequency Magnetic Fields on Chick
Embryos. Dependence o n Incubation Tempera­
ture and Storage o f the Eggs
J . P. J U U T I L A I N E N
1111
A n I m p r o v e d Procedure for the Quantitative Esti­
mation o f the Rust Fungus i n Infected Plant Tissue
G. W I E S E , D . H U G O - W I S S E M A N N ,
and
H . J . GRAM-
BOW
1127
L o w Molecular Mass Inhibitors from Calf Thymus
Selective for T - L y m p h o c y t e Proliferation
H . P. M A T T H I E S S E N and H . R. M A U R E R
1131
The Effect of V o l a t i l e Anesthetics on Giant Neurons
in the L o b u l a Plate i n the Fly
K . KIRSCHFELD
1137
Notes
Preliminary Studies towards a M o n o g r a p h of the
Lichen Family Roccellaceae Che v. V I I . Second­
ary Products and Relationships of the Genera
Combea de N o t . and Schizopelte Τ . M . Fries
( I n German)
G . F O L L M A N N and M . G E Y E R
1117
Microfilament-Supported M a c r o v i l l i in the H i n d g u t
of the Polychaete Dinophilus gyrociliatus
U . OSTER
C o m m e n t on: Is there an E q u i l i b r i u m
Ascorbic and Dehydroascorbic Acids?
H. W . MUELLER
Rearrangement of Glaucolide
canolide 8-O-Methacrylate
M.
MARTINEZ,
A.
P. J O S E P H - N A T H A N
SANCHEZ,
A
G.
LOPEZ,
1145
and
Report
1119
The G l i o - A x o n a l Interaction and the Problem of Re­
generation of Axons i n the Central Nervous Sys­
tem — Concept and Perspectives
H . W O L B U R G , J . N E U H A U S , and A . M A C K
1147
1121
Evidence
for the
Presence of
Glycerophosphonolipids in the L a n d Snail Eobania
Vermiculata
M . C. M O S C H I D I S
between
into V e r n o j a l -
T h i n Layer Chromatographic and I R Spectral E v i ­
dence for the Presence of Phosphonolipids i n
H u m a n Sperm
M . C. M O S C H I D I S
1139
1124
Subject Index
1157
A u t h o r s Index
1181
Protein Phosphorylation — Dephosphorylation in the Cytosol
of Pea Mesophyll Cells
R u t h H r a c k y and J ü r g e n Soll
Botanisches Institut der Universität München, Menzinger Straße 67,
D-8000 München 19, Bundesrepublik Deutschland
Z. Naturforsch. 41c, 856-860 (1986); received July 9/August 5, 1986
Protein Kinase, Protein Phosphatase, Cytosol, Protoplasts, Pisum sativum
Soluble protein kinase and protein phosphatase activities were localized in the cytosol of pea
mesophyll cells using protoplasts fractionation techniques. The molecular weights of the phosphorylated cytosolic proteins, as determined by Polyacrylamide gel electrophoresis, were 68, 55,
46, 38, 36, 30, 22 and 12 kDa. Histone and, to a much lesser extent, casein but not phosvitin were
accepted as exogenous substrates. In every case serine served as acceptor amino acid for the
phosphate residue. The protein phosphorylation activity had an alkaline p H optimum, and
showed no response to varying M g , C a , P cyclo-AMP or calmodulin concentrations. The
kinase activity was competitively inhibited by A D P and pyrophosphate with apparent K values of
0.5 and 0.17 mM, respectively. High ATP concentrations (1—4 mM) resulted in a strong decrease
of radioactivity in the P labeled proteins. It is proposed that the ratio of protein phosphorylation
to protein dephosphorylation is regulated by the ATP to A D P ratio in the cytosol.
2 +
2+
h
}
32
Introduction
M a n y biochemical processes are ultimately controlled by phosphorylation-dephosphorylation o f
regulatory proteins. Even though a lot of information has accumulated from w o r k in animals and
microorganisms (for a review see ref. [1]) much less
information is available for plant tissues. O f the p r o tein kinases described so far i n plant cells most are
membrane bound and located either in the chloroplast [2—4] or at the plasma membrane [ 5 ] . Soluble
protein kinases have been localized in the chloroplast
stroma [6] and in the supernatant of carrot cell culture homogenates [7], T o further study protein
kinase distribution in plant cells we used protoplast
isolation and fractionation techniques. W e report
here on the presence and partial characterization o f
protein kinase and protein phosphatase activities i n
the cytosol o f pea mesophyll cells.
Materials and Methods
3 2
- 1
( γ - Ρ ) Α Τ Ρ (111 T B q m o l ) was from A m e r s h a m Buchler (Braunschweig, F R G ) . Histone I I I - S and
calmodulin from spinach were purchased
from
S I G M A - C h e m i c a l s . Enzymes for the protoplast iso­
lation were either from Calbiochem (Cellulysin) or
from S E R V A (Macerozym R-10). Pea plants (Pisum
sativum, cv. Rosa K r o n e , Bayerische Futtersaatbau,
M u n i c h , F R G ) were grown on vermiculite in the
green house for 10—12 days. A l l other chemicals
were purchased from commercial sources and were
of reagent quality.
Protoplasts were isolated from 20 g of pea leaves
essentially as described in [8] except that 0.2% polyv i n y l p y r r o l i d o n e ^ was used. A f t e r maceration of
the leaves for 2 h at 30 °C in the light, protoplasts
were collected by centrifugation at 100 x g for 5 min
and further purified on a discontinous silicasol gradient (0-, 20-, 3 0 % , v : v , Percoll in isolation m e d i u m ,
200 x g , 10 m i n ) [ 8 ] . Intact purified protoplasts were
collected from the 0 - 2 0 % Percoll gradient interphase. They were washed free of Percoll and recovered by centrifugation at 100 xg for 5 m i n . Cytosol
was obtained by breaking the protoplasts by repeated passages o f the suspension through a small syringe
outlet (aperture 600 μιτι). Chloroplasts were sepa­
rated by centrifugation at 1 0 0 0 x g for 5 m i n . M e m ­
brane particles were removed from the cytosolic frac­
tion by centrifugation at 100,000xg for 1 h. Protein
phosphorylation was assayed by following the incor­
poration o f P from ( γ - Ρ ) Α Τ Ρ into the t r i ­
chloracetic acid and ethanol insoluble fraction as
in [ 4 ] . Standard incubations contained 50 mM
N - T r i s ( h y d r o x y - m e t h y l ) m e t h y l g l y c i n e - K O H p H 7.6,
4 mM
MgCl ,
300 μΜ
ATP
(containing
0 . 5 - 1 x 1 0 B q ( y - P ) A T P ) and where indicated
100 μg Histone I I I - S , i n a final volume o f 100 μΐ.
Enzyme assays were carried out at 22 °C for 15 m i n .
32
3 2
2
Reprint requests to Dr. J. Soil.
Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen
0341-0382/86/0900-0856 $ 01.30/0
5
32
R. Hracky and J. Soll · Protein Phosphorylation
32
P incorporation was either analyzed by S D S - P A G E
on 7.5 — 15% Polyacrylamide gels [4, 9] or by l i q u i d
scintillation counting. A u t o r a d i o g r a p h y was done at
—80 °C using an intensifying screen ( M R - 6 0 0 , AgfaGaevert). Protein was estimated by the method in
[10] using bovine albumin as reference.
Results
I n i t i a l experiments demonstrated high protein
kinase activity i n the cytosolic fraction of pea
mesophyll cells which was different from the activity
in the soluble chloroplast stroma ( F i g . 1). Proteins
phosphorylated in the cytosol had a molecular weight
of 68, 55, 46, 38, 36, 30, 22 and 12 k D a . The phos­
phorylated band at 61 k D a is due to stromal con­
taminations (compare Fig. 1, lane B , D ) . C o n t a m i ­
nation of the cytosolic fraction by soluble proteins
from m i t o c h o n d r i a , chloroplasts and peroxisomes
was assayed using soluble marker enzymes for each
compartment, e.g. isocitrate dehydrogenase ( E . C .
1.1.1.41) reversible glyceraldehyd 3-phosphate de­
hydrogenase ( E . C . 1.2.1.13) and hydroxypyruvate
A
Β
C D
Ε
857
reductase ( E . C . 1.1.26), respectively [ 1 1 , 12, 13].
Usually crosscontamination were about 10% by
mitochondria and chloroplasts, and less than 5% by
peroxisomes. Histone I I I - S (1 mg/ml) can serve as
acceptor protein for the cytosolic kinase activity
(Fig. 1, lane A ) and to a much lesser extent also for
the stromal protein kinase ( F i g . 1, lane C ) . The cyto­
solic protein kinase activity accepts casein (1 mg/ml)
only very poorly and phosvitin (1 mg/ml) not at all.
The presence of histone i n the phosphorylation assay
results i n a 10-fold increase in P incorporation over
the control value (Fig. 1, lane A , B ) (casein 2 - 3 - f o l d
increase). W h e n the m i x t u r e o f phosphorylated
cytosolic proteins is hydrolyzed and the phosphoaminoacids are analyzed by high voltage electro­
phoresis on silica gel plates [14] only phosphoserine
is labeled, no phosphotyrosine and phosphothreonine is found (data not shown). W h e n histone or
casein were included in the incubation mixture
phosphoserine was also the only labeled amino acid.
32
Further attempts were made to characterize the
enzyme properties of the cytosolic protein kinase
activity. Cytosolic protein phosphorylation exhibited
an alkaline p H o p t i m u m i n the broad range between
7.6 and 12 (Fig. 2). I t showed a sharp loss of activity
F
1#
Fig. 1. SDS-PAGE of cytosolic and soluble chloroplast pro­
teins from pea and their phosphorylation products. Lane
A—D autoradiograms of phosphorylated proteins. A ,
150 μg cytosolic protein -I- histone; B , the same as in A histone; C, 136 μg soluble chloroplast protein + histone;
D , the same as in C-histone; E, F, Coomassie brilliant blue
stain of cytosolic proteins (205 μg) and chloroplast soluble
proteins (408 μg). Molecular weight markers (in kDa);
bovine serum albumin (66); egg albumin (45); glycerinephosphate dehydrogenase (36); carbonic anhydrase (29);
trypsinogen (24); soybean trypsin inhibitor (20.1); cyto­
chrome c (12.4).
1
1
1
1
7
8
9
1
ι
•
K)
11
12
pH
Fig. 2. Cytosolic protein phosphorylation activity as
a function of p H .
858
R. Hracky and J. Soll · Protein Phosphorylation
at neutral and acidic p H ( F i g . 2 and data not shown).
Protein phosphorylation was essentially independent
of M g
( 0 - 1 0 m M M g C l ) and inhibited by M n
( 5 0 % i n h i b i t i o n at 1.1 m M M n C l ) . These experi­
ments were performed i n the presence of 0.5 m M
E D T A . Others [7] have described the stimulation of
soluble protein kinases i n plant tissues by C a
and
calmodulin. I n our hands the inclusion o f C a
(3 — 10 μΜ) and calmodulin (7 μΜ) d i d not result i n
any significant increase i n P incorporation (stimula­
t i o n between 10—15%). Furthermore c y c l o - A M P
(1 — 100 μΜ) had no effect o n the P incorporation.
Various nucleoside diphosphates and monophos­
phates were tested to determine their influence on
the P i n c o r p o r a t i o n . A M P , C M P , G M P and U M P
were w i t h o u t effect (0.3 m M A T P , 0.6 m M nucleoside
monophosphates). I n contrast, A D P , G D P and C D P
competively inhibited the phosphorylation to a simi­
lar extent (data not shown). The apparent K value
for A D P is 500 μΜ (Fig. 3 A ) . PPj was a more potent
i n h i b i t o r w i t h an apparent K\ value of 170 μΜ
(Fig. 3 B ) . Phosphate ( 0 . 1 - 1 0 m M ) d i d not affect the
P incorporation.
2 +
2 +
2
2
2 +
2 +
32
32
32
[ADPj, mM
x
32
Protein phosphorylation as a function of A T P con­
centrations increased up to about 0.6 m M A T P .
H i g h e r A T P concentrations o f the same specific
activity produced a strong decrease of P incorpora­
t i o n (Fig. 4 ) . I t was possible to show, that high A T P
concentrations (1—3 m M ) d i d not inhibit the protein
kinase. This was demonstrated using an enzyme prep­
aration which was repeatedly frozen (—20 °C) and
thawn (4 times). The treatment resulted i n an almost
complete loss o f p r o t e i n phosphatase activity while
the p r o t e i n kinase showed a normal saturation be32
[PPi],mM
Fig. 3. Competitive inhibition of cytosolic protein phos­
phorylation by A D P ( A ) and sodium pyrophosphate (B).
The K[ was determined by a Dixon plot using the following
A T P concentrations; A (x, 0.5 mM, Δ , 0.3 m M ;
O, 0.05 m M ) ; Β ( Δ , 0.5 IHM); Ο, 0.3 m M ; χ, 0.05 m M ) .
3 2
1.5
2
[ATP] , mM
3
3.5
Fig. 4. Ρ incorporation into cytosolic pro­
teins in the presence of histone as a function
of ATP concentration. Cytosol was stored at
-196 °C prior to the assay for 2 days (O);
cytosol was stored as above but frozen at
- 2 0 °C and thawn for 4 times prior to the
enzyme test ( Δ ) .
R. Hracky and J. Soll · Protein Phosphorylation
859
haviour w i t h respect to A T P ( S
0.3 ΓΠΜ; V
38 pmol o f Ρ · b o u n d - m g " - m i n " ) ( F i g . 4). The
results in F i g . 4 were further investigated by a differ­
ent approach. Cytosolic proteins were phosphory­
lated for 15 m i n using 0.3 m M A T P , then 3 m M A T P
was added and the dephosphorylation was followed
over a period o f 30 m i n . Analysis of the phosphory­
lation pattern by S D S - P A G E w i t h subsequent auto­
radiography showed an almost complete dephos­
phorylation after 30 m i n i n the presence of 3 m M
A T P (Fig. 5). The phosphatase activity was inhibited
by the simultaneous inclusion o f A D P (3 m M ) i n the
assay (Fig. 5 ) . T o exclude the exchange o f covalently
0 5
3 2
A
A
1 5
A
3 0
1
Β
m a x
Discussion
1
Bis B30
The results presented i n this paper demonstrate
for the first time a protein phosphorylation-dephosphorylation system in the cytosol o f leaf mesophyll
cells. The data do not allow to decide whether only
one protein kinase and one protein phosphatase is
present i n the cytosol or whether multiple kinases
and phosphatases are localized i n this cell compart­
ment. The cytosol used in our study was about 8 0 %
pure and only slightly contaminated by soluble pro­
teins from organelles, as demonstrated i n Fig. 1. Sol­
uble protein kinases have previously been localized i )
in the stroma o f chloroplasts from spinach [ 6 ] , i i ) i n
the supernatant o f homogenates f r o m non green car­
rot tissue cultures [7, 15], i i i ) in the soluble protein
fraction from zucchini hypocotyl hooks and stems
[16], The cytosolic protein kinase activity described
i n this paper has a characteristic phosphorylation
pattern, which is distinct from that o f other soluble
protein kinases (compare Fig. 1, references [6, 7,
16]). I t also differs in its biochemical behaviour, e.g.
it is not C a and calmodulin dependent i n our hands
[7, 16]. Interestingly, the protein kinase shows a
broad alkaline p H o p t i m u m w i t h a strong decrease
in activity in the neutral and acidic range; this
phenomenon is more common for stromal proteins
and indicates a possible regulatory site of the en­
zyme. Furthermore the degree of protein phosphory­
lation is dependent on the A T P / A D P ratio. W e have
therefore determined the characteristics of P incor­
poration at 300 μΜ A T P , an A T P level at which the
protein phosphatase is presumably not active. H i g h
A T P concentrations do not inhibit the protein phos­
phorylation they activate a very potent protein phos­
phatase ( F i g . 5 ) . I n t u r n protein dephosphorylation
is i n h i b i t e d by A D P more strongly than protein phos­
phorylation which shows only very sluggish i n h i b i ­
tion or no inhibition at all at equimolar A T P / A D P
ratios.
2 +
32
Fig. 5. Stimulation of protein dephosphorylation by ATP
and its inhibition by A D P . 190 μg of cytosolic protein were
incubated in the standard incubation mixture (0.3 mM
ATP) for 15 min ( A , B ) . Then 3 mM ATP of the same
specific radioactivity was added and the dephosphorylation
was checked after an additional 15 and 30 min ( A / A ) .
B /B o as above, but 3 mM ATP + 3 mM A D P were added
after the initial phosphorylation period. The phosphory­
lated protein mixture was analyzed by SDS-PAGE and
subsequent autoradiography as described in Material and
Methods.
1 5
15
3 0
3
b o u n d phosphate residues during the course of the
experiment the A T P added to activate the phospha­
tase was o f the same specific radioactivity as for the
preincubation to phosphorylate cytosolic proteins.
I t is not yet clear which regulatory function this
i m p o r t a n t novel system fulfills in the cytosol. Clearly
more w o r k is needed to purify its components and to
elucidate their functions.
Acknowledgements
This w o r k was supported by the Deutsche
schungsgemeinschaft, B o n n .
For­
860
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R. Hracky and J. Soll - Protein Phosphorylation
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