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Unterschiedliche Hemmbarkeit von nicht-cycli-scher und cyclischer Photophosphorylierung war von A V R O N auch in Chloroplasten bei anderen Entkopp-lern beobachtet worden 6.

Methodik

Die Anzucht der Blaualge Anacystis nidulans er-folgte wie bereits beschrieben4. Die Messung der Photosynthese-Aktivität erfolgte in W a r b u r g - Ge-fäßen in 0,1-m. Phosphatpuffer pn 8,0 und Algen mit einem Chlorophyllgehalt von 0,05 mg bei 30 ^C; die Alkyl-benzimidazole wurden in 0,1 ml Methanol zuge-geben. Nach Äquilibrierung mit 8% COa/Luft wurde mit 45 000 Lux (Philips Attralux Lampen) belichtet und die 02-Entwicklung manometrisch verfolgt. Die Photosyntheserate betrug etwa 135 /^Mol Oo/mg Chloro-phyll. 5 B. G E R H A R D T , Ber. dtsch. bot. Ges. 78, 400 [1965] ; B. G E R -

HARDT u. R. SANTO, Z. Naturforschg. 21b , 673 [1966]. 6 M. A V R O N U. N. S H A V I T , in: Photosynthetic Mechanisms of

Green Plants, National Academy of Sciences — National Research Council, Washington 1963; M. A V R O N , in: Bioche-

Die Präparation der zellfreien Partikel von Anacystis durch Gefriertrocknung und anschließende Lysozym-Behandlung erfolgte wie bereits beschrieben 5. Die Re-aktionsansätze zur Bestimmung der photosynthetischen Aktivität in konischen W a r b u r g - Gefäßen von etwa 14 ml Inhalt enthielten in einem Gesamtvolumen von 3 ml: 1 ml Partikel, suspendiert in 5-proz. Saccharose-Lösung/ 0,02-M. Trispuffer PH 7,6 ( = 20 /./Mol/ml) mit einem Chlorophyllgehalt von 0,2 mg, 60 /*Mol Trispuffer pn 8,0, 40 //Mol MgCl2, lO^Mol ADP, 10/^Mol radio-aktives, anorg. Phosphat (mit etwa 105ipm 32P) und die in den Tabellen angegebenen Zusätze. Nach Äqui-librierung der Gefäße mit N2 wurde bei 15 °C mit 35 000 Lux (Philips Attralux Lampen) belichtet. Reinst N2 wurde zur Entfernung von 02-Spuren über einen BTS-Katalysator der BASF geleitet.

ATP wurde nach Fällung des anorg. Phosphates nach S U G I N O und M I Y O S H I 7 über das in organ. Phosphat eingebaute 32P gemessen. Die 02-Entwicklung wurde manometrisch verfolgt.

mical Dimensions of Photosynthesis, Wayne State Univer-sity Press, Detroit 1964.

7 Y. SUGINO U. Y. M I Y O S H I , J. biol. Chemistry 239, 2360 [1964],

Further evidence for the existence of cyclic and non-cyclic photo-phosphorylation in vivo by means of desaspidin and DCMU *

W . U R B A C H a n d W . S I M O N I S

Botanisches Institut der Universität Würzburg

(Z. N a t u r f o r s c h g . 22 b, 5 3 7 — 5 4 0 [19671 ; e i n g e g a n g e n am 18. N o v e m b e r 1966)

The effect of desaspidin and DCMU on photophosphorylation in intact cells under aerobic and anaerobic conditions has been studied. Desaspidin is mainly effective in N2 and inhibits under these conditions the DCMU-insensitive cyclic photophosphorylation in vivo like antimycin A. The inhibi-tion of the phosphorylation in light by DCMU is stronger in N® than in air which suggests a partial existence of oxydative phosphorylation during illumination.

In earlier papers1 6 it has been shown by ex-periments with inhibitors (2,4 dinitrophenol, o-phen-anthroline, D C M U , antimycin A, amytal, HOQNO, salicylaldoxime etc.) that a) besides non-cyclic also a cyclic photophosphorylation exists in vivo, b) oxi-dative phosphorylation in green cells is partially

* Abbreviations used: DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea; HOQNO, 2n-heptyl-4-hydroxylquinoline-N-oxide; TCA- trichloro acetic acid; Po, TCA-soluble organic phosphate compounds.

1 W . SIMONIS U. W . U R B A C H , Z . Naturforschg. 15 b, 816 [I960] .

2 W . URBACH U. W . SIMONIS, in: Vortr. Ges. geb. d. Bot.-N. F. Nr. 1,149, Stuttgart 1962.

3 W . SIMONIS U . W . U R B A C H , in: Studies on Microalgae and Photosynthetic Bacteria, p. 597, Tokyo 1963.

4 W . URBACH and W . SIMONIS, Biochem. biophysic. Res. Com-mun. 17,39 [1964],

inhibited by light and c) a regulation between the different types of photophosphorylation in vivo also seems likely. Some of these results are confirmed recently with different methods by other authors 7 - 1 0 . In investigations with isolated chloroplasts the in-hibitor desaspidin suppressed mainly the cyclic

5 W . SIMONIS, Ber. dtsch. bot. Ges. 77, ( 5 ) , [ 1 9 6 4 ] . 6 W . SIMONIS, in: Currents in photosynthesis, p. 2 1 7 , Donker,

Rotterdam 1966. 7 W . TANNER, L. DÄCHSEL, and O . K A N D L E R , Plant Phvsiol. 4 0 ,

1 1 5 1 [ 1 9 6 5 ] . 8 W . NULTSCH and J E E J I - B A I , Z . Pflanzenphysiol. 5 4 , 8 4

[ 1 9 6 6 ] . FT W . W I E S S N E R , Nature [London] 2 0 5 , 5 6 [ 1 9 6 5 ] ,

IO TANNER, E . Loos, and O . K A N D L E R , in: Currents in photo-synthesis, p. 243, Donker, Rotterdam 1966.

photophosphorylation in argon or N 2 1 1 - 1 3 . More-over, these experiments with desaspidin suggested the existence of two phosphorylation sites in photo-phosphorylation and led to a new hypothesis of electron transport and phosphorylation in photo-synthesis by A R N O N et al .1 3 , 1 4 . Because of this and to verify the existence of cyclic photophosphoryla-tion in vivo it seemed interesting to study the effect of desaspidin in comparison with the effect of DCMU on photophosphorylation with intact cells in a similar way as we have done with antimycin A 4 ' 5 .

Material and Methods

The green alga Ankistrodesmus braunii (Naegeli) used in these experiments was grown in a completely synchronous culture in the medium of P I R S O N and RUPPEL15. A 14-hour light and 10-hour dark schedule was used. Young, recently divided cells were harvested immediately after the start of the light period. They were kept in a phosphate-free medium for 4 hours in the light and for one hour in the dark before the start of the experiments. Cells so treated grew further syn-chronously and became partly deficient in phosphate. For the incorporation of 32P aliquots of the algal sus-pension were used in W a r b u r g vessels with two side arms. One side arm contained 0,5 ml 32P04 (10 — 20 f.iC, carrier-free), the second side arm contained 0,5 ml TCA (65%). The reaction mixture (final volume 5 ml) included algae (70 /ug chlorophyll) and the following in ^moles: tris buffer (PH 3,0) 65; KN0 3 , 24; NaN0 3 , 24; MgS0 4 -7H 2 0 , 3; Ca(N0 3 ) , -4 H,0, 0,3; ZnS0 4 '7 HoO, 0,01; microelements according to P I R S O N

and RUPPEL15; inhibitors as indicated in tables. The experiments were carried out at 25 °C. For the incor-poration in the light (6000 lux) and in the dark 32P was added from the side arm. After 5 min of incor-poration the reaction was stopped by addition of TCA from the second side arm. After 30 min extraction with TCA the labelled Po was obtained from the super-natant liquid by centrifugation and by the fractiona-tion method according to A V R O N 15a. For 14C-fixation (5 min) we used the same method by adding 0,5 ml of a solution of Na214C03 (0,5 fiC//uMo\) from the side arm. The experiments with monochromatic light were carried out in a similar way in small lollipops. In ex-periments in presence of N2 a stream of highly purified nitrogen freed of traces of oxygen by pyrogallol was

1 1 H . BALTSCHEFFSKY and D . Y . D E K I E W I E T , Acta chem. scand. 18,2406 [1964].

1 2 Z . G R O M E T - E L H A N A N and D. I. A R N O N , Plant Physiol. 40, 1060 [1965].

1 3 D . I . A R N O N , H . Y . TSUJIMOTO, and B . C . M C S W A I N , Nature [London] 207, 1267 [1965].

14 D. I. A R N O N , in: Currents in photosynthesis, p. 465, Donker, Rotterdam 1966.

passed through the W a r b u r g vessels for more than 10 min before and during the actual experiment. Desaspidin and DCMU were dissolved in methanol.

Results and Discussion

Tab. 1 shows the effect of different concentrations of desaspidin on the phosphorylation in dark and light under air and N2 as well as on total 14C-fixa-tion. In a N2-atmosphere the phosphorylation in the dark is almost completely inhibited, which demon-

14C-fixa-phosphorylation tion

[%] [%] Treatment [M] dark light light light

(air) (air) (Na) (air)

Control 100 100 100 100 + Desaspidin 6 • 10 - 6 91 95 89 97 + Desaspidin 1 • 10~5 71 83 80 95 + Desaspidin 3 • 10 - 5 6 62 3 65 Control in N 2 7 100,4* — —

Table 1. Effect of desaspidin on 32P incorporation into Po and on total 14C-fixation. * Related to light (air) =100%, average

value of 10 experiments.

strates that the dark phosphorylation we measure is due to oxidative phosphorylation. This phosphory-lation is only slightly influenced by low concentra-tions of desaspidin ( 6 - 1 0 _ 6 M ) , but 3 - 1 0 ~ 5 M desaspidin almost completely uncouples the oxidative phosphorylation16. A similar inhibition of photo-phosphorylation by desaspidin is observed only under N 2 . 3 " 1 0 - 5 M desaspidin inhibits the photo-phosphorylation in N2 completely, while the photo-phosphorylation in air as well as the 14C-fixation is only about 40% suppressed. In comparison the ex-periments with isolated chloroplasts 1 1 - 1 3 show only an inhibition of the cyclic photophosphorylation under argon. The photophosphorylation in air (pseudocyclic type) is not inhibited by desaspidin. Also the non-cyclic photophosphorylation as well as TPN reduction in chloroplasts is not blocked by this inhibitor. The fact that 14C-fixation under our con-ditions in vivo is partially inhibited by 3 ' 1 0 _ 5 M

15 A. PIRSON and H. G. R U P P E L , Arch. Mikrobiol. 4 2 , 299 [1962].

1 5 A M . A V R O N , Biochim. biophysica Acta [Amsterdam] 4 0 , 257 [I960].

1 6 L . RUNEBERG, Socigentas Scientiarum Fennica, Commenta-tiones Biologicae XXVI. 7, Diss., Helsinki 1963.

desaspidin can explain the partial inhibition of the photophosphorylation in air by this concentration of desaspidin. In N2 where by the absence of C0 2

non-cyclic photophosphorylation is suppressed and the cyclic types of photophosphorylation occur pre-dominately, 3 ' 1 0 - 5 M desaspidin inhibits com-pletely. These results in vivo with desaspidin in com-parision to the findings in vitro confirm our earlier conclusion about the existence of a cyclic photo-phosphorylation besides a non-cyclic one in intact cells.

In contrast to desaspidin, DCMU at a concentra-tion of 4 - 1 0 _ 6 M already inhibits 14C-fixation com-pletely (Tab. 2 ) . However under the same condi-tions photophosphorylation is much less suppressed

14C-fixa-phosphorylation tion

[%] [%] Treatment dark light light light

(air) (air) (N2) (air)

Control 100 100 100 100 + Desaspidin 6 • 10~6 M 91 95 89 97 + DCMU 4 • 10-6 M 98 65 48 1,5 + DCMU + Desaspidin

(4 • lO"6) (6 • 10-6 M) 88 55 4 1,4

Table 2. Effects of desaspidin and DCMU on 32P incorpora-tion into Po and on 14C-fixation.

even in a No-atmosphere, where oxidative phos-phorylation is absolutely unable to occur. We al-ready interpreted this result as evidence for the existence of cyclic photophosphorylation in vivo 4. At higher concentrations of DCMU we previously 2

obtained a stronger inhibition of photophosphory-lation, but the oxidative phosphorylation was also partially suppressed under these conditions.

Independent of this possibly unspecific effect of DCMU in higher concentrations in vivo the photo-phosphorylation under N2 is always more strongly inhibited by DCMU than the photophosphorylation in air (Tab. 2 and ref. 2 ' 4 ) . This observation sug-gests that the increased phosphorylation in air com-pared to No in presence of DCMU is due to remain-ing oxidative phosphorylation occurring in light, which is only completely inhibited under strictly anaerobic conditions in No together with DCMU. It is already known that oxidative phosphorylation in

1 7 G . H O C H , 0 . v. O W E N S , and B. K O K , Arch. Biochem. Bio-physics 101, 171 [1962].

1 8 O. K A N D L E R and I. HABERER-LIESENKÖTTER, Z. Naturforschg. 18 b, 718 [1963].

green cells is more or less inhibited in l i g h t 3 - 6 ' 1 7 - 1 9 . Another possible interpretation for the different in-hibition of DCMU in air and No is that besides 0 2 -independent cyclic and DCMU-sensitive non-cyclic photophosphorylation a third type of photophos-phorylation also exists in vivo (pseudocyclic type), which depends on 0 2 20.

The effect of oxygen on photophosphorylation is most noticeable in presence of DCMU and desaspi-din (Tab. 2 ) . In air 6 1 0 _ 6 M desaspidin, which has no effect if added alone, does also not influence the photophosphorylation when desaspidin and DCMU are added together. However, in N2 the same concentration of desaspidin which had only a small effect, when applied alone, suppresses completely the remaining photophosphorylation in presence of DCMU. We conclude, therefore, that this inhibition by desaspidin in N2 in the presence of DCMU is due to inhibition of cyclic photophosphorylation which alone remains in presence of DCMU and N2 .

Experiments in N2 with monochromatic light sup-port these results obtained with DCMU and des-aspidin in N2 . In far-red light of 712 m;w, which preferentially excites photosystem I of photosyn-thesis, the cyclic photophosphorylation occurs pre-dominately. As seeen in Tab. 3 the photophosphory-lation in far-red light under N2 is strongly inhibited by 6 ' 1 0 - 6 M desaspidin which does not really affect

phosphorylation Treatment [M] [%]

712 mft white light Control 100 100 + Desaspidin 6 • 10 - 6 18 89 + DCMU 4 • 10"6 72 48

Table 3. Effects of desaspidin and DCMU on 32P incorpora-tion into Po at 712 m/u and in white light under N2 . Experi-mental conditions as in table 2 ; light intensity of 712 mfi

(6000 e r g - c m - 2 - s e c - 1 ) .

photophosphorylation in white light. In contrast DCMU inhibits photophosphorylation in far-red light only about 30 per cent. In white light, which excites both photosystems I and II, non-cyclic photo-phosphorylation predominates and we obtain a stronger inhibition by DCMU.

1 9 K . A. SANTARIUS and U . H E B E R , Biochim. biophysica Acta [Amsterdam] 1 0 2 , 3 9 [ 1 9 6 5 ] .

2 0 A . R . K R A L L and E . R . B A S S , Nature [London] 196, 7 9 1 [ 1 9 6 2 ] .

The anaerobic cyclic photophosphorylation in vivo, which can be blocked by desaspidin is also sensitive to antimycin A as we have shown earlier 4. Because of the different results with desaspidin ob-tained in presence or absence of oxygen we may not draw further conclusions from our experiments in favour of two phosphorylation sites of photo-phosphorylation in vivo. The behaviour and effect of desaspidin in vivo as well as in vitro in presence of oxygen suggest an inactivation of this inhibitor by photooxydation. Recently several papers 2 1 - 2 4

on the effect of desaspidin on photosynthetic re-actions have been published which indicate a de-

2 1 G . HIND, Nature [London] 2 1 0 , 7 0 3 [ 1 9 6 6 ] . 2 2 Z . GROMET-ELHANAN and M . AVRON, Plant Physiol. 4 1 , 1 2 3 1

[ 1 9 6 6 ] .

struction of desaspidin by photooxydation. Although a series of experiments have been carried out in our laboratory without success to show an irreversible inactivation of desaspidin in algal cells under aero-bic conditions, we mainly refer to our experiments in N 2 , where desaspidin in contrast to the experi-ments in air can be used as a potent inhibitor of DCMU-insensitive cyclic photophosphorylation in vivo.

This investigation was supported by the D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t . The capable tech-nical assistance of Miss S . NEIMANIS and Miss f. OLDEXBÜRGER is gratefully acknowledged. 2 3 G . HIND, Plant Physiol. 4 1 , 1 2 3 7 [ 1 9 6 6 ] . 2 4 R . G . EVERSON, W . COCKBURN, P . W . ELLYARD, a n d M . GIBBS.

Plant Physiol. 4 1 , 1 2 4 0 [ 1 9 6 6 ] .

Ribonucleinsäuren in der Embryogenese von Acheta dornestica L. ** ELISABETH H A N S E N - D E L K E S K A M P , H E L M U T W . SAUER u n d F R A N Z D U S P I V A

Zoologisches Institut der Universität Heidelberg, Physiologischer Lehrstuhl

(Z. Naturforschg. 22 b, 540—545 [1967] ; eingegangen am 29. Dezember 1966)

Herrn Professor Dr. phil. F . SEIDEL zum 70. Geburtstag gewidmet

Aus Grillenkeimen wurde mit der SDS *-Phenolmethode die Gesamt-RNS (ca. 0,1 /<g/Keim) ex-trahiert und mittels Zonengradientenzentrifugation aufgetrennt. Während der synchronen Fur-chungsstadien ist fast keine Markierung der RNS nach 14C02-Inkubation zu beobachten. Ein Einbau in die r-RNS * und in eine m-RNS * (ca. 10 S-Fraktion) setzt gleichzeitig während der heterochro-nen Furchungsteilungen ein und fällt zeitlich mit der Verlängerung der Interphase, dem Auftreten von Nukleolen in den Kernen und der Besiedelung des hinteren Eibereichs mit Furchungskernen zusammen. Eine Nettosynthese von r-RNS erfolgt erst während der Ausbildung der Körpergrund-gestalt. In der beobachteten Entwicklungszeit zeichnete sich regelmäßig die 18 S r-RNS durch eine höhere Einbaurate von 14C02 als die 28 S r-RNS aus.

Eier sind organisierte Systeme mit einer beson-deren Intimstruktur. In ihrem Cytoplasma lassen sich bestimmte Areale abgrenzen, die Träger ent-wicklungsphysiologischer Faktoren sind. Art und Anordnung dieser Faktorenbereiche sind gruppen-spezifisch 1. Die Entwicklungsphysiologie hat am Beispiel der Seeigel-, Amphibien- und Insektenkeime hinsichtlich der Wirkungsart, Lokalisation und In-tensitätsverteilung dieser Faktoren bedeutende Fort-schritte gemacht, konnte aber bezüglich der biochemi-schen Natur derselben noch keine präziseren Anga-ben machen. Ein großes Beobachtungsmaterial legt nahe, daß die Wechselwirkung zwischen den Zell-kernen des jungen Keimes mit gewissen Plasma-

* Folgende Abkürzungen werden verwendet: r-RNS = ribo-somale RNS, m-RNS = „messenger" RNS, s-RNS = lös-liche RNS, SDS = Natriumdodecylsulfat.

bezirken des Eies einer der fundamentalen Mechanis-men der embryonalen Differenzierung ist. Die Vor-aussetzung hierfür liefert die rasche Vermehrung der Zellkerne während der Furchung, die den Ei-raum allmählich immer dichter besiedeln und schließ-lich mit allen Plasmabezirken in engen Kontakt tre-ten. Nicht minder wichtig dürften Bewegungsvor-gänge im Ei sein, die einen systemspezifischen Ver-lauf nehmen und embryonale Zellen bzw. Kerne immer wieder in eine veränderte Umgebung trans-portieren.

In letzter Zeit mehren sich die Hinweise, daß der Modus dieser Kern-Plasma-Interaktionen auf spe-zifischen Hemmungs- bzw. Aktivierungsvorgängen

** Mit Unterstützung der D e u t s c h e n F o r s c h u n g s -g e m e i n s c h a f t und der S t i f t u n g f ü r K r e b s -u n d S c h a r l a c h f o r s c h u n g (STREBEL-Stiftung).