Ryuzi Kanai

Metabolic roles of inorganic polyphosphates in chlorella cells

1. 1. A new type of inorganic polyphosphate (poly-Pi) was detected in Chlorella cells and named Poly-Pi “D”. It is extractable with 2 N KOH at 37° together with Poly-Pi “C” which has been reported earlier. Upon neutralization of the extract with perchloric acid, Poly-Pi “C” is co-precipitated with perchloric acid while Poly-Pi “D” remains dissolved. 2. 2. Using uniformly 32P-labeled Chlorella cells which are subjected to various environmental conditions, the metabolic role of Poly-P1 “D”, as well as those referred to earlier as Poly-Pis “A”, “B” and “C” were investigated. 3. 3. On incubating the 32P-labeled algal cells in a phosphate-free medium under photosynthetic conditions, ribonucleic acid increased and Poly-Pi “D” decreased. When the 32P-labeled algal cells were subcultured in the normal “cold” medium under photosynthetic conditions, a large amount of external phosphate but only a small amount of endogenous 32P was incorporated into the ribonucleic acid fraction. In the meantime, the 32P in Poly-Pis “C” and “A” decreased considerably while that in Poly-Pis “B” and “D” decreased slightly or remained constant. It has been reported that deoxyribonucleic acid and phosphoprotein increased with contamitant decrease of Poly-P1s “A”, “B” and “C” in a phosphate-free medium under photosynthetic condition, and that 32P in the fractions of deoxyribonucleic acid and protein continued to increase when the 32P-labeled algal cells were subcultured in the normal “cold” medium1. It was therefore, inferred that, under photosynthetic conditions, Poly-Pis “C” and “A” serve as intermediates in the transfer of phosphate to the phosphorus compounds synthesized such as deoxyribonucleic acid and phosphoprotein, while Poly-Pis “B” and “D” function as reservoirs of phosphate which are utilized only in the absence of exogenous phosphate source. 4. 4. In the dark the 32P in Poly-Pi “A” continued to increase slowly both in the phosphate-free medium and in the normal cold medium, while no significant change was observed in the levels of 32P or total phosphate of the other phosphorus compounds. This suggests that Poly-Pi “A” accepts phosphate—independently of light— from some intracellular phosphate source.

Einbau von 32P in verschiedene Phosphatfraktionen, besonders Polyphosphate, bei einzelligen Grünalgen (Ankistrodesmus braunii) im Licht und im Dunkeln

SummaryUsing the unicellular green alga, Ankistrodesmus braunii, distribution and turnover of phosphorus in various fractions of cell material were investigated with special reference to the formation of inorganic polyphosphates (Poly-P). 1.The whole P-compounds in Ankistrodesmus cells were fractionated by the modified Schmidt and Thannhauser methods which were applied by Kanaiet al. (1965) for the quantitative separation of various inorganic polyphosphates in Chlorella ellipsoidea. The inorganic polyphosphates in Ankistrodesmus cells were also successfully separated from each other by successive extractions with cold 10% TCA (Poly-P “A”), with cold KOH at pH 9 (Poly-P “B”), and with 2n-KOH (Poly-Ps “C” and “D”; the former precipitates on neutralization, leaving the latter in solution).2.Analysis of the uniformly 32P-labeled algal cells showed that the highest in P-content was four kinds of poluphosphates (“A” + “B” + “C” + “D” = ca. 60% of total P) followed by RNA, lipid, acid soluble organic compounds, inorganic orthophosphate, DNA and protein in decreasing order.3.The pool-size of each polyphosphate changed characteristically during the synchronous growth of Ankistrodesmus cells by changing light and darkness periodically (14 hr L: 10 hr D). The amounts of Poly-Ps “B” and “D” increased soon after the beginning of light period, whereas the increase of Poly-Ps “A” and “C” occurred at the latter stage of light period. In the dark period, the algal cells were divided synchronously. Correspondingly DNA and lipid-P increased, whereas Poly-Ps “A” and “B” (and acid soluble organic P-compounds, as well) decreased. Poly-P “C”, on the other hand, did not show any significant change in darkness.4.Using the Ankistrodesmus cells which were pre-cultured in a P-free medium for about 20 hrs, the rapid incorporation of radioactivity into various P-compounds was followed in the short time-course (0–15 min) by introducing 32PO4≡in light and in darkness. Radioactivity in inorganic orthophosphate within the algal cells and labile nucleotide phosphate increased rapidly and saturated in 5–10 min both in light and in darkness. The rapid increase of 32P in Poly-Ps “C” and “D” and RNA was observed 5–10 min after the addition of 32P; these P-compounds were labeled much faste in light than in darkness.The highest light-enhancement of 32P-incorporation was found in non-nucleotide P-components of the acid soluble organic compounds (probably, in sugar phosphate esters). The radioactivity in these compounds incorporated after 2 min in light was found to be 10 times greater than that in darkness.Poly-Ps “A” and “B” were not labeled in light and in darkness during the course of the short time experiment.2.It is suggested from the short term experiments and others that the synthetic pathways of Poly-Ps “C” and “D” are different from those of Poly-Ps “A” and “B”; the former Poly-Ps are synthesized rapidly in light from inorganic phosphate through the intermediary products such as labile nucleotides and/or sugar phosphates.ZusammenfassungEs wurde die Verteilung und der turnover von Phosphat in verschiedenen aus einzelligen Grünalgen (Ankistrodesmusbraunii) gewonnenen Fraktionen unter besonderer Berücksichtigung der Bildung von anorganischem Polyphosphat (Poly-P) untersucht.1.Die Fraktionierung der gesamten P-Verbindungen in Ankistrodesmus-Zellen wurde mit einer von Kanai u. Mitarb. (1965) veränderten Methode nach Schmidt u. Thannhauser vorgenommen. Auch die anorganischen Polyphosphate wurden in aufeinanderfolgenden Extraktionen in 4 Fraktionen (Poly-P “A” bis “D”) aufgetrennt (vgl. Methodik).2.Die 4 Poly-P-Fraktionen besaßen mit etwa 60% den größten Phosphatanteil. Mit abfallendem Mengenanteil folgten RNS, Lipoid-Phosphat, säurelösliche organische P-Verbindungen, anorganisches Orthophosphat, DNS und Proteinphosphat.3.Die Pool-Größen der 4 Poly-P-Fraktionen änderten sich charakteristisch während des synchronen Wachstums (14 Std L: 10 Std Du) der Ankistrodesmus-Zellen. Poly-P “B” und “D” erhöhten sich gleich nach Beginn der Lichtperiode, “A” und “C” erst später. In der Dunkelperiode, während der synchronen Teilung der Zellen, stieg DNS und Lipoid-P an, während Poly-P “A” und “B” abfiel. Poly-P “C” zeigte während der Dunkelperiode keine Änderung.4.Weiterhin wurde in Kurzzeitversuchen (0–15 min) die Einlagerung von 32PO4≡in die verschiedenen Fraktionen aus den P-frei vorkultivierten Algen (20 Std) verfolgt. Unter anderem war die Markierung von Poly-P “C”, “D” und RNS 5–10 min nach 32P-Zugabe im Licht viel stärker als im Dunkeln. Poly-P “A” und “B” wurden während der Kurzzeitversuche weder im Licht noch im Dunkeln markiert.5.Es kann angenommen werden, daß die Synthesewege der Poly-P “A” und “B” von denen der Poly-P “C” und “D” verschieden sind. “C” und “D” werden offenbar im Licht auf den Weg über Intermediärprodukte, wie labile Nucleotide und/oder Zuckerphosphate, gebildet.

Über den Einfluß der Vorbelichtung auf die anschließende Phosphorylierung im Dunkeln bei einzelligen Grünalgen (Ankistrodesmus braunii)

SummaryPreilluminated unicellular green algae (Ankistrodesmus braunii) were treated in the subsequent darkness with 32PO4. The post-illumination dark incorporation was considerably increased compared with the control in continuous dark. The labeling of the separated phosphate-fractions was similar to that of continuous light. The light-induced dark incorporation depended from the light intensity as well as from the time of preillumination. A preillumination of 7 min was required for a maximal enhancement of this preillumination effect. On the other hand the effect diminished in darkness with a half life of approximately 4 min. Finally the enhancement was found to be greater in the absence of CO2 than in the presence of CO2.The experiments demonstrate the light-induced formation of a state in the algae, which permits the enhancement of 32P-incorporation into several phosphate-fractions for a limited time during subsequent darkness.It is discussed, that this may be performed through the formation of a light-reduced substance “R” maintaining for a limited time a cyclic electron transport in darkness, coupled with phosphorylation. On the other hand it seems possible, that preillumination induces a high energy intermediate “XE”—this could also be a pool of protons—formed in the course of energy-transfer from electron transport to ATP-formation. But we must consider also the possibility that light accellerates the ATP-Pi exchange on chloroplast-membranes for a time after preillumination.ZusammenfassungWird einzelligen Algen (Ankistrodesmus braunii) nach Vorbelichtung in anschließender Dunkelheit 32P-markiertes Phosphat geboten, so tritt gegenüber Dauerdunkel eine erhebliche Förderung der 32P-Einlagerung auf. Die nach Vorbelichtung bestimmte Markierung der aufgetrennten Phosphatfraktionen ähnelt sehr derjenigen im Dauerlicht. Die erhöhte Dunkelphosphorylierung nach Vorbelichtung hängt von der CO2-Konzentration, von der Lichtintensität und der Zeit der Vorbelichtung ab. Unter den vorliegenden Bedingungen waren 7 min Vorbelichtung zur maximalen Förderung nötig. Die Halbwertzeit des Abklingens betrug etwa 4 min.Aus den Experimenten geht hervor, daß durch die Belichtung der Algen auch in vivo ein Zustand gebildet wird, der noch nach Belichtung eine Zeitlang im Dumkeln eine Erhöhung der 32P-Einlagerung erlaubt.Es wird diskutiert, ob es sich einerseits um die Bildung einer im Licht reduzierten Substanz „R” handeln könnte, die für eine begrenzte Zeit in Dunkelheit noch einen cyclischen, mit Phosphorylierung gekoppelten Elektronentransport aufrechterhalten kann. Andererseits könnte durch die Vorbelichtung ein energiereiches Zwischenprodukt „XE” — oder auch ein Protonenpool — gebildet werden, das bei dem Energietransfer vom Elektronentransportsystem zur ATP aufgebaut wird. Schließlich muß berücksichtigt werden, daß durch die Vorbelichtung an den Chloroplastenmembranen ein verstärkter ATP-Pi-Austausch zustande kommen könnte, der nach Belichtung nur langsam abklingt.

Fixation of carbon dioxide in Hydrogenomonas facilis as induced by preliminary oxyhydrogen reaction.

Summary1.Using a Knallgas bacterium, Hydrogenomonas facilis, dependence of its CO2-fixing capacity upon the process of Knallgas reaction was investigated under various experimental conditions.2.The CO2-fixation coupled with the Knallgas reaction occurred strongly in bacterial cells which had been grown autotrophically in an oxyhydrogen atmosphere, while it was almost nil when the bacterium was grown heterotrophically using lactate as carbon source. In both autotrophically and heterotrophically grown cells lactate was found to accelerate the CO2-fixation in air (i. e., in the absence of oxyhydrogen), indicating the utilization of the energy of lactate oxidation for the CO2-fixation.3.With the autotrophically grown cells, it was demonstrated that by the pre-incubation of cells in an oxyhydrogen atmosphere in the absence of CO2, the cells acquired a capacity of fixing CO2 on being brought in contact with CO2 in nitrogen or in air with cessation of oxyhydrogen reaction. The maximum level of this capacity in question was found to be attained in about 30 minutes of pre-incubation in the oxyhydrogen atmosphere (at 30°).4.On removal of hydrogen from the gas mixture after a sufficient period of pre-incubation in the oxyhydrogen atmosphere, there occurred a decay in the acquired CO2-fixing capacity, the decay being more rapid in nitrogen atmosphere than in air.