Ulrich Heber

Changes in the intracellular levels of ATP, ADP, AMP and Pi and regulatory function of the adenylate system in leaf cells during photosynthesis

1. 1. Leaves were exposed to periods of light and dark, killed in liquid air and freeze-dried. The dry material was fractionated by a non-aqueous method into a chloroplastic and a residual fraction. These were analyzed for ATP, ADP, AMP, pyruvate and orthophosphate and for adenylate kinase (EC 2.7.4.3). 2. 2. The concentrations of the adenylates in chloroplasts vary between 1.5 · 10−4 and 5 · 10−4M. ATP shows a rapid increase in the light and a rapid decrease in the dark in both chloroplasts and cytoplasm. The changes in ADP are opposite to those in ATP. The changes in AMP are the same as those for ADP in Spinacia and Beta, while in Coleus an increase is observed even in the light. This may be explained by a low activity of adenylate kinase in Coleus and by high activities in Spinacia and Beta. Less than 50% of the adenylate kinase is located in the chloroplasts of the investigated species. 3. 3. The levels of inorganic phosphate in chloroplasts are relatively high (4 · 10t3 to 25 · 10−3M); they decrease rapidly in the light, sometimes by as much as 30% of the initial value, and increase slowly in a subsequent dark period. 4. 4. Pyruvate decreases in the light and increases in the dark. 5. 5. From the results it is concluded that the controlling factor in the inhibition of glycolysis and respiration by light is the increased ratio of ATP to ADP rather than a drop in the orthophosphate. 6. 6. Mass action ratios and the behaviour of AMP point to the existence of a photosynthetic reaction generating AMP.

Compartmentation and reduction of pyridine nucleotides in relation to photosynthesis

1. 1. Leaves were exposed to periods of light and dark, killed in precooled light petroleum (at approx. −120°) or liquid air and freeze-dried. The dry material was fractionated by a non-aqueous method into a chloroplastic and a residual fraction. These were analyzed for oxidized and reduced pyridine nucleotides. 2. 2. In the dark about 25% of the total chloroplastic NADP(H2) and 2–5% of the total NAD(H2) occurred in the reduced state. Upon illumination a rapid reduction of pyridine nucleotides takes place in the chloroplasts. After 15–30 sec in the light NADPH2 and NADH2 reach a maximum and then decrease, first rapidly and then slowly. Levels of chloroplastic NADP show changes opposite to those of NADPH2. Owing to the rather high content of NAD and the low content of NADH2, no light-dependent changes of NAD could be detected in chloroplasts. 3. 3. No light-dependent changes in the concentrations of pyridine nucleotides similar to those observed in the chloroplasts for NADP, NADPH2 and NADH2 have been found in the cytoplasm. Contrary to the situation in the chloroplasts, most of the cytoplasmic NADP(H2) occurred in the reduced state (70–100%). These observations strongly suggest a compartmentation of pyridine nucleotides between chloroplasts and cytoplasm. 4. 4. Aqueously isolated chloroplasts, which are suspended in an isotonic sucrose buffer, reduce exogenous NADP at a very low rate in the light. Brief ultrasonication stimulates NADP reduction by a factor of 10–20. Osmotic shock shows the same effect. Osmotically ruptured chloroplasts cannot be stimulated further by ultrasound provided that no ferrodoxin is added to the chloroplast system. These results are further evidence of a compartmentation of pyridine nucleotides between chloroplasts and cytoplasm of the leaf cell. 5. 5. The maximum observed in the chloroplastic levels of NAD(P)H2 and of ATP after 15–30 sec illumination corresponds closely to a minimum in PGA. The subsequent decrease in NAD(P)H2 and ATP in the light is accompanied by an increase in PGA. Darkening leads to a rapid rise in PGA and a concomitant fall of NAD(P)H2 and ATP levels. Thus the kinetics of PGA, NAD(P)H2 and ATP suggest a rapid reduction of PGA by reduced pyridine nucleotides and ATP in the chloroplasts of illuminated leaf cells.

Sites of synthesis and transport of photosynthetic products within the leaf cell

Abstract 1. 1. After illumination of leaves in the presence of 14CO2 for various times and subsequent freeze drying, chloroplasts were isolated using a nonaqueous procedure. The time-course of the distribution of a number of compounds between chloroplasts and the remainder of the cell was calculated from the 14C-incorporation into the fractions obtained. 2. 2. Labelled ribulose diphosphate, sedoheptulose diphosphate and sedoheptulose monophosphate occurred, at least during the first minutes of photosynthesis, solely in the chloroplasts. At the beginning of photosynthesis phosphoglyceric acid, fructose diphosphate, fructose monophosphate and glucose monophosphate appeared first in the chloroplasts, but were found later also in the non-chloroplastic part of the cell. The major part of glucose diphosphate, uridine diphosphoglucose, sucrose, malic acid and citric acid was always located in the non-chloroplastic part of the cell. 3. 3. From the results it is concluded that the photosynthetic carbon cycle operates exclusively in the chloroplasts. Sugar phosphates, which are not needed in the cyclic regeneration of the CO2-acceptor, are directly translocated into the cytoplasm. The synthesis of uridine diphosphoglucose takes place mainly in the cytoplasm. Glucose diphosphate and possibly also sucrose seem to be formed in the cytoplasm of the leaf cell.

Über das Denaturieren pflanzlicher Eiweisse durch Ausfrieren und seine Verhinderung

ZusammenfassungZucker zeigen eine starke Schutzwirkung bei der Frostdenaturierung pflanzlicher Eiweiße. Die Stabilisierung geht zumindest bei langdauerndem Gefrieren auf eine direkte Wechselwirkung Protein-Zucker zurück, wobei allerdings die Frage offen bleibt, ob an Zucker fest gebundene Wassermoleküle an der Frostschutzwirkung beteiligt sind. Die Schutzwirkung beruht nicht auf der Bildung einer stabilen Zucker-Proteinverbindung. Sie setzt erst bei Beladung der Proteinteilchen mit einer bestimmten Anzahl von Zuckermolekülen ein. Der Mechanismus der Zuckerschutzwirkung wird diskutiert und es wird eine Hypothese zur Erklärung der Wechselwirkung entwickelt. Außer Zuckern besitzen auch andere hydroxylhaltige Stoffe gegen einen Wasserentzug durch Eisbildung schützende Eigenschaften.

Verteilung und Wanderung von Phosphoglycerat zwischen den Chloroplasten und dem Cytoplasma während der Photosynthese

The distribution of phosphoglyceric acid (PGA) * between chloroplasts and cytoplasm of leaf cells during transients from dark to light and vice versa has been investigated. The data indicate that pools of PGA in the chloroplasts and cytoplasm are interchangeable and that PGA may function as a transport metabolite in actively metabolising leaf cells. These views are supported by the following results: 1. In the presence of 14CO2 and light, labelled PGA rapidly appears in the cytoplasm, even though the carboxydismutase reaction, in which 14C enters into PGA, proceeds in the chloroplasts. 2. After less than 1 min. illumination in the presence of 14CO2, the distribution of labelled PGA between chloroplasts and cytoplasm reaches an equilibrium, which is then maintained. The same distribution is to be found by enzymatic analyses of the total pools of PGA in chloroplasts and cytoplasm. When equilibrium is reached, the percentages of both 14C labelled and of total PGA to be found in the chloroplasts of Spinach and Elodea are approximately 75% and 35 —40% respectively. 3. In both the chloroplasts and the cytoplasm, the levels of PGA first decrease after illumination to a fraction of the original dark levels and then show a concomitant slow increase. On darkening a further very rapid increase in PGA occurs in chloroplasts and cytoplasm. 4. In photosynthetically active leaf material the rate of decrease in the level of cytoplasmic PGA, as observed after 12 —15 secs. illumination, is higher than the turnover rate of PGA in respiration. 5. Upon illumination, aqueously isolated chloroplasts, suspended in isotonic sucrose buffer, reduce added PGA to dihydroxyacetone phosphate and other products far faster than they reduce added NADP. Whereas PGA reduction is not increased by ultrasonic disintegration of the chloroplasts, the reduction of NADP is stimulated. This indicates that whereas the movement of NADP is prevented by a permeability barrier, the transferance of PGA across the chloroplast membrane occurs easily. 6. In illuminated Elodea shoots the inhibition of metabolism by cyanide after 15 secs. photosynthesis in the presence of Η14CO3⊖ leads to a rapid decrease in PGA. This applies to both the 14C labelled PGA and the total PGA to a similar extent. The decrease in PGA amounts from 70 — 85% of the original dark levels. Since the chloroplasts of Elodea contain only 35—40% of the total PGA of the cell, a fall in the level of PGA as a result of the cyanide poisoning obviously occurs not only in the chloroplasts, but also in the cytoplasm. Since cyanide effectively inhibits cytochrome oxidase, while PGA reduction in the chloroplasts is relatively resistant, the large decrease in PGA suggests that part of the cytoplasmic PGA is transferred into the chloroplasts and reduced there.

Die Verteilung des Orthophosphates auf Plastiden, Cytoplasma und Vacuole in der Blattzelle und ihre Veränderung im Licht-Dunkel-Wedisel

The distribution of orthophosphate between chloroplasts and the nonchloroplast parts of leaf cells has been investigated. Upon illumination, the percentage of the total orthophosphate located in the chloroplasts decreases rapidly, while darkening results in a slow increase of that percentage to the original dark value. If, in the dark, comparable concentrations of orthophosphate in the chloroplasts and in the cytoplasm are assumed, the concentration of orthophosphate in the vacuole can be calculated from the available data. Values are then obtained, which are considerably lower than the corresponding values in the protoplasm; this indicates that a concentration gradient for orthophosphate is maintained between the cytoplasm and the vacuole. Leaves of spinach and Elodea, which were fed 32P for 2 to 4 hours and then left to stand for two or three days, showed higher specific activities of orthophosphate in the chloroplasts than in the nonchloroplast parts of the cells. Since there is reasn to assume a fairly rapid exchange of orthophosphate between chloroplasts and cytoplasm, the differences in the specific activities which were observed several days after feeding with 32P, point to a very slow exchange of orthophosphate across the tonoplast membrane.

Ribonucleinsäuren in den Chloroplasten der Blattzelle

ZusammenfassungDas Vorkommen von Nucleinsäuren in Chloroplasten wurde untersucht. Um Artefakte durch eine Sekundärverlagerung von Nucleinsäuren während der Präparation auszuschließen, die bei der üblichen Chloroplastenisolierung in Saccharose- oder NaCl-Puffer möglich sind, wurden chloroplasten nach Gefriertrocknung von Blättern in einem nicht-wäßrigen Medium isoliert. Es ergab sich, daß reine Chloroplasten von Bohnen, Roggen, Tabak und Spinat 2–5 mg RNS/100 mg Protein enthalten. Der nicht-plastische Plasmaanteil wies demgegenüber einen durchschnittlichen RNS-Gehalt von 10–15 mg RNS/100 mg Protein auf. Der RNS-Spiegel von Chloroplasten liegt somit wesentlich niedriger als der des restlichen Plasmas. Der Anteil der Chloroplasten an der Gesamt-RNS der Blattzelle ist im Regelfalle höher als 25–35%.Ein beträchtlicher Anteil der Chloroplasten-RNS ist mit dem Lamellarsystem der Chloroplasten assoziiert. Aufgrund des Sedimentationsverhaltens kann weiter auf das Vorhandensein von hochmolekularer, „ribosomaler”, und niedermolekularer, „löslicher” RNS geschlossen werden.Die Chloroplasten enthalten eine hitze-, alkohol- und säurestabile Ribonuclease, die RNS schnell abbaut.SummaryThe occurrence of nucleic acids in chloroplasts has been investigated. Chloroplasts were isolated from freeze-dried leaves in a non-aqueous procedure, since isolation techniques in aqueous media do not exclude the possibility of artifacts due to leaching of nucleic acids from or adsorption onto the chloroplasts. Chloroplasts of rye, broad bean, tobacco and spinach leaves were found to contain, after suitable corrections for cytoplasmic contaminations of the chloroplast fraction were made, between 2 and 5 mg RNA/100 mg protein. The RNA content of the non-chloroplast part of the cell plasm was about 10 to 15 mg/100 mg protein. Since nuclei and cytoplasm are supposed to contain approximately equal amounts of RNA, this figure also reflects the RNA content of the cytoplasm. Thus, chloroplasts possess, on a unit protein basis, considerably less RNA than the surrounding cytoplasm. However, since the major part of the cell proteins is located in the chloroplasts (50 to 60%), still at least 25 to 35% of the total RNA of the leaf cell are contained in the chloroplasts.A considerable part of the chloroplastic RNA, possibly of ribosomal nature, is associated with the lamellar system of the chloroplast. From ultracentrifugal studies it is concluded, that chloroplasts also contain soluble and ribosomal RNA.The investigation of chloroplastic RNA is complicated because of the presence of an alcohol- and heat-resistant ribonuclease in the chloroplasts.

ber eine neue Methode zur quantitativen Stoffbestimmung direktam Papierchromatogramm, insbesondere zur Analyse von Aminosure- und Zuckergemischen

ZusammenfassungEs wird ein neues, leicht herstellbares Gerät beschrieben, mit dem Transmissions- und Remissionsmessungen an papierchromatographischen Flecken zur quantitativen Auswertung durchgeführt werden können. Das Gerät ist auch für andere Zwecke brauchbar, ist aber vor allen Dingen für genaue Serienuntersuchungen an biologischem Material entwickelt worden. Die Fehlergrenze liegt bei etwa ± 3% (bei 100 μg Galaktose und Anilinphthalatentwicklung). Theoretische Grundlagen werden aufgezeigt und diskutiert. An Stelle des Lambert-Beerschen Gesetzes wird eine Beziehung mit weiterem Gültigkeitsbereich für die Auswertung von Transmissionsmessungen an Papieren erörtert.

Photosynthese und Phosphathaushalt

The formation of labelled phosphate esters in cytoplasm and chloroplasts of leaf cells of Elodea densa has been followed in feeding experiments with radioactive bicarbonate and phosphate in the light and in the dark. From the kinetic analysis of the distribution of label between chloroplasts and cytoplasm it is concluded that the incorporation of 14C into sugar phosphates and phosphoglyceric acid proceeds in the light exclusively, or almost exclusively, in the chloroplasts. On the other hand, in the light and in the dark incorporation of 32P into sugar phosphates, ATP and ADP occurs predominantly in the cytoplasm of the leaf cell. This can only be explained in terms of low permeability of the chloroplast membrane to inorganic phosphate. Due to this a specific activity of inorganic phosphate, which is higher in the cytoplasm than in the chloroplasts, is maintained during the feeding experiments. This results in higher incorporation of 32P into organic compounds in the cytoplasm, although phosphate turnover is slower there than in the chloroplasts. In consequence, 32ΡΟ43Θ appears unsuited for many types of in vivo experiments on the phosphate metabolism of photosynthesis. Contrary to other phosphate esters, phosphoglyceric acid becomes labelled with 32P, as with 14C, in the light predominantly in the chloroplasts. This indicates a light inhibition of phosphoglyceric acid formation in the cytoplasm in addition to the inhibition of the citric acid cycle. ATP levels of the cell increase markedly upon illumination and decrease rapidly after darkening. That the response of the ATP/ADP system to light and dark is much faster than that of inorganic phosphate strongly suggests a control of respiration in photosynthesizing cells by the decreased steady state ratio of ADP to ATP rather than by lack of inorganic phosphate.

ber die Verteilung und das Verhalten der Pyridinnukleotide in der Blattzelle im Licht-Dunkel-Wechsel

1) TOLBERT, N.E. : J. Biol. Chem. 235, 475 ( t 9 6 0 ) . 2)WIRWlLLE, J .W. , and J .W. MITCHELL: Sot. Gaz. 111, 491 (1950). 3) PRESTON, W.H. , and C.B. LINK: Plant Physiol. 33, IX (1958). 4) KREWSOX, C.F., J .W. WOOD, W.C. WOLFE, J .W. MITCHELL, and P.C. MARTH: J. Agr. Food Chem. 7, 264 (1959). ~) KEND~, H., H. NINNEMANN, and A. LANO: Naturwissensehaften SO, 599 (1963).-~) MUEAKAMI, Y.: Botan. Mag. (Tokyo) 72, 36 (1959). 7) IKEKAWA, N., T. KAGAWA and Y. SuMI~I: Proc. Japan Acad. 39, 507 (1963).