A mass spectrometer with a membrane inlet system was used to monitor directly gaseous components in a suspension of algae. Using labeled oxygen, we observed that during the frrst 20 seconds of illumination after a dark period, when no net 02 evolution or CO2 uptake was observed, 02 evolution was normal but completely compensated by 02 uptake. Similarly, when CO2 uptake was totally or partially inhibited, 02 evolution proceeded at a high (near maximal) rate. Under all conditions, 02 uptake balanced that fraction of the 02 evolution which could not be accounted for by CO2 uptake.From these observations we concluded that 02 and CO2 are in direct competition for photosynthetically generated reducing power, with 02 being the main electron acceptor during the induction process and under other conditions in which CO2 reduction cannot keep pace with 02 evolution. The After a period of darkness, photosynthesis has a distinct induction phase in which there is a lag (zero rate) of net 02 evolution and its associated CO2 uptake (14,17,19). It is unlikely that the lag is due to the O2-evolving system; in isolated chloroplasts, 02 evolution is virtually maximal from the onset of illumination. In this communication we will describe experiments in which a mass spectrometer with a special inlet and a fast mass stepper system was used to monitor simultaneously 02 evolution, 02 uptake, and CO2 uptake by algae during and after the induction period. A preliminary report of some of these experiments was presented earlier (15). MATERIALS AND METHODSThe mass spectrometer inlet system used in these experiments is similar to the one developed earlier in this laboratory (5). The reaction vessel is a 1. trometer vacuum system through a circular window (-16 mm2) of 1 mil silicone rubber membrane (MEM 213, General Electric Co.) that forms part of the wall of the cavity; it transmits only dissolved gases and some water vapor from the liquid phase.Since this sampling system does not require equilibration between a gas and a liquid phase, the mass spectrometer signals rather accurately reflect the gaseous components in the reaction vessel. The time response of this system is limited by the mass spectrometer, its associated vacuum system, and the transport characteristics of the membrane; the response time of our present system end-to-end is 2 to 3 sec.The quadropole mass spectrometer (Extranuclear Laboratories, Inc.) is programmed using a peak selection stepper system developed in-house. This system monitors up to eight masses, stepped cyclically. In the experiments described below we moni- Algae suspensions were illuminated using a 1000-w projector lamp focussed on the reaction vessel. To minimize heating effects, the light beam passed through a 30-cm water bath containing copper sulfate and an OG-3 (Schott) filter.The usual assay procedure (unless stated otherwise) was as follows. Five ml of cells (cell density 0.3%, v/v) were concentrated to 0.1 ml by centrifugation and transferred to the reaction vessel using a Pasteur pipette (15...

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The time course of light-induced 02 exchange by isolated intact chloroplasts and cells from spinach was determined under various conditions using isotopicaUy labeled 02 and a mass spectrometer. In dark-adapted chloroplasts and ceUs supplemented with saturating amounts of bicarbonate, 02 evolution began immediately upon ilumination. However, this initial rate of 02 evolution was counterbalanced by a simultaneous increase in the rate of 02 uptake, so that little net 02 was evolved or consumed during the first 1 I minute of illumination. After this induction (lag) phase, the rate of 02 evolution increased 3 to 4-fold while the rate of 02 uptake diminished to a very low level. Inhibition of the Calvin cycle, e.g. with DLglyceraldehyde or iodoacetamide, had negligible effects on the initial rate of 02 evolution or 02 uptake; both rates were sutained for several minutes, and about balanced so that no net 02 was produced. Uncouplers had an effect similar to that observed with Calvin cycle inhibitors, except that rates of 02 evolution and photoreduction were stimulated 40 to 50%.These results suggest that higher plant phostosynthetic preparations which retain the ability to reduce CO2 also have a significant capacity to photoreduce 02. With (5,6,12,14,15,17). Here, we determined the magnitude and kinetics of 02 evolution and reduction in isolated intact chloroplasts and whole cells obtained from spinach using a mass spectrometer with a fast mass stepper system. Significant rates of 02 reduction are shown to occur principally when CO2 fixation reactions are suboptimal. MATERIALS AND METHODSIntact chloroplasts were isolated from greenhouse-grown spinach as described previously (26). Preparations contained more than 70%/o intact chloroplasts as determined by the ferricyanide reduction method (13) and fixed CO2 at rates in excess of 100 ,umol/mg Chl * h in saturating light. Chloroplasts were routinely assayed in a 0.33 M sorbitol, 50 mm Hepes-KOH (pH 8.0) medium containing 10 mm NaHCO3, 5 mm Na4P207, 2 mm EDTA, 0.25 mM K2HPO4, and catalase (195 units/ml). Other additions are indicated in the figure legends.Intact cells were prepared from freshly harvested spinach leaves sliced into small (0.5 x 2.0 cm) strips. Approximately 2 g of leaf strips were vacuum-infiltrated in 20 ml of media containing 0.8 M sorbitol, 20 mm Mes (pH 5.8) buffer, 12.5 mm K2SO4, and 0.75% Macerase (obtained from Calbiochem). Leaf strips were digested in 75 ml of the infiltration medium (maintained at 15 C) in an apparatus similar to that described by Servaites and Ogren (23

A mass spectrometer with a membrane inlet was used to monitor lightdriven 02 evolution, 02 uptake, and CO2 uptake in suspensions of algae (Scenedesmus obHlquus MATERIALS AND METHODSThe mass spectrometer inlet system, data acquisition system, and assay procedures used in these experiments were similar to those described earlier (7,8). Algae were suspended in 0.1 M phosphate buffer (pH 6). The pertinent cell densities are given in the figure legends.The values for 02 uptake (Uo) and evolution (Eo) were obtained from the expressions: Cultures of Scenedesmus obliquus (Gaffron strain D3) were grown under the conditions described previously in this laboratory (2). Ten mm NH4' was used as the nitrogen source to avoid interference due to nitrate reduction. Due to an unavoidable mass spectrometer background at m/e = 44 ('2CO2), measurements of low CO2 concentrations necessitated the use of 13C-labeled algae.To this end, Scenedesmus cells were grown in a closed culture vessel containing 13CO2 generated from 13C-labeled barium carbonate (the initial concentration of CO2 in the gas phase was 5%). Chl concentrations were determined by the methods described in (2). RESULTS AND DISCUSSIONSubstrate Affinity of 03 Cycle (Apparent Kin). As described earlier (7), in the presence of the Calvin cycle inhibitor, iodoacetamide, 02 uptake replaces CO2 uptake and balances 02 evolution, so that there is no net change in 02 concentration. This inhibitor offers the opportunity to study the 02 uptake reaction unemcumbered by the varying and complicating effects of CO2. Figure 1, which is a compilation of a series of experiments similar to those of Figure 2 in reference 7, shows the net rates of 02 uptake (and 02 evolution, the two are mirror images) by Scenedesmus D3 in the presence of iodoacetamide. Note that at 02 concentrations above 30% the rate is maximal and invariant within experimental error (in Fig. 1, Vm. = 33.5 cell vol/hr). Half this rate is observed at an atmospheric abundance of 8% (indicated as Ki, Fig. 1). Five repetitions ofthis set ofexperiments (all made at room temperature and at intensities inducing greater than half-maximal rates) gave similar results.The linear plot (v versus v/02) in the inset of Figure I

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