Bean leaves were supplied 14CO2 in light or darkness. After a brief flush, the total and specific radioactivity of respired CO2 were measured when leaves were kept in light or darkness either in air or in CO2-free air. Changes in the specific radioactivity of respired CO2 show that photorespiration and dark respiration are different processes having different and separate substrates. Little, if any, CO2 production by the process of photorespiration could be detected in darkness. The process of dark respiration was about 75% suppressed in light. Measurements of ATP levels in light or darkness and in air or CO2-free air suggest that dark respiration is controlled by photosynthetically produced ATP. This is supported by the fact that isolated intact spinach chloroplasts transfer most of their photosynthetically produced ATP to the medium.

A bstract. A chloroplast fraction isolated from Acetabularia mediterrania carries on photosynthesis at rates essentially equal to those of whole cells. Electron WVe have recently prepared chloroplasts from the giant algal cell, Acetabularia mediterrania, which are ajble to carry otut normal photosynthesis for several hr under laboratory conditions (15). Preliminary gas analysis of photosynthesis indicated that the rate of photosynthesis of this chloroplast preparation was comparable with that of intact cells on a chlorophyll content basis. The products of 14CO2 fixation by chloroplasts were normal and indistinguishable from those of cells. Mitochondrial contamination in the preparation was minimal; the addition of Kreb's cycle substrates and ADP did not stimulate the low rate of dark CO., evolution. Oxygen evolution was proportional to bicarbonate concentration and was not affected by the addition of glycerate-3-P, glycolic acid, ribulose-1,5-diP, fructose-I`6-diP, ferredoxin, NADP or ADP, when adequiate bicarbonate was present. Glycerate-3-P supported oxygen evolution only when the concentration of bicarbonate was limiting. All these observations (15) indicate that a normal photosynthetic process occurs in these chloroplasts, which therefore form an excellent system for the study of photosynthesis and related metabolic processes. This paper describes the characteristics of light and dark CO., exchange in the presence of varying levels of oxygen. By this means the photosynthetic, photorespiratory and respiratory behavior of chloroplast preparations is compared with wvhole Acetabnlaria cells and bean leaves.

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The chloroplast fraction isolated from Acetabularia mediterranie was exposed to 14CO2 as NaH'4C03 in light and darkness, and soluble radioactive compounds were analyzed at frequent intervals. The behavior of Calvin cycle intermediates on the chloroplasts isolated from 2 to 3 g of cells, about 2 cm long, each preparation containing 600 to 750 j,g of total chorophyll (1). The chloroplasts were suspended in 5 ml of the A medium described earlier (6), except that the pH was reduced to 7.2 in order to reduce the equilibrium bicarbonate concentration of the medium (15). As a result, the bicarbonate concentration was low enough that it probably became rate-limiting after about 10 min of photosynthesis (6), but it ensured maximum specific activity of fixed 4CO2 .Experimental. Chloroplast preparations were illuminated by fluorescent lights (2500 ft-c), and 100 ,uc of NaH14CO3 (specific radioactivity about 40 mc/mmole) were added. Sampling began immediately. Samples of 25 Al were withdrawn with a Schwartz Biopette automatic pipette and were quickly applied to the corner of 8-inch X 11-inch chromatography sheets (Whatman 3MM). These were immediately placed in liquid nitrogen until the end of the experiment (30-60 min). A few chromatograms were made with 50 or 100 ,ul. These did not have any spots which could not be seen on the 25 Al chromatograms, showing that all major compounds were being detected. With this technique, samples could be taken at intervals as short as 8 sec, although the sample size was apt to be erratic because of pipetting errors at this speed. Much of this problem was later eliminated by cutting the tips of the polyethylene pipettes at a sharp angle. Samples taken at longer intervals were in close agreement. The time from withdrawal of the sample to the freezing of the chromatograms was about 4 sec. In the dark fixation experiment (Table I), all conditions and procedures were identical except that the experiment was run in darkness.Chromatography and Radioautography. At the conclusion of the experiment chromatograms were removed singly from the liquid nitrogen, and the origins were fixed by 1-to 2-min exposure to the hot vapors of vigorously boiling ethanol. This procedure avoids the problems of extraction and concentration and reduces the nonbiological breakdown of labile intermediates to a minimum (5). Two-dimensional chromatograms were run in 80% phenol at pH 5.4 and butanol-acetic acid-water (12:3:5) and radioautographed. Representative radioautographs are shown in Figure 1. The spots were cut out and counted on both sides in an ultrathin window gas flow Geiger-Mueller counter. The counts from both sides were averaged (4) and corrected for coincidence. All results are presented as counts per minute per sample as determined from a 25 MAl chromatogram, and they are strictly comparable within each experiment. The sum of radioactivities of all the compounds that moved from the origin was called "soluble radioactivity," and the radioactivity which did not move from the origin was termed "insolu...

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with 14CO2 for periods of 6–8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of 14CO2 into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with 14CO2 in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.