Intermediates Of Photosynthesis in Acetabularia mediterranie Chloroplasts

Abstract

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 Samples of 25 Al were withdrawn with a Schwartz Biopette automatic pipette and were quickly applied to the corner of 8-inch X 11-inch chromatography sheets (Whatman 3MM).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...

BIOSYNTHESIS IN ISOLATED ACETABULARIA CHLOROPLASTS

“…to detect any appreciable utilization of added amino acids, and recently synthesized amino acids seem to be used preferentially for protein synthesis Our results do not contradict previous studies (8,11) where it was reported that Acetabula, ia chloroplasts utilize 14C-amino acids. These authors were able to detect the incorporation of 20 ng of total amino acid per mg of protein in the sample (Goffeau, personal communication).…”

“…Serine, glyclne, alanine, and aspartic acid appear as free pools in the chloroplasts and are rapidly labeled from photosynthetic intermediates (8). The simplest explanation for their origin is by transamination or reducdve amination of the respective a-keto acids as typical in photosynthetic tissue.…”

“…Two-dimensional chromatography of photosynthetic products was carried out as previously described (8,40), except that cellulose thin-layer plates were used instead of paper.…”

“…Their in vitro photosynthetic activities are normal for many hours (7,8,39,40). This isolate should provide an excellent material for studying organelle biosynthetic capacity.…”

“…Such redundancy would seem more reasonable the more independent the chloroplasts are in providing the amino acids and other requirements to support their own protein synthesis. Free pools of several amino acids (glycine, serine, alanine, and aspartic and glutamic acids) appear as early products of photosynthesis in most chloroplasts (8,21,23,41), but the numerous enzymes of the pathways for the rest of the amino acids are not usually considered chloroplast constituents.…”