A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with 4C-starch i... more A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with 4C-starch is described. These intact chloroplasts were incubated serobically in the dark for 30 minutes. Radioactivity in starch declined and glyceric acid 3-phosphate and maltose were the major radioactive products. It is proposed that starch is degraded within the chloroplast to glyceric acid 3-phosphate and to maltose.
On etudie le catabolisme de l'amidon en conditions anaerobies et aerobies chez 5 Algues verte... more On etudie le catabolisme de l'amidon en conditions anaerobies et aerobies chez 5 Algues vertes et une Cyanobacterie, cultivees a la lumiere sur milieu mineral ou sur milieu enrichi en glucose ou en acetate
Dark H2 metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas... more Dark H2 metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas, and mosses. A time variable and temperature-sensitive anaerobic incubation was required prior to 112 evolution. H2 evolution was sensitive to disalicylidenepropanediamine. An immediate H2 uptake was observed in these algae. Immediate dark H2 uptake but no evolution was observed in the mosses. A cell-free hydrogenase preparation was obtained from anaerobically adapted Chlamydomonas reinhardii by means of sonic oscillation. The hydrogenase was not sedimented at 100,000g. It catalyzed the reduction of methylene blue, p-benzoquinone, NAD, NADP, but not spinach ferredoxin. H2 evolution was noted with dithionite and with reduced methyl viologen as donors but not with reduced spinach ferredoxin. Similarly, hydrogenase activities were not affected by disalicylidenepropanediamine. The pH optima for H2 evolution and for H2 uptake were 7.2 and 7.5 to 9.5, respectively. Extracts prepared from the anaerobically adapted red alga, Chondrus crispus, and the moss, Leptobryum pyriforme, consumed but did not evolve 112. Uptake was slightly stimulated by methylene blue. It is proposed that red algae and mosses appear to metabolize H2 by a different pathway than Chlamydomonas.
The Emerson enhancement effect was evaluated in the intact spinach (Spinacia oleracea var. Long S... more The Emerson enhancement effect was evaluated in the intact spinach (Spinacia oleracea var. Long Standing Bloomsdale) chloroplast by monitoring the uptake of "CO2 during illumina- tion by 640 nm and 720 nm lights. Low levels (about 10 /AM)
Biochemical and Biophysical Research Communications, 1976
The effects of orthophosphate (Pi) on starch synthesis and degradation in the isolated intact spi... more The effects of orthophosphate (Pi) on starch synthesis and degradation in the isolated intact spinach chloroplast are investigated. During CO 2 fixation in the light increasing levels of Pi (O-io0 mM), which have little effect on the rate of fixation, prolong the lag of starch synthesis and reduce its maximal rate from 14o5 to 6o8 ~g-atom C incorporated into starch per milligram chlorophyll per hour. The label in dihydroxyacetone phosphate and other sugar phosphates, especially sedoheptulose-l,7-bisphosphate, increases up to lO-fold under these conditions. Starch degradation in the dark is monitored in the chloroplast by following the breakdown of the 14C labeled starch. Pi concentrations in the order of 2.5-5o0 mM are optimal for starch mobilization at a rate of about I gg-atom C per milligram chlorophyll per hour. INTRODUCTION: Over I00 years ago, Sachs (I) established that carbon assimilated during photosynthesis can be stored in the chloroplast as starch° Starch is synthesized from well-characterized intermediates of the photosynthetic carbon reduction cycle. Recently, Levi and Gibbs (2) reported that starch degradation occurs in the isolated intact spinach chloroplast by a pathway similar to classical glycolysis. Metabolic pathways for the synthesis and the utilization of starch involve phosphorylated compounds and, therefore, require Pi. We report upon the regulatory effect of Pi on carbon flow into starch during photosynthesis and on starch degradation in the dark° i) This research was supported by NSF (BMS-71-O0978) and by ERDA (E(II-I)-3231). 2
A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with 4C-starch i... more A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with 4C-starch is described. These intact chloroplasts were incubated serobically in the dark for 30 minutes. Radioactivity in starch declined and glyceric acid 3-phosphate and maltose were the major radioactive products. It is proposed that starch is degraded within the chloroplast to glyceric acid 3-phosphate and to maltose.
Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour... more Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia olracca) chloroplast which had been preloaded with "4C-starch photosynthetically from 14CO2. Starch breakdown was dependent upon inorganic phosphate and the "4C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of 14C from triose-phosphate to glycerate 3-phosphate. lodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was conduded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic deavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate. In 1862, Sachs (14) demonstrated that carbon assimilated during photosynthesis by the chloroplast accumulated in the form of starch. The synthesis of this polyglucan from well characterized intermediates of the photosynthetic carbon reduction cycle has been amply documented. In recent publications from this laboratory, preliminary evidence was presented indicating that in the intact spinach chloroplast starch synthesis and degra
Dark H2 metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas... more Dark H2 metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas, and mosses. A time variable and temperature-sensitive anaerobic incubation was required prior to 112 evolution. H2 evolution was sensitive to disalicylidenepropanediamine. An immediate H2 uptake was observed in these algae. Immediate dark H2 uptake but no evolution was observed in the mosses. A cell-free hydrogenase preparation was obtained from anaerobically adapted Chlamydomonas reinhardii by means of sonic oscillation. The hydrogenase was not sedimented at 100,000g. It catalyzed the reduction of methylene blue, p-benzoquinone, NAD, NADP, but not spinach ferredoxin. H2 evolution was noted with dithionite and with reduced methyl viologen as donors but not with reduced spinach ferredoxin. Similarly, hydrogenase activities were not affected by disalicylidenepropanediamine. The pH optima for H2 evolution and for H2 uptake were 7.2 and 7.5 to 9.5, respectively. Extracts prepared from the anaerobically adapted red alga, Chondrus crispus, and the moss, Leptobryum pyriforme, consumed but did not evolve 112. Uptake was slightly stimulated by methylene blue. It is proposed that red algae and mosses appear to metabolize H2 by a different pathway than Chlamydomonas.
The marine chlorophyte Dunaliella tertiolecta was grown in continuous cultures under NH4+-N, N02-... more The marine chlorophyte Dunaliella tertiolecta was grown in continuous cultures under NH4+-N, N02-N, N03-N, and urea-N limitations. The effect of the nitrogen cell quota (Qn) on the steady-state growth rate (,) was the same regardless of the N source. The relationship between ,u and Q. was well described by the Droop equation, but only up to the true maximum growth rate ,i (= cell washout rate). The ratio between the minimum cell quota (kQ) and the maximum cell quota (Qm) was 0.19. Hence, there is no substitute for determining ,u experimentally. That there was no difference in growth response to different N sources suggests that no intemal pooling of inorganic nitrogen occurred. Both the carbon
A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with 4C-starch i... more A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with 4C-starch is described. These intact chloroplasts were incubated serobically in the dark for 30 minutes. Radioactivity in starch declined and glyceric acid 3-phosphate and maltose were the major radioactive products. It is proposed that starch is degraded within the chloroplast to glyceric acid 3-phosphate and to maltose.
On etudie le catabolisme de l'amidon en conditions anaerobies et aerobies chez 5 Algues verte... more On etudie le catabolisme de l'amidon en conditions anaerobies et aerobies chez 5 Algues vertes et une Cyanobacterie, cultivees a la lumiere sur milieu mineral ou sur milieu enrichi en glucose ou en acetate
Dark H2 metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas... more Dark H2 metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas, and mosses. A time variable and temperature-sensitive anaerobic incubation was required prior to 112 evolution. H2 evolution was sensitive to disalicylidenepropanediamine. An immediate H2 uptake was observed in these algae. Immediate dark H2 uptake but no evolution was observed in the mosses. A cell-free hydrogenase preparation was obtained from anaerobically adapted Chlamydomonas reinhardii by means of sonic oscillation. The hydrogenase was not sedimented at 100,000g. It catalyzed the reduction of methylene blue, p-benzoquinone, NAD, NADP, but not spinach ferredoxin. H2 evolution was noted with dithionite and with reduced methyl viologen as donors but not with reduced spinach ferredoxin. Similarly, hydrogenase activities were not affected by disalicylidenepropanediamine. The pH optima for H2 evolution and for H2 uptake were 7.2 and 7.5 to 9.5, respectively. Extracts prepared from the anaerobically adapted red alga, Chondrus crispus, and the moss, Leptobryum pyriforme, consumed but did not evolve 112. Uptake was slightly stimulated by methylene blue. It is proposed that red algae and mosses appear to metabolize H2 by a different pathway than Chlamydomonas.
The Emerson enhancement effect was evaluated in the intact spinach (Spinacia oleracea var. Long S... more The Emerson enhancement effect was evaluated in the intact spinach (Spinacia oleracea var. Long Standing Bloomsdale) chloroplast by monitoring the uptake of "CO2 during illumina- tion by 640 nm and 720 nm lights. Low levels (about 10 /AM)
Biochemical and Biophysical Research Communications, 1976
The effects of orthophosphate (Pi) on starch synthesis and degradation in the isolated intact spi... more The effects of orthophosphate (Pi) on starch synthesis and degradation in the isolated intact spinach chloroplast are investigated. During CO 2 fixation in the light increasing levels of Pi (O-io0 mM), which have little effect on the rate of fixation, prolong the lag of starch synthesis and reduce its maximal rate from 14o5 to 6o8 ~g-atom C incorporated into starch per milligram chlorophyll per hour. The label in dihydroxyacetone phosphate and other sugar phosphates, especially sedoheptulose-l,7-bisphosphate, increases up to lO-fold under these conditions. Starch degradation in the dark is monitored in the chloroplast by following the breakdown of the 14C labeled starch. Pi concentrations in the order of 2.5-5o0 mM are optimal for starch mobilization at a rate of about I gg-atom C per milligram chlorophyll per hour. INTRODUCTION: Over I00 years ago, Sachs (I) established that carbon assimilated during photosynthesis can be stored in the chloroplast as starch° Starch is synthesized from well-characterized intermediates of the photosynthetic carbon reduction cycle. Recently, Levi and Gibbs (2) reported that starch degradation occurs in the isolated intact spinach chloroplast by a pathway similar to classical glycolysis. Metabolic pathways for the synthesis and the utilization of starch involve phosphorylated compounds and, therefore, require Pi. We report upon the regulatory effect of Pi on carbon flow into starch during photosynthesis and on starch degradation in the dark° i) This research was supported by NSF (BMS-71-O0978) and by ERDA (E(II-I)-3231). 2
A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with 4C-starch i... more A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with 4C-starch is described. These intact chloroplasts were incubated serobically in the dark for 30 minutes. Radioactivity in starch declined and glyceric acid 3-phosphate and maltose were the major radioactive products. It is proposed that starch is degraded within the chloroplast to glyceric acid 3-phosphate and to maltose.
Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour... more Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia olracca) chloroplast which had been preloaded with "4C-starch photosynthetically from 14CO2. Starch breakdown was dependent upon inorganic phosphate and the "4C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of 14C from triose-phosphate to glycerate 3-phosphate. lodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was conduded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic deavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate. In 1862, Sachs (14) demonstrated that carbon assimilated during photosynthesis by the chloroplast accumulated in the form of starch. The synthesis of this polyglucan from well characterized intermediates of the photosynthetic carbon reduction cycle has been amply documented. In recent publications from this laboratory, preliminary evidence was presented indicating that in the intact spinach chloroplast starch synthesis and degra
Dark H2 metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas... more Dark H2 metabolism was studied in marine and fresh water red algae, the green alga, Chlamydomonas, and mosses. A time variable and temperature-sensitive anaerobic incubation was required prior to 112 evolution. H2 evolution was sensitive to disalicylidenepropanediamine. An immediate H2 uptake was observed in these algae. Immediate dark H2 uptake but no evolution was observed in the mosses. A cell-free hydrogenase preparation was obtained from anaerobically adapted Chlamydomonas reinhardii by means of sonic oscillation. The hydrogenase was not sedimented at 100,000g. It catalyzed the reduction of methylene blue, p-benzoquinone, NAD, NADP, but not spinach ferredoxin. H2 evolution was noted with dithionite and with reduced methyl viologen as donors but not with reduced spinach ferredoxin. Similarly, hydrogenase activities were not affected by disalicylidenepropanediamine. The pH optima for H2 evolution and for H2 uptake were 7.2 and 7.5 to 9.5, respectively. Extracts prepared from the anaerobically adapted red alga, Chondrus crispus, and the moss, Leptobryum pyriforme, consumed but did not evolve 112. Uptake was slightly stimulated by methylene blue. It is proposed that red algae and mosses appear to metabolize H2 by a different pathway than Chlamydomonas.
The marine chlorophyte Dunaliella tertiolecta was grown in continuous cultures under NH4+-N, N02-... more The marine chlorophyte Dunaliella tertiolecta was grown in continuous cultures under NH4+-N, N02-N, N03-N, and urea-N limitations. The effect of the nitrogen cell quota (Qn) on the steady-state growth rate (,) was the same regardless of the N source. The relationship between ,u and Q. was well described by the Droop equation, but only up to the true maximum growth rate ,i (= cell washout rate). The ratio between the minimum cell quota (kQ) and the maximum cell quota (Qm) was 0.19. Hence, there is no substitute for determining ,u experimentally. That there was no difference in growth response to different N sources suggests that no intemal pooling of inorganic nitrogen occurred. Both the carbon
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