Chloroplast and cytoplasmic enzymes II. Pea leaf triose phosphate isomerases

Self Cite

Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. In mammalian systems fructose-1,6-diP aldolases from different tissues have kinetic properties related to the function (synthetic or degradative) of the enzyme. Differences in the ratio V.a. fructose-1,6-diP cleavage to Vma. fructose-1,6-diP synthesis led Rutter and coworkers (15) to conclude that muscle aldolase is "more effective in cleaving fructose-1 ,6-diP whereas liver aldolase is relatively more effective in synthesis." Since in green plants chloroplast and cytoplasmic aldolases might also be expected to have opposing functions, it seemed of interest to compare the properties of the plant aldolases. Although spinach whole leaf (8, 14) and chloroplast (7) aldolases and a pea seed aldolase (10,19,20) had been studied prior to the present investigation, no higher plant aldolase known to be cytoplasmic had been characterized. The purpose of the experiments reported here was to characterize the pea leaf chloroplast and cytoplasmic aldolases and to compare the physical and kinetic properties of the isoenzymes.The results indicate that the chloroplast and cytoplasmic aldolases are exceedingly similar enzymes, the pH optima and Michaelis constants for fructose-1 ,6-diP, fructose-1-P, and sedoheptulose-1,7-diP being almost identical. Isoelectric Focusing. Chloroplast and cytoplasmic extracts, prepared as described previously (5) (except that the chloroplast extract was centrifuged 45 min at 500,000g), and containing 10 to 15 mg of protein, were subjected to electrophoresis for 1.5 days in pH 3 to 6 ampholytes at 450 v or pH 4 to 6 ampholytes at 500 v, in a 100-ml LKB isoelectric focusing column at 10 C. Ampholyte and electrode solutions were made up with 5 mm 2-mercaptoethanol. The anode solution (made with 0.5 N H2SO4) was at the bottom of the column. The cathode solution was 0.25 N NaOH. The density gradient was usually made with sucrose. Sorbitol was used in place of sucrose in experiments where aldolase activity was measured as fructose-1,6-diP formation. Fractions (9 drops, 0.7 ml) were collected and analyzed for enzyme activity. The pH of the individual fractions was measured at 0 C using a Radiometer pH meter 26.Purification of Chloroplast and Cytoplasmic Aldolases. Chloroplast and cytoplasmic extracts were prepared as described previously (5) except that the procedure was scaled up for 1 kg of tissue, and the entire shoot of the plant was used. Chloroplasts were usually prepared beforehand and stored at -20 C. The method of Fluri et al. (8) for purification of spinach aldolase was followed, with some modifications. The chloroplast extract, in 10 mm potassium phosphate buffer, pH 7.5, was brought to pH 5.5 with 3.5 M acetic acid, and the supernatant fractio...

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Chloroplast and Cytoplasmic Enzymes

Anderson

Pacold

1972

Self Cite

“…Inhibition of photosynthesis in presence of BHB probably indicates that either nonmetabolized phosphoglycolate or glycolate is toxic for this metabolism. Phosphoglycolate, which is known to inhibit the Calvin cycle enzyme triose phosphate isomerase (1), is likely to be responsible for the observed toxicity. This conclusion is consistent with the fact that mutants of Arabidopsis thaliana lacking phosphoglycolate phosphatase are not viable in air (28).…”

Section: Discussionmentioning

confidence: 99%

Photorespiratory Properties of Mesophyll Protoplasts of Nicotiana plumbaginifolia

Rey¹,

Peltier²

1989

The photorespiratory activity of mesophyll protoplasts of Nicotlana plumbaglnhfolla has been clearly demonstrated by the presence of a Warburg-effect, the occurrence of an important C02-sensitive 02 uptake and the effect of some photorespiratory inhibitors on photosynthetic activity. At a nonsaturating dissolved inorganic carbon (DIC) concentration (0.1 millimolar), we observed that the rate of CO2 fixation was 60% lower at 50% 02 compared to that measured at 2% 02. Using 1802 and mass spectrometry, we measured 02 exchange as a function of light intensity and of DIC concentration. Oxygen uptake measured at the CO2 compensation point (47.4 micromoles 02 per hour per milligram chlorophyll) was three-fold higher than that measured at a saturating CO2 concentration. Cyanide or iodoacetamide, inhibitors of the Calvin cycle, were found to reduce the 02 uptake to the same extent as CO2 saturation. We conclude from these results that the major part of the C02-sensitive 02 uptake is due to photorespiration. Further, we investigated the effect on net photosynthesis of some inhibitors of the glycolate pathway. At CO2 saturation (10 millimolar DIC), 5 millimolar aminoacetonitrile (AAN), and I millimolar aminooxyacetate (AOA) did not cause any significant decrease in net photosynthesis. However, when these two inhibitors were added under a period of active photorespiration (10 minutes at the CO2 compensation point at 20% 02), we observed a decrease in the rate of net photosynthesis at 10 millimolar DIC measured afterward (respectively, 18 and 29%).This inhibition did not appear at 2% 02, but was stronger at 50% 02 (40% for AAN and 47% for AOA). With 0.05 millimolar butyl 2-hydroxy-3-butynoate (BHB) or 0.5 millimolar L-methionine-DL-sulfoximine (L-MSO), rates of net photosynthesis at 10 millimolar DIC were decreased by 10 to 15%. Additional decreases were observed after a period at the CO2 compensation point at 20% 02 (30% for BHB and 20% for L-MSO). From the sites of action of the four inhibitors tested, we suggest the inhibition of photosynthesis occurring after a period of active photorespiration to be due to the toxic accumulation of nonmetabolized phosphoglycolate. tion between 02 and CO2 at the active site of Rubisco,' the enzyme catalyzing the initial reactions ofboth photosynthetic and photorespiratory metabolisms. In other experiments, the activity of the photosynthetic carbon oxidation cycle was deduced from the effect on photosynthesis of some photorespiratory inhibitors. AAN, an inhibitor of the conversion of glycine to serine, has been shown to cause a decrease in the photosynthetic activity in protoplasts of Triticum aestivum (29), only when added in conditions of active photorespiration. Such an inhibition is usually attributed to the accumulation of nonmetabolized photorespiratory intermediates. According to Servaites and Ogren (27), the inhibition of photosynthesis would result from a deficiency in the recycling of carbon in the Calvin cycle, but Creach and Stewart (5) did not observe a decrease in th...

“…Only enzymes specific for the Calvin cycle were restricted to the chloroplasts (12,16,27). When enzyme activities are present in both cell compartments, they are due to isoenzymes, one located in the chloroplasts and the other in the cytosol (1,2,4). This distribution also applies to the two key enzymes of the oxidative pentose phosphate cycle, glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase (11,25,26).…”

mentioning

confidence: 98%

Development and Intracellular Distribution of Enzymes of the Oxidative Pentose Phosphate Cycle in Radish Cotyledons

Schnarrenberger¹,

Tetour²,

Herbert³

1975

Developmeental and compartmenitation studies were used to evaluate the relative roles of the oxidative pentose phosphate cycle, the Calvin cycle, and the glycoly-sis in cotvledons of radish (Raphanus sativus L.).Glucose-6-P dehydrogenase, 6-P-gluconate dehydrogenase, glucose-6-P isomerase, and the NAD-dependent glyceraldehyde-3-P dehydrogenase were present in high activity in ungerminated seeds, increased about 2-fold during germination in the dark, and were slightly enhanced by light. In contrast, NADP-dependent glyceraldehvde-3-P dehydrogenase was developed to only a small degree in the dark, but increased severalfold in continuous white or far red light. The activity of phosphofructokinase was low throughout germinationi.The separation of cell conmpartment-specific isoenzymes showed that, except in ungerminated seeds, the plastid enzyme accouinted for 40 to 45% of the total activity of 6-Pgluconate dehydrogenase and for 13 to 20% of glucose-6-P isomerase. The remaining activity was due to the cytosolic isoenzymes. The presence of gltucose-6-P dehydrogenase and 6-P-gluconate dehydrogeniase in plastids was also established by their presence in the isolated organelle. The NAD -dependenit glyceraldehyde-3 -P dehydrogeniase was mostly due to the cytosolic isoenzyme, whereas the NADdependent activity associated with the NADP-dependent glyceraldehyde-3-P dehydrogenase was very small.The data indicate that the en-zynmes of the oxidative penitose phosphate cycle are present in the cytosol throughotut germiniation. IIn the plastids these enzy-iies already becanie fully developed duiring early germninationi in the dark, whereas entzymes of the Calvin cycle increased onlly in the light. Glycolvsis seemned to be of m-linor importanice.Enzymes involved in the sugar phosphate metabolism of plants have been found in two subcellular compartments, the chloroplasts and the cytosol (15, 25). Only enzymes specific for the Calvin cycle were restricted to the chloroplasts (12,16,27). When enzyme activities are present in both cell compartments, they are due to isoenzymes, one located in the chloroplasts and the other in the cytosol (1, 2, 4). This distribution also applies to the two key enzymes of the oxidative pentose phosphate cycle, glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase (11,25,26).At present only little evidence is available about the intra-'This investigation was supported by the Deutsche Forschungsgemeinschaft. cellular distribution of enzymes in etiolated plant tissues, since most work has been performed using green leaves. In general, one can assume the same intracellular distribution of enzymes in etiolated tissues as in green leaves. In addition to changing levels of total enzyme activities in etiolated tissues (10, 28) it is possible that the quantitative distribution may be different in the two cellular compartments. Previous studies have measured only total enzyme activities from crude extracts, so that the extent of compartmentation of enzyme activities in the plastids and in the cytosol wa...