Chloroplast and Cytoplasmic Enzymes: Three Distinct Isoenzymes Associated with the Reductive Pentose Phosphate Cycle
TL;DR: Three pea (Pisum sativum) leaf chloroplast enzymes-triose phosphate isomerase, glyceric acid 3-phosphate kinase, and fructose 1,6-diphosphate aldolase-have been separated from the corresponding cytoplasmic enzymes by isoelectric focusing.
Abstract: Three pea (Pisum sativum) leaf chloroplast enzymes—triose phosphate isomerase, glyceric acid 3-phosphate kinase, and fructose 1,6-diphosphate aldolase—have been separated from the corresponding cytoplasmic enzymes by isoelectric focusing. These three enzymes of the reductive pentose phosphate cycle are therefore distinct proteins, not identical with the analogous enzymes of the Embden-Meyerhof-Parnas pathway.
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TL;DR: Many enzymes in plants have isozymes because the same catalytic reaction is often present in several subcellular compartments, most frequently the plastids and the cytosol, but gene duplication in diploid species and the addition of genomes in polyploids species have increased the number of isoz enzymes.
Abstract: Many enzymes in plants have isozymes because the same catalytic reaction is often present in several subcellular compartments, most frequently the plastids and the cytosol. The number and subcellular locations of the isozymes appear to be highly conserved in plant evolution. However, gene duplication in diploid species and the addition of genomes in polyploid species have increased the number of isozymes.
610 citations
TL;DR: Fractionation of protoplasts and purification of subcellular organelles has shown that enzyme activity is associated with the chloroplasts, mitochondria and cytosol, and distinct multiple isoforms of the enzyme, which differed in isoelectric point and were compartment-specific, were resolved from purified mitochondria
Abstract: On sodium-dodecyl-sulfate polyacrylamide gels, purified glutathione reductase (GR; EC 1.6.4.2) from the leaves of two- to three-week-old pea (Pisum sativum L. cv. Birte) seedlings was represented by a single band with an apparent molecular weight of 55 kilodaltons. This polypeptide was resolved to multiple isoforms by two-dimensional electrophoresis. Fractionation of protoplasts and purification of subcellular organelles has shown that enzyme activity is associated with the chloroplasts, mitochondria and cytosol (in this order, approx. 77%, 3%, and 20% of the total activity). Distinct multiple isoforms of the enzyme, which differed in isoelectric point and were compartment-specific, were resolved from purified mitochondria and chloroplasts. The latency of the glutathione reductase activity which co-purified on Percoll gradients with the mitochondrial marker enzyme, cytochrome-c oxidase (EC 1.9.3.1.), indicated that this enzyme was within the mitochondrion. The mitochondrial glutathione reductase activity was strongly dependent on NADPH and not NADH.
334 citations
Cites background from "Chloroplast and Cytoplasmic Enzymes..."
...Higher-plant metabolism appears to be regulated in part by subcellular compartmentat ion (see Oaks and Bidwell 1970 for a review) and there is evidence for duplication of metabolic pathways which differ in their subcellular location and expression during various physiological states (Anderson and Advani 1970; Schnarrenberger and Oeser 1974)....
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TL;DR: The number and nature of these compounds suggests that metabolic intermediates exert a cumulative effect on the activity of the triose-P isomerases, acting concertedly to modulate the activityof these enzymes in the green leaf.
208 citations
TL;DR: The results suggest that most, if not all, the nitrite reductase and acetolactate synthetase activity of the cell is in the plastids, and some evidence was obtained for low levels of glutamate dehydrogenase activity in chloroplasts.
Abstract: Density gradient separation of plastids from leaf and root tissue was carried out The distribution in the gradients of the activity of the following enzymes was determined: nitrite reductase, glutamine synthetase, acetolactate synthetase, aspartate aminotransferase, catalase, cytochrome oxidase, and triosephosphate isomerase The distribution of chlorophyll was followed in gradients from leaf tissue The presence of plastids that have retained their stroma enzymes was denoted by a peak of triosephosphate isomerase activity Coincidental with this peak were bands of nitrite reductase, acetolactate synthetase, glutamine synthetase, and aspartate aminotransferase activity The results suggest that most, if not all, the nitrite reductase and acetolactate synthetase activity of the cell is in the plastids The plastids were found to contain only part of the total glutamine synthetase, aspartate aminotransferase, and triosephosphate dehydrogenase activity in the cell Some evidence was obtained for low levels of glutamate dehydrogenase activity in chloroplasts
203 citations
TL;DR: Light-generated vicinal-dithiols seem to be involved in modulation of the activity of the enzymes included in this study, and apparently involves a component preceding ferredoxin in the photosynthetic electron transport chain.
Abstract: Inhibitor experiments indicate that photosynthetic electron transport is required for light activation of the pea (Pisum sativum) leaf chloroplast enzymes NADP-linked glyceraldehyde-3-phosphate dehydrogenase, NADP-linked malic dehydrogenase, ribulose-5-phosphate kinase and sedoheptulose-1,7-diphosphate phosphatase, and for inactivation of glucose-6-phosphate dehydrogenase. Modulation of the activity of the dehydrogenases and kinase apparently involves a component preceding ferredoxin in the photosynthetic electron transport chain; activation of the phosphatase involves an electron transport component at the level of ferredoxin. Modulation of enzyme activity can be obtained in a broken chloroplast system consisting of membrane fragments and stromal extract. The capacity for light regulation in this system is reduced or eliminated when the membrane fraction is exposed to arsenite in the light or to sulfite in light or dark. Light-generated vicinal-dithiols seem therefore to be involved in modulation of the activity of the enzymes included in this study.
172 citations
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TL;DR: The turbidity produced when protein is mixed with low concentrations of any of the common protein precipitants can be used as an index of protein concentration, and this advantage is used to eliminate the interference of nucleic acids in the estimation of protein.
Abstract: Publisher Summary The turbidity produced when protein is mixed with low concentrations of any of the common protein precipitants can be used as an index of protein concentration. The resulting turbidity is maximum after about 10 minutes and may be measured spectrophotometrically in the wavelength region of 600 m. Standardization may be effected by comparison with the turbidity produced by a suspension of a dried protein precipitate, or reference may be had to the methyl acrylate-styrene polymer. Turbidimetric techniques are rapid and convenient, but they yield different values with different proteins. They do not permit differentiation between protein and acid-insoluble compounds such as nucleic acids. Protein estimation with the Folin-Ciocalteu reagent include (1) biuret reaction of protein with copper ion in alkali, and (2) reduction of the phosphomolybdic-phosphotungstic reagent by the tyrosine and tryptophan present in the treated protein. Protein estimation by ultraviolet absorption takes advantage of the fact that nucleic acid, however, absorbs much more strongly at 260 mμ than at 280 mμ, whereas with protein the reverse is true. This advantage is used to eliminate, by calculation, the interference of nucleic acids in the estimation of protein.
3,391 citations
TL;DR: Phosphopyruvate carboxylase was apparently the major photosynthetic carbon dioxide-fixing enzyme in the tropical grasses, although malic enzyme may contribute to a lesser extent.
Abstract: 1. The activity per unit of chlorophyll of certain carboxylases, and of other enzymes involved in photosynthesis, was determined in leaf extracts of the tropical grasses, sugar-cane, maize and sorghum, and compared with the activities for wheat, oat and silver-beet. Maximum rates of photosynthetic carbon dioxide uptake were also measured for comparison with enzyme activities. 2. Phosphopyruvate carboxylase activity was about 60 times greater in the tropical grasses than in wheat, oat and silver-beet and was severalfold higher than the rates of photosynthetic carbon dioxide uptake. Most of the enzyme was located in the chloroplast fraction of cell extracts. 3. Phosphopyruvate carboxylase was apparently the major photosynthetic carbon dioxide-fixing enzyme in the tropical grasses, although malic enzyme may contribute to a lesser extent. 4. Tropical grasses contained less than one-tenth of the ribulose diphosphate carboxylase activity present in wheat, oat and silver-beet. For the tropical grasses this activity, determined with a saturating concentration of bicarbonate, was approx. 10% of the rate of photosynthesis. 5. The fraction-1 protein content of leaf extracts paralleled the ribulose diphosphate carboxylase activity. 6. In contrast, the activity of several other enzymes of the Calvin cycle was similar in the different species examined.
225 citations
159 citations
TL;DR: A simplified procedure permits the separation of spinach chloroplasts which retain the ability to catalyze the photosynthetic assimilation of carbon dioxide and its associated oxygen evolution.
Abstract: A simplified procedure (involving disruption in sorbitol-pyrophosphate mixtures) permits the separation of spinach chloroplasts which retain the ability to catalyze the photosynthetic assimilation of carbon dioxide and its associated oxygen evolution
151 citations