NADH and NADPH Dependent Malate Dehydrogenases of <i>Phaseolus vulgaris</i>

Vidal, Jean; Gadal, Pierre; Cavalié, G.; Cailliau-Commanay, Lucienne

doi:10.1111/j.1399-3054.1977.tb04034.x

Cited by 13 publications

(9 citation statements)

References 16 publications

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“…The second optimum at pH 8.0 was associated with a chloroplastic enzyme which required DTT for activation. Similar results were obtained for NADP-malate dehydrogenase extracted from leaves of Phaseolus vulgaris (22). These authors showed that the activity at the lower pH was due to an NAD-malate dehydrogenase which also exhibited NADP-dependent activity.…”

Section: Methodssupporting

confidence: 80%

Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C₃ Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism

Winter

Foster²,

Edwards³

et al. 1982

Plant Physiol.

149

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Mesembryarnhemnam crystafiinm, a halophilic, inducible Crassulacean acid metabolism (CAM) species, was grown at NaCI concentrations of 20 and 400 milimlar in the rooting medium. Plants from the low salinity treatment showed exclusively C3-photosynthetic net CO2 fixation, whereas plants exposed to the high salinity level exhibited net CO2 dark fixation involving CAM. Mesophyll protoplasts, isolated from both tissues, were gently ruptured, and the intracelular localization of enzymes was studied following differential centrifugation and Percoll density gradient centrifugation of protoplast extracts. Both centrifugation techniques resulted in the separation of intact chloroplasts, with up to 90% yield, from other organelles and the nonparticulate fraction of cels. Enzymes were identified by determination of activity and by sodium dodecyl sulfate gel electrophoresis of enzyme protein.Experiments established the extraorganelar (cytoplasmic) location of phosphoenolpyruvate carboxylase, enolase, phosphoglyceromutase, and NADP-malic enzyme; the mitochondrial location of NAD-malic enzyme; and the chloroplastic location of pyruvate, Pi dikinase. NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphohexose isomerase, and phosphoglycerate kinase were associated with both cytoplasm and chloroplasts.NADP-dependent malate dehydrogenase activity was found in both the chloroplastic and extrachloroplastic fractions; the activity in the chloroplast showed an optimum at pH 8.0 and was dependent upon preincubation of enzyme with dithiothreitol. The extrachloroplastic activity showed an optimum at pH 6.5 and was independent ofpretreatment with dithiothreitol. In the paper presented here, the intracellular distribution of 16 enzymes was studied in the halophilic, inducible CAM plant Mesembryanthemum crystallinum (26). Leaves of M. crystallinum are apparently low in phenolic compounds, and high extractable activities of several enzymes have been reported for this species (9). Experimental plants were manipulated to show either exclusively C3-photosynthetic net CO2 fixation during the light (growth at low salinity levels in the rooting medium) or to exhibit nocturnal net CO2 uptake involving CAM (growth at high salinity levels). Mesophyll extracts with almost all chloroplasts intact were routinely obtained from plants of both salinity treatments by gentle rupture of enzymically isolated protoplasts. Two techniques, differential centrifugation and Percoll density gradient centrifugation, were used to study the intracellular localization of enzymes. Chloroplast integrity was preserved during these procedures, and the two methods gave identical results. MATERIALS AND METHODS Growth

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Section: Methodssupporting

confidence: 80%

Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C₃ Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism

Winter

Foster²,

Edwards³

et al. 1982

Plant Physiol.

149

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“…The relatively high activity of the NADP-MDH of guard cells in the dark can be ascribed to an incomplete specificity of the NAD-MDH, as has been reported for leaf and root extracts of P. vulgaris (28), and confirmed by us with pea leaf extracts and extracts of pea leaf guard cells. It was possible to separate a DTT-dependent activity from that detected without preincubation with DTT, the latter comigrated with the NADdependent activities (Fig.…”

Section: Amounts Of Nadp-mdh In Extracts Of Guard Cells and Mesophyllsupporting

confidence: 81%

“…However, the ratio between the DTT-independent and the DTT-stimulated NADP-MDH activities was lower than in guard cell extract. Vidal et al (28) had obtained an MDHactivity profile comparable to that shown in Fig. 3A, when they separated a leaf extract of P. vulgaris on a DEAEcellulose column.…”

Section: Separation Of Mdh Activities In Epidermal Extractsmentioning

confidence: 59%

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Malate Dehydrogenases in Guard Cells of Pisum sativum

Scheibe

Reckmann²,

Hedrich³

et al. 1990

Plant Physiol.

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Guard cell protoplasts of Pisum sativum show considerable NADP-dependent malate dehydrogenase (MDH) activity in darkness which can be enhanced severalfold by illumination or treatment with dithiothreitol (DTT). The question arose whether guard cells possess an NADP-MDH different from that present in the chloroplasts of the mesophyll (which is inactive in darkness or in the absence of DTT). MDH activities were determined in extracts of isolated protoplasts from mesophyll and epidermis, and in mechanically prepared epidermal pieces (with guard cells as the only living cells and no interference from proteases originating from the cell wall digesting enzymes). Guard cells possessed NAD-dependent MDHs of high activity and incomplete exclusion of NADP as a coenzyme. This NADP-dependent activity of the NAD-MDH(s) could not be stimulated by DTT or, inferentially, by light. The DTT-(and light-) dependent NADP-MDH represented 0.05% of the total protein of the guard cells and had a specific activity of 0.1 unit per milligram protein; both values are in the same range as the corresponding ones of the mesophyll cells.Agreement was also found in the extent of light activation, in subunit molecular weight, immunological cross-reactions, and in the behavior on an ion exchange column. The activity of the chloroplastic NADP-MDH in guard cells barely suffices to meet the malate requirement for stomatal opening in the light. It is therefore likely that NAD-MDHs residing in other compartments of the guard cells supplement the activity of the chloroplastic NADP-MDH particularly during stomatal opening in darkness.the contributions made to malate formation in guard cells made by the NADP-and NAD-depending MDHs, while paying attention to their participation in producing the observed light-dark differences in malate synthesis.Of the various difficulties, which arise when measuring enzyme activities in guard cells (and cells of other types) and which might give rise to contradictory results, we wished to address two: (a) Isoenzymes are located in several cell compartments and exhibit distinctly different properties as to coenzyme specificity, pH optimum, etc. In crude extracts the sum of all these activities is determined; and (b) Cell extracts may contain proteases liberated from the vacuoles, or being added through the cell-wall digesting media during protoplast preparation; these proteases may severely interfere with enzyme determinations.Of course, enzymes could be identified in resin-embedded sections by immunolocalization and evaluation of electron micrographs (22). However, such a procedure does not permit the measurement of the enzymatic activity of an investigated protein. We therefore chose the judicious determination of enzyme activities in extracts and of protoplasts of guard cells and the common epidermal cells as well as in extracts of epidermal pieces. The results were compared with those obtained with mesophyll cell protoplasts and leaf extracts. MATERIALS AND METHODS Plant Material

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“…Activity of MDH was estimated according to the method of Vidal et al (1977). The assay mixture contained lOmM oxaloacetate and 5 mM EDTA in 50 mM Tris-HCl buffer, pH 7'6.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Discrimination of some African Armillaria species by isozyme electrophoretic analysis

Agustian¹,

Mohammed

Guillaumin

et al. 1994

New Phytologist

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SUMMARYTen Armillaria isolates, collected from various host plants and widespread geographical origins in tropical Africa, were cultivated on orange fragments in the presence of water and two different culture media in order to optimize enzyme and mycelial cord production. Seven enzymes involved in the primary metabolism of nitrogen and carbon (glutamate dehydrogenases, aspartate aminotransferase, malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phospbogluconatt dehydrogenase and isocitrate dehydrogenase) were extracted from the mycelial cords and analyzed by polyacrylamide gel electrophoresis. Cluster analy.sis based on calculated similarity i-alues derived from isozyme banding patterns separated the isolates into fi\'e groups. Two isolates considered as belonging to A. metlea ssp. africana (an African species closely related to European A. metlea) were present in a clearly separated cluster when compared to the other isolate groups. Two Kenyan isolates, belonging to an as yet unnamed biological species, which w ere characterized by the production of few slow growing mycelial cords, were also found in a separate cluster with slightly greater similarit\' coefficients to the other isolates. The six other isolates, referred to as isolates of A. heimii (a highly variable species with different sexual systems) fell into three sub^clusters of variable homology. The two bomothallic heimii isolates from Tanzania and Malawi, which were \ery closely related, displaying 100 "" isozyme similarity, exhibited a higher degree of similarit>' with the two other homothalhc heimii isolates from Zimbabwe and Congo, than with the two heterothailic unifactorial heimti isolates from Cameroon and Gabon. The value of isozymes in the classification of African Armtllaria spp. is discussed.

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NADH and NADPH Dependent Malate Dehydrogenases of Phaseolus vulgaris

Cited by 13 publications

References 16 publications

Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C₃ Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism

Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C₃ Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism

Malate Dehydrogenases in Guard Cells of Pisum sativum

Discrimination of some African Armillaria species by isozyme electrophoretic analysis

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NADH and NADPH Dependent Malate Dehydrogenases of Phaseolus vulgaris

Cited by 13 publications

References 16 publications

Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C3 Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism

Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C3 Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism

Malate Dehydrogenases in Guard Cells of Pisum sativum

Discrimination of some African Armillaria species by isozyme electrophoretic analysis

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Product

Resources

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Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C₃ Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism

Intracellular Localization of Enzymes of Carbon Metabolism in Mesembryanthemum crystallinum Exhibiting C₃ Photosynthetic Characteristics or Performing Crassulacean Acid Metabolism