The control of NAD specific malic enzyme from cauliflower bud mitochondria by metabolites

Davies, Daniel; Patil, K. D.

doi:10.1007/bf00388963

Cited by 43 publications

(22 citation statements)

References 17 publications

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“…The mol wt of this form is in close accord with the reported mol wt of NAD malic enzyme isolated from animal sources (7,12). Mol wt of approximately 300,000 to 400,000 have been reported for impure preparations of NAD malic enzyme from plant tissues (4,10,14). As we also find that the purified NAD malic enzyme from potato can exist in a higher mol wt form (477,000 D) it would seem that the plant NAD malic enzymes are capable of existing in higher states of aggregation than the enzyme from animal sources.…”

Section: Discussionsupporting

confidence: 67%

Kinetic Ramifications of the Association-Dissociation Behavior of NAD Malic Enzyme

Grover¹,

Wedding²

1982

Plant Physiol.

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Our studies on the aggregation state of NAD malic enzyme arose from observations of complex substrate saturation curves having two or more distinct phases. These results suggested that multiple forms of the enzyme, each with its own kinetic properties, might be present under assay conditions. In addition, the kinetic parameters of the enzyme were found to change with enzyme concentration, suggesting that the multiple forms may simply be different oligomeric states of the enzyme. Oligomerization of enzymes, like conformational change, can be an important regulatory mechanism (6). We have pursued the question ofoligomeric changes using enzyme which we have purified to homogeneity from potato tuber mitochondria.MATERIALS AND METHODS Enzyme. NAD malic enzyme was purified from red potato (Solanum tuberosum var. Chieftan) tubers as previously described trophotometrically by following the reduction ofNAD (or NADP, as indicated) at 340 nm in 1.0 ml of assay medium at 25°C. For routine assays of column fractions, the following assay mix was used: 4 mm NAD, 100 mm malate, 5 mm DTT, 20 mm MgC12, 50 mM Tes (pH 7.0). Because the enzyme does not utilize metalsubstrate complexes, detailed kinetic evaluations were performed using assays in which malate2-, Mg2+, and NAD+ concentrations were all expressed in terms of free, uncomplexed species, as previously described (3,8). Assays for malate saturation series contained 6 mm free NAD, 8 mm free Mg2+ and 50 mm Tes (pH 6.8). Assays were run for a period of time ranging from 8 to 30 min in order to ensure that the reaction velocity had obtained a constant value. Kinetic parameters were obtained by fitting the data from a substrate saturation curve (usually comprising data from 16 individual assays) to the equationwhere n is the Hill coefficient and Vm is the maximal velocity under the given experimental conditions. The data were fitted to this model by means of an iterative nonlinear fitting program, based on the Marquart algorithm, as previously described (3). This program was written in BASIC, and was used with a TRS-80 Model II computer. Gel Filtration. Molecular weights were estimated by gel filtration according to the method of Andrews (1) using a 1.6 x 75 cm or 1.6 x 79 cm column of Bio-Gel A-0.5m at 5°C. As the mol wt calibration of the column is sensitive to the ionic strength of the eluting buffer, mol wt standards were run in each buffer system used with malic enzyme. The standard column buffer comprised 100 mm Tes (pH 7.0), 2 msm Mg2+, and 5 mm DTT. The Mg was supplied as MgO rather than as a salt to avoid complications caused by most of the common anions such as Cl-(which causes progressive irreversible inactivation of malic enzyme with prolonged exposures) and S04 (which activates the enzyme). When additional ligands were added to the elution buffer, the Mg2+ and ligand levels were corrected for any depletion caused by metal complexation and all concentrations expressed in terms of free, uncomplexed species, as with the assay medium. Filtration was carried out in an upwa...

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Section: Discussionsupporting

confidence: 67%

Kinetic Ramifications of the Association-Dissociation Behavior of NAD Malic Enzyme

Grover¹,

Wedding²

1982

Plant Physiol.

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“…In addition to the NAD malic enzyme, enzymes which produce pyruvate from malate using NADP as a cofactor have been reported from plant sources (7,8,15). We have found that the oxidation of malate by extracts of sweet potato mitochondria using NADP as a cofactor is somewhat variable, but in several different preparations, the activity with NADP was found to be 16.4 ± 1.5% of the activity with NAD.…”

Section: Resultsmentioning

confidence: 80%

“…The NAD malic enzyme has been isolated from a number of plant mitochondria (3,8,9,14,15 (8,9) also seems to indicate the probable importance of its role as part of a crucial switching point in plant metabolism.…”

Section: Resultsmentioning

confidence: 96%

Malate Dehydrogenase and NAD Malic Enzyme in the Oxidation of Malate by Sweet Potato Mitochondria

Wedding¹,

Black²,

Pap³

1976

Plant Physiol.

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Over a range of concentrations from less than 0.1 mM to more than 70 mM, sweet potato root mitochondria display a bimodal substrate saturation isotherm for malate. The high affinity portion of the isotherm has an apparent Km for malate of 0.85 mm and fits a rectangular hyperbolic function. The low affinity portion of the isotherm is sigmoid in character and gives an apparent S0.5 of 40.6 mm and a Hill number of 3.7.Extracts of sweet potato mitochondria contain both malate dehydrogenase and NAD malic enzyme. The malate dehydrogenase, assayed in the forward direction at pH 7.2, shows typical Michaelis-Menten kinetics with a Km for malate of 0.38 mm. The NAD malic enzyme shows pronounced sigmoidicity in response to malate with a Hill number of 3.5 and an S0 5 of 41.6 mM.On the basis of the normal kinetics, the Km, and the fact that oxaloacetate production from malate by mitochondria appears most active at low malate concentrations, the high affinity portion of the malate isotherm with mitochondria is attributed to malate dehydrogenase. The low affinity portion of the malate isotherm with mitochondria is thought, on the basis of the similarity of SO.5 values, the Hill numbers, and the greater production of pyruvate from malate at high malate concentrations, to represent the activity of the NAD malic enzyme.The response of plant mitochondria to malate is often variable and erratic (6,10,12,13,15). Saturation with the substrate is difficult to achieve, coupling with phosphorylation is sometimes less than with other tricarboxylic acid cycle substrates using NAD as a cofactor (16) and the response to exogenously added cofactors such as NAD may be at variance with that of other substrates (3,6,15). In part, these unexpected responses may arise from the characteristics of the enzyme, malate dehydrogenase, usually assumed to be responsible for the oxidation of malate in the tricarboxylic acid cycle. Malate dehydrogenase is strongly inhibited by its product, oxaloacetate, at pH values in the range likely to be found in the mitochondria (10,17,18), and is subject to allosteric regulation by a number of effectors (1).We have observed in mitochondria from a variety of plant tissues, including sweet potato, beet roots, cauliflower florets, and Arum spadix, a failure of 02 uptake rates to be saturated by levels of malate greatly in excess of the concentrations which would be expected to elicit no further response on the basis of the observed Km for malate of malate dehydrogenase in mitochondrial extracts. In addition, it is frequently difficult to obtain smooth curves in the 02 uptake response of plant mitochondria to malate, and where sufficient numbers of careful determinations are made, the malate response curve appears to be inflected. Similar results have been obtained by others (3, 13).This report details the characteristics of 02 uptake by sweet potato mitochondria in response to malate, and shows that the saturation isotherm for malate is bimodal, with an intermediary plateau, and provides evidence that this resp...

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“…Enzymes implicated in synthesis and catabolism of this substrate are widely distributed in the cellular compartments. If these enzymes have become well known in herbaceous plants (Macrae, 1971;Davis and Patil, 1975;Wiskich and Dry, 1985;Artus and Edwards, 1985), very few studies have been made on woody plants Cheliak, 1985, 1986;Weimar and Rothe, 1987 (Gerant et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

“…Enzymatic activities were measured according to Hatch (1978) for fumarase (EC 4.2.1.2), Davis and Patil (1975) for NADmalic enzyme (EC 1.1.1.39), Queiroz (1968) (Fig. 1A) and June 1987-March 1988 for spruce (Fig.…”

Section: Introductionmentioning

confidence: 99%

Extraction and study of enzymes linked to malate metabolism in tree leaves

Gérant¹,

Citerne²,

Fabert³

et al. 1989

Ann. For. Sci.

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The control of NAD specific malic enzyme from cauliflower bud mitochondria by metabolites

Cited by 43 publications

References 17 publications

Kinetic Ramifications of the Association-Dissociation Behavior of NAD Malic Enzyme

Kinetic Ramifications of the Association-Dissociation Behavior of NAD Malic Enzyme

Malate Dehydrogenase and NAD Malic Enzyme in the Oxidation of Malate by Sweet Potato Mitochondria

Extraction and study of enzymes linked to malate metabolism in tree leaves

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