A possible plasma membrane particle containing malic enzyme activity

Pupillo, Paolo; Grosso, Elena Del

doi:10.1007/bf00387427

Cited by 9 publications

(4 citation statements)

References 20 publications

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“…Soluble NADP-malic enzyme is constantly present as contaminant of washed mitochondrial preparations, as documented above (Fig. 1) and elsewhere (16). Due to the similarity of size, this activity would co-purify with the NAD-linked protein, as discussed below.…”

Section: Discussionmentioning

confidence: 70%

“…On the other hand, the NADP-linked activity occurs as a mitochondrial peak and, in addition, in lighter fractions of the gradient banding around 32 to 35% sucrose (w/ w). This nonmitochondrial activity, characterized by high affinity for NADP+ (Km = 10-20 ,uM), has been attributed to an enzyme fraction associated with the plasma membrane (16). Therefore, the NADP-dependent activity of crude mitochondrial sediments is heterogeneous.…”

Section: Resultsmentioning

confidence: 99%

“…Triton was omitted in experiments with purified protein. CCO2 and CCR activities were determined as described elsewhere (16 EDTA, and 0.1 mm 2-mercaptoethanol. The gradient was constructed above a 2-ml cushion of 60%o sucrose (w/w), by mixing 14 ml of a 55% sucrose solution and 15.5 ml of a 15% solution with the aid of a mechanic stirrer.…”

Section: Methodsmentioning

confidence: 99%

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Activation Kinetics of NAD-Dependent Malic Enzyme of Cauliflower Bud Mitochondria

Valenti¹,

Pupillo²

1981

Plant Physiol.

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NAD-dependent malic enzyme (EC 1.1.1.39) was obtained from isolated mitochondria of cauliflower buds (Brassica okracca L., var. botrytis). The NAD-linked activty is accompanied by a minor NADP-linked activity. Some contaminant NADP-malc enzyme from the supernatant and the plasma membrane is usually present in crude mitochondrial preparations.NAD-dependent malic enzyme has been purified 38-fold by ammonium sulfate fractionation and gel permeation chromatography, to a specific activity up to 2 micromoles per minute per millgram.

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Section: Discussionmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 99%

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Activation Kinetics of NAD-Dependent Malic Enzyme of Cauliflower Bud Mitochondria

Valenti¹,

Pupillo²

1981

Plant Physiol.

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“…Other buffers (MesNaOH, Hepes-NaOH, Tricine-NaOH) were used as needed at suitable pH values. Marker enzymes for gradient fractions were assayed as described in previous publications (5,19) (18). On the other hand, DQ behaves as a recycling cofactor, that never becomes exhausted even when supplied at low concentrations.…”

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confidence: 99%

Kinetic Characterization of Reduced Pyridine Nucleotide Dehydrogenases (Duroquinone-Dependent) in Cucurbita Microsomes

Pupillo¹,

Valenti²,

Luca³

et al. 1986

Plant Physiol.

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Some properties of microsomal electron transfer chains, dependent for oxidase activity on addition of NADH or NADPH, duroquinone, and oxygen (L. De Luca et al., 1984, Plant Sci Lett 36: 93-98) are described. Activity is characterized by negatively cooperative kinetics toward reduced pyridine nucleotides, with limiting K,, of 10 to 50 micromolar at pH 7.0 (increasing at lower pH), as well as toward duroquinone with limiting K, of 100 to 400 micromolar regardless of pH. Molecular oxygen is reduced by the enzyme complex with S_.s of about 30 micromolar and production of H20 and H202, without superoxide involvement. The ratio NAD(P)H:02 averages 1.35 in the presence of KCN and 1.85 in its absence. The pyridine nucleotide specificity of the dehydrogenases has been investigated by kinetic competition experiments. Some enzyme heterogeneity was established for all preparations. At least two enzymes are detectable in plasma membrane-enriched fractions: a major NAD(P)H dehydrogenase having an acid pH optimum, and an NADPH dehydrogenase active around neutrality. Addition of Triton X-100 strongly enhances the activity over most of the pH scale, but depresses it increasingly at pH values higher than 8.0, to the effect that pH profile shows, under these conditions, a major peak at about pH 5.8 for both NADH and NADPH oxidase. Results with endoplasmic reticulum preparations are similar, except that they suggest the presence of still more activities at and above pH 7. The results are interpreted in terms of different complexes catalyzing electron transfer from NAD(P)H to 02 without release of intermediates.Recent studies (1,3,7,21,22,25) indicate that reducing equivalents can be transferred from the inside of plant cells to the outer space through the plasma membrane, for control of the neighboring environment, uptake of nutrients, and intracellular regulation. An NADH oxidase ofroot cell surfaces, possibly coupled to a transport system (12,14), has been isolated and seems to contain quinone (15). Some typical redox chain components such as flavins and a light sensitive b-type Cyt are present in the plasmalemma (8,10,13,27 (16), and may not be related to the bulk of DQ-dependent oxidases of the plasma membrane.Because of the uncertainties, we have undertaken a detailed biochemical characterization of these oxidases. This paper deals mainly with the nature ofthe reactions. It also shows that among different components a pyridine nucleotide-nonspecific dehydrogenase is responsible for most of the activity.MATERIALS AND METHODS Plant Material and Tissue Fractionation (5, 9). Cucurbita pepo L. was grown from seeds (hybrid 'Senator', Asgrow Co.) for 5 d in the dark at 24°C on vermiculite. Hypocotyl segments 2 to 3 cm below the hook were harvested. All subsequent procedures were carried out on ice in daylight.Hypocotyl sections were chopped with razor blades in two volumes of extraction medium (0.25 M sucrose, 50 mM Tris-HCl pH 7.5, 1 mm Na2EDTA, 1 mm Na2EGTA), then ground in a mortar and squeezed through a nylon cloth. The...

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Plasma Membranes

Larsson¹

1985

Cell Components

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A possible plasma membrane particle containing malic enzyme activity

Cited by 9 publications

References 20 publications

Activation Kinetics of NAD-Dependent Malic Enzyme of Cauliflower Bud Mitochondria

Activation Kinetics of NAD-Dependent Malic Enzyme of Cauliflower Bud Mitochondria

Kinetic Characterization of Reduced Pyridine Nucleotide Dehydrogenases (Duroquinone-Dependent) in Cucurbita Microsomes

Plasma Membranes

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