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1

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Light activation and molecular-mass changes of NAD(P)-glyceraldehyde 3-phosphate dehydrogenase of spinach and maize leaves (1993)

Scagliarini, Sandra ; Trost, Paolo ; Pupillo, Paolo ; [et al.]

Springer

Planta 190 (1993), S. 313-319

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ISSN: 1432-2048

Keywords: Calvin cycle ; Glyceraldehyde 3-phosphate dehydrogenase, NAD(P)-dependent ; Photosynthesis ; Spinacia ; Zea

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Light modulation of chloroplast glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) has been investigated. Complete activation of NADPH-dependent activity is achieved at 25 W.m−2 photosynthetically active radiation in spinach (Spinacia oleracea L.) and 100 W.m−2 in maize (Zea mays L.) leaves. Light activation is stronger in spinach (fivefold on average) than in maize (twofold), which shows higher “dark” activity. The NADH dependent activity does not change appreciably. Several substrate activators can simulate in vitro the light effect with recovery of latent NADPH-dependent activity of spinach enzyme, but they are almost inactive with maize enzyme. A mixture of activators has been devised to fully activate the spinach enzyme under most conditions. The NAD(P)-GAPDH protein can be resolved by rapid gel filtration (fast protein liquid chromatography) into three conformers which have different molecular masses according to the light conditions. Enzyme from darkened leaves or chloroplasts, or dichlorophenyl-1,1-dimethylurea-treated chloroplasts is mainly a 600-kDa regulatory form with low NADPH-dependent activity relative to NADH-activity. Enzyme from spinach leaves or chloroplasts during photosynthesis is mainly a 300-kDa oligomer, which along with the 600-kDa form also occurs in leaves of darkened maize. The conformer of illuminated maize leaves is mainly a 160-kDa species. Results are consistent with a model of NAD(P)-GAPDH freely interconvertible between protomers of the 160-kDa (or 300-kDa intermediate) form with high NADPH-activity, produced in the light by the action of thioredoxin and activating metabolites (spinach only), and a regulatory 600-kDa conformer with lower NADPH-activity produced in darkness or when photosynthesis is inhibited. This behavior is reminiscent of the in-vitro properties of purified enzyme; therefore, it seems unlikely that NAD(P)-GAPDH in the chloroplast is part of a stable multienzyme complex or is bound to membranes.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00196959

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2

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The NADH-dependent Fe3+-chelate reductases of tomato roots (1997)

Bagnaresi, Paolo ; Basso, Barbara ; Pupillo, Paolo

Springer

Planta 202 (1997), S. 427-434

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ISSN: 1432-2048

Keywords: Key words: Fe3+-chelate reductase ; Ferricyanide re ductase ; Iron starvation ; Lycopersicon (Fe starvation)

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract. The NADH-dependent Fe3+-chelate reductase (NFCHR) of tomato (Lycopersicon esculentum L.) roots, a strategy I species, was investigated. The Fe3+-citrate reductase (FeCitR) assay was strongly inhibited by p-hydroxymercuribenzoic acid (PHMB); moreover, the inhibitor was found to be more specific to the FeCitR assay than to the Fe3+-EDTA reductase assay, which was catalyzed by at least another reductase of 46 kDa. After high-speed centrifugation of tomato root membranes, high FeCitR activities were detected in pellets and lower activities in supernatants. After two-phase partitioning of microsomes, FeCitR activity (91 nmol · min−1 · mg−1) was less active in the upper phase (plasma membrane) than in the lower phase (277 nmol · min−1 · mg−1). However, only the activity of the plasma-membrane-associated NFCHR (FeCitR) was significantly enhanced (2.6-fold) in iron-deficient tomato plants, whereas that of NFCHR in non-plasma-membrane rich fractions was unaffected by this treatment. The NFCHR obtained from lysophosphatidylcholine-solubilized plasma membrane was present as a 200-kDa protein complex following fast protein liquid chromatography on Superdex 200, or as a 28-kDa form following Blue Sepharose CL-6B chromatography. Both preparations were more active following iron starvation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the 28-kDa protein purified from solubilized tomato microsomes or supernatant fractions by a final Mono Q step consisted of a single band of 32 kDa. Tomato root NFCHR resembled the NFCHR of maize (a strategy II plant, P Bagnaresi and P Pupillo, 1995, J Exp Bot 46: 1497–1503) in several properties: relative molecular mass, hydrophilicity, chromatographic behaviour, sensitivity to mercurials, specificity for electron donors and acceptors (e.g. cytochrome c), and a ferricyanide reductase-to-FeCitR ratio of 2.5. Preincubation with NADH partially protected NFCHR from PHMB-induced inactivation. Our data show that strategy I and II plants seem to share similar NFCHR proteins, which appear to belong to the cytochrome b 5 reductase flavoprotein group.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s004250050146

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3

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Activation of spinach chloroplast glyceraldehyde 3-phosphate dehydrogenase: effect of glycerate 1,3-bisphosphate (1993)

Trost, Paolo ; Scagliarini, Sandra ; Valenti, Vincenzo ; [et al.]

Springer

Planta 190 (1993), S. 320-326

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ISSN: 1432-2048

Keywords: Calvin cycle ; Glyceraldehyde 3-phosphate dehydrogenase, NAD(P)-dependent ; Glycerate 1,3-bisphosphate ; Photosynthesis ; Spinacia

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Spinach (Spinacia oleracea L.) chloroplast NAD(P)-dependent glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) was purified. The association state of the protein was monitored by fast protein liquid chromatography-Superose 12 gel filtration. Protein chromatographed in the presence of NADP+ and dithiothreitol consisted of highly NADPH-active protomers of 160 kDa; otherwise, it always consisted of a 600-kDa oligomer (regulatory form) favoured by the addition of NAD+ in buffers and with low NADPH-dependent activity (ratio of activities with NADPH versus NADH of 0.2–0.4). Glycerate 1,3-bisphosphate (BPGA) was prepared enzymatically using rabbit-muscle NAD-GAPDH, and purified. Among known modulators of spinach NAD(P)-GAPDH, BPGA is the most effective on a molar basis in stimulating NADPH-activity of “dark” chloroplast extracts and purified NAD(P)-GAPDH (activation constant, K a= 12 μM). It also causes the enzyme to dissociate into 160-kDa protomers. The K m of BPGA both with NADPH or NADH as coenzyme is 4–7 μM. NAD+ and NADH are inhibitory to the activation process induced by BPGA. This compound, together with NADP(H) and ATP belongs to a group of substrate-modifiers of the NADPH-activity and conformational state of spinach NAD(P)-GAPDH, all characterized by K a values three- to tenfold higher than the K m. Since NADP(H) is largely converted to NAD(H) in darkened chloroplasts Heineke et al. 1991, Plant Physiol. 95, 1131–1137, it is proposed that NAD+ promotes NAD(P)-GAPDH association into a regulatory conformer with low NADPH-activity during dark deactivation. The process is reversed in the light by BPGA and other substrate-modifiers whose concentration increases during photosynthesis, in addition to reduced thioredoxin.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00196960

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4

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Two forms of NADP-dependent malic enzyme in expanding maize leaves (1979)

Pupillo, Paolo ; Bossi, Patrizia

Springer

Planta 144 (1979), S. 283-289

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ISSN: 1432-2048

Keywords: C4 Species ; Isozymes ; Malic enzyme ; Zea

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Etiolated maize leaves (Zea mays L.) contain a major isozyme of NADP-dependent malic enzyme (L-malate dehydrogenase, decarboxylating, EC 1.1.1.40) having an isoelectric point of 5.28±0.03, a Km (L-malate) 0.3–0.6 mM at pH 7.45; a broad pH optimum around pH 6.9 under the conditions of assay; a molecular weight of 280,000 (sometimes accompanied by a minor component of 150,000); and an NAD-dependent activity about 1/50 the NADP-dependent activity. This isozyme, resembling the NADP-malic enzyme of vertebrates, is labeled type 1. The dominant isozyme of young green leaves (type 2) has, however, a pI 4.90±0.03, a Km (L-malate) 0.10–0.15 mM, a pH optimum of 8, and a molecular weight of 280,000. It is also more stable and exhibits an appreciable NAD-dependent activity (1/5–1/7 the NADP activity). Both isozymes show linear kinetics, dependence on Mn or Mg ions, similar Km (NADP+), and the typical increase of Km for L-malate with increasing pH values. Type 1 isozyme of maize is assumed to be cytosolic. Type 2 corresponds in each property to the chloroplast enzyme of bundle-sheath cells. It is present at a low level in etiolated leaves and develops to a high specific activity (up to 100 nmol min-1 mg protein-1 by 150 h illumination) during photosynthetic differentiation, replacing the type 1 form.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00388771

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5

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A possible plasma membrane particle containing malic enzyme activity (1981)

Pupillo, Paolo ; Grosso, Elena

Springer

Planta 151 (1981), S. 506-511

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ISSN: 1432-2048

Keywords: Cell fractionation ; Cucurbita ; Malic enzyme ; Plasma membrane ; Subcellular localization ; Zea

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract A definite membrane fraction from Cucurbita hypocotyls, maize coleoptiles, and other plant tissues contains a NADP-dependent malic enzyme activity, up to 10% of overall tissue activity, and probably other soluble proteins. This “malic enzyme particle” is identified as plasmalemma on the basis of sedimentation behavior, density distribution in sucrose gradients, in comparison with enzyme markers, and sluggish penetration by the sugar Metrizamide. Enzyme binding to the plasma membrane is stable and scarcely sensitive to salts and EDTA, although all activity is released to the supernatant in the presence of Triton-X-100 or under hypotonic conditions. The properties of bound enzyme are similar to those of free enzyme in cell extracts. It is proposed that osmotically sensitive plasma membrane vesicles, containing cytoplasm fragments, are formed during homogenization. Low malic enzyme activities are also associated with Cucurbita proplastids.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00387427

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