Regulation of ‘malic’ enzyme of Solanum tuberosum by metabolites

NADP:malic enzyme from com (Zea mays L.) leaves was purified by conventional techniques to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies raised against this protein in rabbits were purified, coupled covalently to protein A-Sepharose CL-4B, and used as an immunoaffinity resin to purify the NADP:malic enzymes of the C3 plants spinach (Spinacia oleracea L.) and wheat (Triticum aestivum L.), of the Crassulacean acid metabolism (CAM) plant Bryophyllum daigremontianum R. Hamed et Perr. de la Bathie and the C4 plants corn, sugarcane (Saccharum officinarum L.), and Portulaca grandHflora L. Such procedures yielded homogeneous protein preparations with a single protein band, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for P. grandiflora L. with two bands. The specific activities of the purified proteins ranged between 56 and 91 units (milligrams per protein). NADP:malic enzyme represented up to 1% of the total soluble protein in C4 plants, 0.5% in the CAM plant, and less than 0.01% in C3 plants. In immunotitration tests involving immunoprecipitation and immunoinhibition of activity by an antiserum against the corn leaf enzyme, the NADP:malic enzymes of com and sugarcane shQwed virtually full identity of epitopes, while the NADP:malic enzymes of the C3 and CAM plants exhibited a cross-reaction of one-twentieth and one-fourth by these tests, respectively. The NADP:malic enzyme of P. grandlflora exhibited characteristics more closely related to the enzymes of C3 and CAM plants than to those of C4 plants.

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confidence: 99%

Purification by Immunoadsorption and Immunochemical Properties of NADP-Dependent Malic Enzymes from Leaves of C₃, C₄, and Crassulacean Acid Metabolism Plants

Fathi

Schnarrenberger²

1990

“…Unless otherwise specified the enzyme was assayed using the coupled assay method of Buicher (9). Each cuvette contained 10 umoles of ATP, 10 /moles of MgC12, 9 /,moles of 3-P-glycerate, 4.15 ,umoles of NADH, 50 jumoles of tris, HCl, pH 7.5, 100 /tg of glyceraldehyde-3-P dehydrogenase, and enzyme in a total volume of 1 ml. The rate of NADH disappearance was followed at 340 nm using a Gilford 2400 recording spectrophotometer.…”

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confidence: 99%

“…Determination of Nucleoside Triphosphate Specificity. Various ribonucleoside 5'-triphosphates were substituted for ATP in the assay which contained 10 tumoles of 3-P-glyceric acid, 0.1 Mmole of NADH, 50 tris-HCl, pH 7.5, 10 Mmoles of MgCl1, 100 jug of glyceraldehyde-3-P dehydrogenase, 2.5 ,umoles of either ATP, GTP, CTP, ITP, TIP, or UTP, and 3-P-glyceric acid kinase in a total volume of 1.0 ml. After measuring the activity with substituted nucleotides, 2.5 ,umoles of ATP (0.1 ml) were added into the reaction mixture to determine whether those nucleoside triphosphates had an inhibitory effect on the ATP-utilizing activity of 3-P-glyceric acid kinase.…”

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confidence: 99%

Chloroplast and Cytoplasmic Enzymes

Pacold¹,

Anderson²

1975

Pea (Pisum sativum) leaf chloroplastic and cytoplasmic 3-phosphoglycerate kinases (ATP: D-3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3) have similar Michaelis constants for ATP, 0.7 and 0.55 mM, for ADP, 0.18 and 0.22, and for 3-P-glycerate, 0.59 and 0.54 mM at low substrate concentrations, and 1.6 and 1.25 mM at high substrate concentrations. Both enzymes are inhibited by ADP and AMP in the ATP-utilizing direction and by ATP and AMP in the ATPgenerating direction and are controlled by energy charge. Apparently, whether the cytoplasmic and chloroplastic kinases in the plant cell will participate in the reductive pentose phosphate cycle and gluconeogenesis or in glycolysis will be determined by the environment in the cell compartment and not by the differential properties of the enzymes themselves.3-Phosphoglycerate kinase (ATP: D-3-phosphoglycerate 1-phosphotransferase, EC 2.7.2.3) catalyzes the reversible interconversion of 3-P-glycerate and 1,3-diP-glycerate with the concomitant utilization or generation of ATP. This enzyme participates in the reductive pentose phosphate cycle and in glycolysis and gluconeogenesis in green plants. Although the chloroplastic and cytoplasmic forms of the enzyme have been separated by isoelectric focusing (4), further characterization of the two forms has been lacking. The purpose of the experiments described here was to determine and compare the properties of the two forms of 3-P-glycerate kinase.The pea (Pisum sativum L.) chloroplastic and cytoplasmic 3-P-glycerate kinases have identical pH optima, with Pglycerate as substrate, and similar or identical Michaelis constants for ATP, ADP, and 3-P-glycerate. Both forms are inhibited by AMP and ADP and the Ki values differ by a factor of 1.5. The only naturally occurring nucleoside triphosphate which serves as a phosphate donor is ATP. The pea leaf 3-P-glycerate kinases are apparently very similar enzymes and also resemble in many, but not all, respects the enzymes from pea seeds (8), yeast (13)(14)(15), rabbit muscle (13), and the chemosynthetic bacterium Hydrogenomonas facilis (19).

Section: Introductionmentioning

confidence: 99%

“…In plants, two forms of this enzyme are known to occur and have important metabolic roles. The cytosolic form is thought to participate in the regulation of intracellular pH (3,20) and/or in the provision of reducing power that can be used in processes requiring NADPH (8 OPBM Radiochemical titration of sulfhydryl groups of malic enzyme preincubated with 0.2 mm OPBM in the presence and absence of 10 mm NADP was performed using the same conditions as described above. When 1.5 mg of purified malic enzyme modified by OPBM in the absence of NADP was inactivated to 20% of initial activity, the reaction mixture was passed twice through a small column of Sephadex G-50 (17), and 2.5 mm [3H]NEM was added.…”

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confidence: 99%

Evidence for the Existence of Two Essential and Proximal Cysteinyl Residues in NADP-Malic Enzyme from Maize Leaves

Drincovich

Spampinato²,

Andreo³

1992

Incubation of maize (Zea mays) leaf NADP-malic enzyme with monofunctional and bifunctional N-substituted maleimides results in an irreversible inactivation of the enzyme. Inactivation by the monofunctional reagents, N-ethylmaleimide (NEM) and N-phenylmaleimide, followed pseudo-first-order kinetics. The maximum inactivation rate constant for phenylmaleimide was 10-fold higher than that for NEM, suggesting a possible hydrophobic microenvironment of the residue(s) involved in the modification of the enzyme. In contrast, the inactivation kinetics with the bifunctional maleimides, ortho-, meta-, and para-phenylenebismaleimide, were biphasic, probably due to different reactivities of the groups reacting with the two heads of these bifunctional reagents, with a possible cross-linking of two sulfhydryl groups. plants such as maize. The enzyme plays a key role in C4 photosynthetic metabolism because it generates reducing power and CO2 in the bundle sheath chloroplasts where Rubisco and the Calvin cycle operate (7,8).Thiol groups play a key role in the activity of the enzyme obtained from different sources. The importance of sulfhydryl residues in pigeon liver malic enzyme has been investigated extensively (10). Studies of the inactivation of this enzyme with reagents selective for sulfhydryl groups, e.g. 5,5'-dithiobis(2-nitrobenzoic acid) or NEM2, confirmed the presence of one sulfhydryl group near each substrate site on the enzyme tetramer (24). In contrast, evidence for the presence of two essential sulfhydryl residues per monomer of the enzyme from maize leaves has been recently reported (4).To obtain further information concerning the function and microenvironment of the active site thiol group(s), the reaction of NADP-malic enzyme from maize leaves with different N-substituted maleimides was studied. Bifunctional maleimides were used to obtain further evidence about the existence of two proximal sulfhydryl groups near the NADP-binding site of the C4 enzyme (4). Several isomers of phenylenebismaleimide with different estimated cross-linking distances, as well as maleimides that differ in hydrophobic properties, were all found to modify the enzyme in an irreversible fashion.NADP-dependent malic enzyme (L-malate: NADP oxidoreductase/decarboxylase; EC 1.1.1.40) acts in a wide range of metabolic pathways in both animals and plants. The enzyme catalyzes the reversible oxidative decarboxylation of L-malate in the presence of NADP and a bivalent metal ion to produce pyruvate, NADPH, and CO2. In animals, cytosolic malic enzyme generates reducing power for the biosynthesis of fatty acids (9, 28). In plants, two forms of this enzyme are known to occur and have important metabolic roles. The cytosolic form is thought to participate in the regulation of intracellular pH (3,20) and/or in the provision of reducing power that can be used in processes requiring NADPH (8).The chloroplast stromal form is found specifically in the bundle sheath chloroplasts of NADP-malic enzyme type C4