Malic Enzyme Activity in Bacteroids from Soybean Nodules

Copeland, Les; Quinnell, Rosanne; Day, David A.

doi:10.1099/00221287-135-7-2005

Cited by 24 publications

(33 citation statements)

References 19 publications

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“…PEPC fixes the "C in the C-4 position of OAA. The obvious route to alanine via malic enzyme, which is known to be present in the bacteroids (6,9), would release the label from the C-4 position, generating unlabeled pyruvate and, assuming that alanine is formed by transamination of pyruvate, unlabeled alanine. f3-Decarboxylation of aspartate would also yield unlabeled alanine.…”

Section: Discussionmentioning

confidence: 99%

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with ¹⁴CO₂

Salminen¹,

Streeter²

1992

Plant Physiol.

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The aim of the work reported here was to ascertain that the patterns of labeling seen in isolated bacteroids also occurred in bacteroids in intact nodules and to observe early metabolic events following exposure of intact nodules to 4CO2. Intact nodules of soybean (Glycine max L. Merr. cv Ripley) inoculated with Bradyrhizobium japonicum USDA 110 and pea (Pisum sativum L. cv Progress 9) inoculated with Rhizobium leguminosarum bv viciae isolate 128C53 were detached and immediately fed 14CO2 for 1 to 6 min. Bacteroids were purified from these nodules in 5 to 7 min after the feeding period. In the cytosol from both soybean and pea nodules, malate had the highest radioactivity, followed by citrate and aspartate. In peas, asparagine labeling equaled that of aspartate. In B. japonicum bacteroids, malate was the most rapidly labeled compound, and the rate of glutamate labeling was 67% of the rate of malate labeling. Aspartate and alanine were the next most rapidly labeled compounds. R. leguminosarum bacteroids had very low amounts of 14C and, after a 1-min feeding, malate contained 90% of the radioactivity in the organic acid fraction. Only a trace of activity was found in aspartate, whereas the rate of glutamate and alanine labeling approached that of malate after 6 min of feeding. Under the conditions studied, malate was the major form of labeled carbon supplied to both types of bacteroids. These results with intact nodules confirm our earlier results with isolated bacteroids, which showed that a significant proportion of provided labeled substrate, such as malate, is diverted to glutamate. This supports the conclusion that microaerobic conditions in nodules influence carbon metabolism in bacteroids. Conclusions from studies of carbon uptake and metabolism by isolated bacteroids need to be confirmed with bacteroids in their natural environment, i.e. in the intact nodules. A noninvasive method to establish the form of carbon received by bacteroids would be to feed "4CO2 to the leaves and analyze the compounds labeled in the nodule and bacteroids.Results from experiments of this type (15,21,22) have yielded valuable information. However, due to the variation in rates of photosynthesis, translocation, and subsequent metabolism, the method does not allow clear resolution of the order of metabolic events in nodules.The problem can also be approached by taking advantage of high PEPC2 activity in the nodules (4, 16). This approach is biased to some degree because metabolism of labeled carbon begins at OAA and malate rather than sucrose (31). However, the malate pool in the cytosol of nodules is a relatively large carbon pool (29), and the fact that PEPC activity may be equivalent to as much as 14% of nodule respiration (1 1) makes it possible rapidly to label the relatively large pool. These facts combined with the established importance of dicarboxylic acids in bacteroid carbon nutrition make this an attractive approach for the study of carbon metabolism in intact nodules. The approach has been employed by others, b...

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Section: Discussionmentioning

confidence: 99%

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with ¹⁴CO₂

Salminen¹,

Streeter²

1992

Plant Physiol.

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“…The assays were started with the addition of extract. Potential interference from malate dehydrogenase activity was controlled for by leaving out MnCl2 from the initial reaction mixture (10). The activity of NADP-malic enzyme of "leaky organelles" in the leucoplast fraction was also determined.…”

Section: Malic Enzyme Assaymentioning

confidence: 99%

“…Enolase activity was measured using an enzymatic-coupled assay and a dual wavelength spectrophotometer (Sigma ZFP22). The assay mixture contained 50 mM Tes (pH 7.5), 0.4 M sorbitol, 10 ,uL) and the absorbance at 340 nm was followed. The proportion of "leaky" leucoplasts was taken as the ratio of the initial enolase activity to that following the disruption of the leucoplasts by adding 10 10 min, pelleted, and resuspended in 0.1 N NaOH.…”

Section: Determination Of Leucoplast Intactnessmentioning

confidence: 99%

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Malate- and Pyruvate-Dependent Fatty Acid Synthesis in Leucoplasts from Developing Castor Endosperm

Smith¹,

Gauthier²,

Dennis³

et al. 1992

Plant Physiol.

155

102

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Leucoplasts were isolated from the endosperm of developing castor (Ricinis communis) endosperm using a discontinuous Percoil gradient. The rate of fatty acid synthesis was highest when malate was the precursor, at 155 nanomoles acetyl-CoA equivalents per milligram protein per hour. Pyruvate and acetate also were precursors of fatty acid synthesis, but the rates were approximately 4.5 and 120 times less, respectively, than when malate was the precursor. When acetate was supplied to leucoplasts, exogenous ATP, NADH, and NADPH were required to obtain maximal rates of fatty acid synthesis. In contrast, the incorporation of malate and pyruvate into fatty acids did not require a supply of exogenous reductant. Further, the incorporation of radiolabel into fatty acids by leucoplasts supplied with radiolabeled malate, pyruvate, or acetate was reduced upon coincubation with cold pyruvate or malate. The data suggest that malate and pyruvate may be good in vivo sources of carbon for fatty acid synthesis and that, in these preparations, leucoplast fatty acid synthesis may be limited by activity at or downstream of the acetyl-CoA carboxylase reaction.or leucoplast glycolytic enzymes are more than adequate to account for the triacylglycerol synthesis of castor endosperm (17). However, glycolytic intermediates have not been well characterized as carbon sources for fatty acid synthesis (18). In this study, we present data regarding the kinetics and exogenous cofactor requirements for fatty acid synthesis in isolated leucoplast preparations using pyruvate, malate, and acetate as carbon sources. We show that malate and pyruvate support much higher rates of fatty acid synthesis than does acetate and the metabolism of both these substrates alleviates any requirement for externally added reductant. Based on the measurement of leucoplast-associated NADP+-malic enzyme activity, we suggest that cytosolic malate may be a source of carbon for fatty acid synthesis in vivo. MATERIALS AND METHODS Plant MaterialCastor plants (Ricinus communis L. cv Baker 296) were greenhouse grown under natural light supplemented with 16 h fluorescent light.Storage lipids make up 45 to 50% of the dry weight of castor bean (Ricinus communis) endosperm (9, 15). During development, the conversion of sucrose to triacylglycerols is the major metabolic activity of this tissue (9). Dennis (11) presented a model indicating the possible pathways of triacylglycerol synthesis from sucrose. The model is primarily based on enzyme compartmentation studies (12,17,22,23,27) and suggests that glycolytic intermediates may cross the leucoplast membrane and serve as carbon skeletons for fatty acid synthesis. Acetate is another possible precursor.Acetate incorporation into fatty acids has been used to study the cofactor requirements for fatty acid biosynthesis (6,16,18,25,26). It is unlikely that acetate is the in vivo carbon precursor of fatty acid synthesis because of its relatively low cellular concentration and the low efficiency of acetate utilization for de novo fat...

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“…In this respect, the partial purification of NADP-ME in B. japaonicum J50l from soybean nodules has been reported by this laboratory.5) Some properties of MEs in B. japonicum USDAIIO from soybean nodules have also been reported by Copeland el al. 6) In the present study, NADP-ME was first purified to homogeneity from B. japonicum AlO17, which had high efficiency for nitrogen fixation, and a detailed survey of the molecular characteristics became possible. The enzyme characteristics were surveyed to elucidate the role of this enzyme in nitrogen-fixing nodules, and the N-terminal amino acid sequence was analyzed for gene cloning.…”

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confidence: 99%

Purification and Characterization of the NADP-Malic Enzyme fromBradyrhizobium japonicumA1017

Fan

Okabe

Osano

et al. 1997

Bioscience, Biotechnology, and Biochemistry

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Malic Enzyme Activity in Bacteroids from Soybean Nodules

Cited by 24 publications

References 19 publications

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with ¹⁴CO₂

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with ¹⁴CO₂

Malate- and Pyruvate-Dependent Fatty Acid Synthesis in Leucoplasts from Developing Castor Endosperm

Purification and Characterization of the NADP-Malic Enzyme fromBradyrhizobium japonicumA1017

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Malic Enzyme Activity in Bacteroids from Soybean Nodules

Cited by 24 publications

References 19 publications

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with 14CO2

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with 14CO2

Malate- and Pyruvate-Dependent Fatty Acid Synthesis in Leucoplasts from Developing Castor Endosperm

Purification and Characterization of the NADP-Malic Enzyme fromBradyrhizobium japonicumA1017

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Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with ¹⁴CO₂

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with ¹⁴CO₂