6-Phosphogluconate Dehydrogenase Isoenzymes from the Developing Endosperm of Ricinus communis L

The cytosolic and proplastid isoenzymes of 6-phosphogluconate dehydrogenase were purified from the developing endosperm of the castor bean (Ricinis communis L.). No differences in physical or kinetic properties were found for the purified isoenzymes. Each was composed of two identical 55,000 subunits. They had identical pH optima of 7.8 to 8.0 and similar MgCl₂ stimulation for the oxidative decarboxylation of 6-phosphogluconate. The Km values for 6-phosphogluconate were 12 and 9.6 micromolar and for NADP⁺ were 4.1 and 5.4 micromolar for the cytosolic and proplastid isoenzymes, respectively. Therefore, the synthesis of two distinct 6-phosphogluconate dehydrogenase isoenzymes does not appear to have any kinetic significance for the developing seed. However, changes in the proplastid contribution toward carbohydrate metabolism occur in the developing seed and may necessitate independent gene expression to allow for a unique and flexible subcellular distribution of isoenzymes during development.     

Enzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinus communis L

The metabolism of sucrose to long chain fatty acids in the endosperm of developing castor bean (Ricinus communis L.) seeds requires a combination of cytosolic and proplastid enzymes. The total activity and the subcellular distribution of the intermediate enzymic steps responsible for the conversion of sucrose to pyruvate have been determined. Hexose phosphate synthesis from sucrose occurs in the cytosol along with the first oxidative step in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase. The proplastids contain the necessary complement of glycolytic enzymes to account for the in vivo rates of acetate synthesis from glucose 6-phosphate. These organelles also contain the majority of the cellular 6-phosphogluconate dehydrogenase, transketolase, and transaldolase activities.     

Isoenzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinis communis L

Two isoenzymes each of hexose-P isomerase, aldolase and 6-P-gluconate dehydrogenase have been found in the endosperm of developing castor beans (Ricinus communis L.). One isoenzyme for each activity is present in the proplastid fraction. Only one form of glucose-6-P dehydrogenase was found. It is suggested that the partition of an enzyme activity between cytosol and plastid is regulated by the synthesis of isoenzymes which are subcellular site specific. In addition, this report describes the use of diethylaminoethyl-Sephadex A-25 sievorptive chromatography for the preparation of plant enzymes.     

Orientation of electron transport activities in the membrane of intact glyoxysomes isolated from castor bean endosperm

Intact glyoxysomes were isolated from castor bean endosperm on isometric Percoll gradients. The matrix enzyme, malate dehydrogenase, was 80% latent in the intact glyoxysomes. NADH:ferricyanide and NADH:cytochrome c reductase activities were measured in intact and deliberately broken organelles. The latencies of these redox activities were found to be about half the malate dehydrogenase latency. Incubation of intact organelles with trypsin eliminated NADH:cytochrome c reductase activity, but did not affect NADH:ferricyanide reductase activity. NADH oxidase and transhydrogenase activities were negligible in isolated glyoxysomes. Mersalyl and Cibacron blue 3GA were potent inhibitors of NADH:cytochrome c reductase. Quinacrine, Ca²⁺ and Mg²⁺ stimulated NADH:cytochrome c reductase activity in intact glyoxysomes. The data suggest that some electron donor sites are on the matrix side and some electron acceptor sites are on the cytosolic side of the membrane.     

Localization and properties of ribulose diphosphate carboxylase from castor bean endosperm

A substantial portion of the ribulose 1,5-diphosphate carboxylase activity in the endosperm of germinating castor beans (Ricinus communis var. Hale) is recovered in the proplastid fraction. The partially purified enzyme shows homology with the enzyme from spinach (Spinacia oleracea) leaves, as evidenced by its reaction against antibodies to the native spinach enzyme and to its catalytic subunit. The enzyme from the endosperm of castor beans has a molecular weight of about 500,000 and, with the exception of a higher affinity for ribulose 1,5-diphosphate, has similar kinetic properties to the spinach enzyme. The castor bean carboxylase is inhibited by oxygen and also displays ribulose 1,5-diphosphate oxygenase activity with an optimum at pH 7.5.     

Uricase and allantoinase in glyoxysomes

In fat-degrading tissues of seedlings of seven different plant species examined, uricase activity (urate:O₂ oxidoreductase, EC 1.7.33) was associated with particulate fractions. After equilibrium density centrifugation on sucrose density gradients the enzyme activity was recovered in the glyoxysomal band (density: 1.25 grams per cubic centimeter). Allantoinase is also present in glyoxysomes but, equally, in the proplastid region (density: 1.22 grams per cubic centimeter). Xanthine oxidase, xanthine dehydrogenase, allantoicase, and urease were not detected in glyoxysomes from castor bean endosperm. Uricase in these particles shows its maximal activity at pH 8.9. The apparent K_m is 7.4 μm. Urate concentrations greater than 120 μm as well as certain other purine compounds inhibit the enzyme. Cyanide at a concentration of 10 μm is a potent inhibitor. 2,6-Dichlorophenolindophenol did not substitute for oxygen as electron acceptor.     

Glutamate dehydrogenase in developing endosperm, chloroplasts, and roots of castor bean

All the glutamate dehydrogenase activity in developing castor bean endosperm is shown to be located in the mitochondria. The enzyme can not be detected in the plastids, and this is probably not due to the inactivation of an unstable enzyme, since a stable enzyme can be isolated from castor bean leaf chloroplasts. The endosperm mitochondrial glutamate dehydrogenase consists of a series of differently charged forms which stain on polyacrylamide gel electrophoresis with both NAD⁺ and NADP⁺. The chloroplast and root enzymes differ from the endosperm enzyme on polyacrylamide gel electrophoresis. The amination reaction of all the enzymes is affected by high salt concentrations. For the endosperm enzyme, the ratio of activity with NADH to that with NADPH is 6.3 at 250 millimolar NH₄Cl and 1.5 at 12.5 millimolar NH₄Cl. K_m values for NH₄⁺ and NAD(P)H are reduced at low salt concentrations. The low K_m values for the nucleotides may favor a role for glutamate dehydrogenase in ammonia assimilation in some situations.     

Nicotinamide cofactors (NAD and NADP) in glyoxysomes,mitochondria, and plastids isolated from castor bean endosperm

Glyoxysomes, mitochondria, and plastids were separated from the cytosol of germinating castor bean endosperm by sucrose gradient centrifugation in a vertical rotor (25 min, 50,000g_av). The amounts of nicotinamide cofactors, NAD(H) and NADP(H), retained in the isolated organelle fractions were measured by enzyme cycling techniques. The NAD(H) was equally distributed between the cytosol and the mitochondria with a small amount in the glyoxysomes. The mitochondria retained 4 pmol of NAD(H)/ μg protein, about seven times as much as the glyoxysomes. Most of the NADP(H) was in the cytosol. However, the glyoxysomes and plastids retained significant amounts, both having 0.3 pmol NADP(H)/μg protein, twice that in the mitochondria. The subcellular distribution of NADP(H) was compared to the location of dehydrogenases capable of using this cofactor. The cytosol and plastids contained 6-phosphogluconate dehydrogenase. NADP isocitrate dehydrogenase was found in the glyoxysomes, in mitochondria, and in an unidentified subcellular fraction obtained at 1.16 g/ml in the density gradients. Knowledge of the quantities of NADP(H) and NAD(H) retained in the isolated organelles should make it possible to investigate their reduction and reoxidation in intact organelles.     

Changes in stability of isocitrate dehydrogenase (NADP+) during germination of castor bean seeds

In crude extract of castor bean endosperm, isocitrate dehydrogenase (NADP⁺) (EC 1.1.1.42) was stable at 57°C at the beginning of seed germination as well as in maturing and dry seeds. The enzyme gradually became less thermostable as germination proceeded and became unstable after 4 days. Extract from 5-day-old endosperm reduced the thermostability of the thermostable enzyme. The destabilizing factor accumulated in the endosperm as germination progressed and was identified as ricinoleate. Ricinoleate destabilized the purified enzyme which was stabilized by isocitrate and Mg²⁺, but ricinoleate did not affect the activity of NADP⁺-isocitrate dehydrogenase itself. Stearate, oleate, palmitate and myristate were similar to ricinoleate in their effect on the thermostability of the enzyme. The thermolabile enzyme in the crude extract of 5-day-old endosperm was readily inactivated by trypsin and in low concentrations of buffer. The thermostable enzyme in the crude extract of 2-day-old endosperm was not affected by these treatments. The thermostable enzyme treated with ricinoleate showed the same instabilities as the thermolabile enzyme. The role of ricinoleate in ther germinating castor bean endosperm is discussed.     

A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment

A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863–871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.). Received: 20 December 1997 / Accepted: 18 March 1998     

Increased hexokinase levels in endosperm of mutant maize

Hexokinase activity was measured in endosperms of shrunken-2 (sh₂) and starchy maize. Initial increases in hexokinase were observed for developing endosperms of both genotypes, and the enzyme declined in both as the seeds matured. A higher level of hexokinase was observed in developing sh₂ than in starchy endosperm. This difference persisted throughout maturation and occurred also in germinating seeds. Soluble hexokinase activity per endosperm continued to increase in sh₂ for about 8 days (22–30 days after pollination) after the enzyme in starchy endosperm had attained maximum activity and begun to decline. Hexokinase was predominantly soluble in both genotypes so the differences observed are not due to altered distribution of enzyme between particulate and soluble fractions.     

Characterization of the activity of fatty-acid epoxide hydrolase in seeds of castor bean (Ricinus communis L.)

Epoxide hydrolase (EC 3.3.2.3) activity was measured with [1-¹⁴C]cis-9,10-epoxystearic acid as the substrate. Homogenates were prepared from the endosperm tissue of germinating seeds of castor bean (Ricinus communis L. zanzibariensis). The activity of fatty-acid epoxide hydrolase was characterized with respect to dependence on time, amount of protein, pH and temperature. Analyses of enzyme distribution in endosperm, cotyledons, root and hypocotyl showed the highest total activity in the endosperm, less in the cotyledons and low activity in the root and hypocotyl. The specific activity was similar for cotyledons and endosperm. Analysis of the temporal expression of the enzyme in the endosperm during germination revealed high activity already in the imbibed seed. Activity was maximal between days four to six and then decreased at the end of one week. Subcellular fractionation of endosperm revealed a dual distribution of activity between the glyoxysomal and the cytosolic fractions.     

Low-potential cytochrome b as an essential electron-transport component of menaquinone reduction by formate in Vibrio succinogenes

Incorporation of the electron-transport enzymes of Vibrio succinogenes into liposomes was used to investigate the question of whether, in this organism, a cytochrome b is involved in electron transport from formate to fumarate on the formate side of menaquinone. (1) Formate dehydrogenase lacking cytochrome b was prepared by splitting the cytochrome from the formate dehydrogenase complex. The enzyme consisted of two different subunits (M_r 110 000 and 20 000), catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone by formate, and could be incorporated into liposomes. (2) The modified enzyme did not restore electron transport from formate to fumarate when incorporated into liposomes together with vitamin K-1 (instead of menaquinone) and fumarate reductase complex. In contrast, restoration was observed in liposomes that contained formate dehydrogenase with cytochrome b (E_m = ?224 mV), in addition to the subunits mentioned above (formate dehydrogenase complex). (3) In the liposomes containing formate dehydrogenase complex and fumarate reductase complex, the response of the cytochrome b of the formate dehydrogenase complex was consistent with its interaction on the formate side of menaquinone in a linear sequence of the components. The low-potential cytochrome b associated with fumarate reductase complex was not reducible by formate under any condition. It is concluded that the low-potential cytochrome b of the formate dehydrogenase complex is an essential component in the electron transport from formate to menaquinone. The low-potential cytochrome b of the fumarate reductase complex could not replace the former cytochrome in restoring electron-transport activity.     

Entwicklung und Struktur der Proplastiden

In this study the proplastid development in embryonic cells is described for the apical meristem of Elodea canadensis, embryo sacs from Lilies, and Begonia leaf buds. The formation of these cell organelles originates with submicroscopical particles which consist of a homogeneous stroma with a surrounding double membrane. When these proplastids reach an average size of 1 µ, the inner layer of the membrane begins to invaginate into the stroma. This process is comparable to tubuli formation in mitochondria. Under growth conditions with sufficient exposure to light, the development of the grana and stroma lamellae proceeds without interruption. If the plants are kept in the dark, small vesicles are formed which accumulate in the prolamellar body of the proplastids. After illumination these elementary vesicles merge to form membranes which evolve into grana and stroma lamellae. The structural similarity of the early proplastid stages with the mitochondria seems to indicate that there exists some phylogenetic relationship between the two cell organelles.     

Long chain fatty acid synthesis in developing castor bean seeds IV. The synthetic system in proplastids

The proplastid fraction containing no cytosol and mitochondrionwas isolated from developing castor bean endosperm by stepwisesucrose density centrifugation. This fraction possesses thecapacity to synthesize LFAs from [u-14C]sucrose, [u-¹⁴C]-glucose,[u-¹⁴C]G-1-P, [u-¹⁴C]G-6-P, [2-¹⁴C]pyruvate and [1-¹⁴C]acetate.Little was incorporated from [1-¹⁴C]pyruvate into LFAs, butmuch into ¹⁴CO_a. Addition of cytosol to the proplastid fractiondid not enhance the LFA synthesis. From these data, the wholepath from sucrose to LFAs through glycolytic path and pyruvatedecarboxylation seems to be located within the proplastid indeveloping castor bean endosperm. The difference in utilizationof substrates indicates that the rate of LFA synthesis in castorbean proplastids is limited at a step between sucrose and hexosephosphate. In addition, experiments with CO₂ output and LFAsynthesis from [1-¹⁴C]glucose, [6-¹⁴C]glucose and [u-¹⁴C]G-6-Pstrongly suggest that the path flow branches actively throughG-6-P to the pentose phosphate path and little through acetylCoAto the TCA cycle. (Received May 12, 1975; )     

Anaerobic stress in germinating castor bean, ethanol metabolism, and effects on subcellular organelles

Endosperms from castor beans (Ricinus communis) germinated for 0 to 6 days were exposed to anoxia for 0 to 15 hours. Ethanol, the only alcohol detected by gas chromatography in the tissue, accumulates to a concentration of 15 millimolar during the first 2 to 4 hours of anoxia and subsequently decreases. The absolute amount of ethanol varies from 10 micromoles per 5-day endosperm after 4 hours anoxia to less than 1 micromole in 2-day endosperm after 4 hours. Lactate content is 2 micromoles or less per endosperm. Alcohol dehydrogenase and pyruvate decarboxylase activities, which are localized in cytosolic fractions, are not greatly affected by anoxia. The recoveries of the marker enzymes and protein in endoplasmic reticulum (ER) and mitochondrial fractions decrease during anoxia. After 15 hours, the recovery of NADPH cytochrome c reductase is 15% of that in controls, fumarase is 50%, and catalase is 75%.

Glyoxysomes and ER are capable of converting ethanol to acetaldehyde which was measured using the fluorogenic reagent, 5,5-dimethyl-1,3-cyclohexanedione. The glyoxysomal activity is dependent on a hydrogen peroxide-generating substrate and the ER is dependent on NADPH. However, these activities are less than 3% of the alcohol dehydrogenase activity.

     

Capacity for NADPH/NADP turnover in the cytosol of barley seed endosperm: The role of NADPH-dependent hydroxypyruvate reductase

Barley (Hordeum vulgare L.) endosperm from developing seeds was found to contain relatively high activities of cytosolic NAD(P)H-dependent hydroxypyruvate reductase (HPR-2) and isocitrate dehydrogenase (ICDH). In contrast, activities of peroxisomal NADH-dependent hydroxypyruvate reductase (HPR-1) and glycolate oxidase as well as cytosolic NAD(P)H-dependent glyoxylate reductase were very low or absent in the endosperm both during maturation and seed germination, indicating the lack of a complete glycolate cycle in this tissue. In addition, activities of cytosolic glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase were low or absent in the endosperm. The endosperm HPR-2 exhibited similar properties to those of an earlier described HPR-2 from green leaves, e.g. activities with both hydroxypyruvate and glyoxylate, utilization of both NADPH and NADH as cofactors, and a strong uncompetitive inhibition by oxalate (K_i in the order of micromolar). In etiolated leaves, both HPR-1 and HPR-2 were present with the same activity as in green leaves, indicating that the lack of HPR-1 in the endosperm is not a general feature of non-photosynthetic tissues. We conclude that the endosperm has considerable capacity for cytosolic NADP/NADPH cycling via HPR-2 and ICDH, the former being possibly involved in the utilization of a serine-derived carbon.