A 2,4-D-inducible glutathione S-transferase from soybean (Glycine max). Purification, characterisation and induction

Flury, Thomas; Adam, Daniel; Kreuz, Klaus

doi:10.1034/j.1399-3054.1995.940219.x

Cited by 10 publications

(18 citation statements)

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“…In all previous determinations of kinetic parameters for purified plant GSTs, initial-velocity data have been analyzed using the standard Michaelis-Menton equation for a unireaction (O'Connell et al, 1988;Williamson and Beverley, 1988;Singhal et al, 1991;Irzyk and Fuerst, 1993;Droog et al, 1995;Flury et al, 1995). However, kinetic models describing bireactant mechanisms are better suited for the determination of kinetic constants for GSTs, which utilize two substrates.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of GlutathioneS-Transferase Isozymes from Sorghum1

1998

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Two glutathione S-transferase (GST) isozymes, A1/A1 and B1/B2, were purified from etiolated, O-1,3-dioxolan-2-yl-methyl-2,2,2,-trifluoro-4 -chloroacetophenone-oxime-treated sorghum (Sorghum bicolor L. Moench) shoots. GST A1/A1, a constitutively expressed homodimer, had a subunit molecular mass of 26 kD and an isoelectric point of 4.9. GST A1/A1 exhibited high activity with 1-chloro-2, 4,dinitrobenzene (CDNB) but low activity with the chloroacetanilide herbicide metolachlor. For GST A1/A1, the random, rapidequilibrium bireactant kinetic model provided a good description of the kinetic data for the substrates CDNB and glutathione (GSH). GST B1/B2 was a heterodimer with subunit molecular masses of 26 kD (designated the B1 subunit) and 28 kD (designated the B2 subunit) and a native isoelectric point of 4.8. GST B1/B2 exhibited low activity with CDNB and high activity with metolachlor as the substrate. The kinetics of GST B1/B2 activity with GSH and metolachlor fit a model describing a multisite enzyme having two binding sites with different affinities for these substrates. Both GST A1/A1 and GST B1/B2 exhibited GSH-conjugating activity with ethacrynic acid and GSH peroxidase activity with cumene hydroperoxide, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid and 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid. Both GST A1/A1 and GST B1/B2 are glycoproteins, as indicated by their binding of concanavalin A. Polyclonal antibodies raised against GST A1/A1 exhibited cross-reactivity with the B1 subunit of GST B1/B2. Comparisons of the N-terminal amino acid sequences of the GST A1, B1, and B2 subunits with other type I -GSTs indicated a high degree of homology with the maize GST I subunit and a sugarcane GST.GSTs (EC 2.5.1.18) are dimeric enzymes found in mammals, insects, plants, and microbes that catalyze nucleophilic attack by the thiolate anion of GSH at electrophilic centers of hydrophobic molecules (Mannervik and Danielson, 1988). In addition to catalyzing GSH conjugation, GSTs also exhibit GSH peroxidase activity and ligandbinding functions (Mannervik and Danielson, 1988;Marrs, 1996). Mammalian GSTs compose a multigene family; in rat liver at least 13 different cytosolic GST subunits are found as either heterodimers or homodimers . Mammalian cytosolic GSTs have been divided into four classes (␣, , , and ) based on immunological, biochemical, and sequence similarities (Buetler and Eaton, 1992). It is well established that mammalian GSTs play an important role in the detoxification of electrophilic xenobiotics (Mannervik and Danielson, 1988). Although endogenous substrates for mammalian GSTs have not been clearly defined, there is evidence that ␣-GSTs protect against oxidative stress by detoxifying reactive products generated by lipid peroxidation (Ålin et al., 1985;Ketterer and Coles, 1991;Singhal et al., 1992).In general, plant GSTs have not been as well characterized as mammalian GSTs. Plant cytosolic GSTs belong to the archaic class of GSTs (Meyer et al., 1991;Marrs, 1996). This class, which is very heterog...

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Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of GlutathioneS-Transferase Isozymes from Sorghum1

1998

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“…Enzyme purification was performed as previously described (Flury et al, 1995) with minor modifications. In brief, hypocotyl tissue was pulverized under liquid nitrogen with a mortar and pestle.…”

Section: Purification Of Cstmentioning

confidence: 99%

“…For one-dimensional SDS-PAGE, tissue samples were extracted with electrophoresis sample buffer and subjected to SDS-PAGE in 12% acrylamide gels as previously described (Flury et al, 1995), using the buffer system of Laemmli (1970). For two-dimensional PAGE, protein was extracted by the method of Barent and Elthon (1992) and subjected to first-dimension IEF according to the procedure of OFarrell (1975) but using 3-[(3-~holamidopropyl)dimethylammoniol-1-propanesulfonate as the detergent and piperazine diacrylamide instead of bis-acrylamide.…”

Section: Electrophoresis and Lmmunoblottingmentioning

confidence: 99%

An Inducible Glutathione S-Transferase in Soybean Hypocotyl Is Localized in the Apoplast

Flury¹,

Wagner²,

Kreuz³

1996

Plant Physiology

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Clutathione S-transferases (CSTs) with additional activities as fatty acid hydroperoxidases were investigated in soybean (Glycine max 1.) hypocotyls. Aside from the GSTs present in total soluble tissue extracts, enzyme activities and distinct immunoreactive GST polypeptides were also detected in the intercellular washing fluid. Whereas the intracellular isoenzymes were both constitutive and inducible, apoplastic CST and glutathione peroxidase was detectable only in tissues treated with the known GST inducer 2,3,5-triiodobenzoic acid. Monensin inhibited the induced accumulation of apoplastic CST but did not affect the intracellular isoforms. The discovery of apoplastic inducible CST will be discussed in light of the putative function of these enzymes in plants.The GSTs (EC 2.5.1.18) are a family of proteins with severa1 activities (Wilce and Parker, 1994;Marrs, 1996). GSTs catalyze the nucleophilic attack of the thiol of GSH to electrophilic substrates, typically resulting in the formation of GSH conjugates. GSTs are usually dimeric proteins with subunit molecular masses of 24 to 30 kD; they are organized in gene families that produce multiple isoenzymes in eukaryotic organisms. GSTs are mostly soluble cytoplasmic enzymes, but microsomal isoforms are also known in both plants and animals. GSTs conjugate GSH to various xenobiotics, e.g. drugs and pesticides, which is often a key step in their metabolic detoxification and elimination from the cytoplasm (Lamoureux and Rusness, 1989;Kreuz et al., 1996;Reinemer et al., 1996).The multitude of GST isoenzymes in plants and their inducibility by diverse biotic and abiotic factors has been the primary focus of research in recent years. Yet, knowledge about the physiological substrate(s) and function(s) of plant GSTs is only slowly beginning to emerge. Thus, GSTs have been implicated in the conjugation of cinnamic acid and of anthocyanins and in the binding of IAA (Edwards and Dixon, 1991;Marrs, 1996). Some GST isoenzymes display additional activities; for example, the selenium-independent GSH peroxidases catalyze the reduction of fatty acid hydroperoxides with concomitant formation of GSSG (Bartling et al., 1993). Such hydroperoxides are formed by the action of active oxygen species that are generated both as normal by-products of aerobic metabolism and as the result of pathogen infection or exposure to certain abiotic * Corresponding author; e-mail klaus-eugen.kreuz@chbs.mhs. ciba.com; fax 41-61-697-8455.agents. Organic hydroperoxides are potentially cytotoxic, and their removal by GSH peroxidase activity has thus been implicated in the protection of tissues against oxidative stress (Ketterer et al., 1990).In this report we describe the isolation and characterization of GSTs from soybean (Glycine max L.) that display high GSH peroxidase activity toward hydroperoxides of linolenic acid and arachidonic acid. In addition to the soluble intracellular isoenzymes, GST and GSH peroxidase were also found in the intercellular washing fluid of hypocotyls. These apoplastic enzyme ac...

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“…These protective enzymes, however, cannot catabolize lipid-hydroperoxides directly. Another enzyme, GTase, which also exhibits POX activity (Ketterer et al, 1990;Bartling et al, 1993) and is inducible by a wide range of environmental stress conditions (Flury et al, 1995), efficiently catabolizes lipidhydroperoxides that form during the propagation phase of lipid peroxidation (Haenen, 1989).…”

Section: Discussionmentioning

confidence: 99%

“…Age-enhanced, free-radical-mediated lipid peroxidation and increases in activities of enzymes that catabolize free radicals (SOD, POX, catalase, GTase) are not necessarily mutually exclusive processes. These enzymes are substrate-inducible (Bowler et al, 1989;Edreva et al, 1989;Flury et al, 1995), and higher activities are indicative of higher rates of production of superoxide and H 2 O 2 in tissues. Similarly, in the absence of increased enzyme per unit of cytosolic protein, higher GTase activity reflects a greater availability of lipid-hydroperoxide substrate.…”

Section: Sds-pagementioning

confidence: 99%

Oxidative Stress Results in Increased Sinks for Metabolic Energy during Aging and Sprouting of Potato Seed-Tubers

Kumar

Knowles

1996

Plant Physiology

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Glutathione-mediated free-radical-scavenging and plasma membrane ATPase activities increase as sinks for metabolic energy with advancing tuber age. Plasma membrane ATPase activity from 19-month-old tubers was 77% higher than that from 7-month-old tubers throughout sprouting. The higher activity was not attended by an increase in the amount of ATPase per unit plasma membrane protein.Concentrations of oxidized (CSSC) and reduced glutathione more than doubled as tuber age advanced from 6 to 30 months, but the proportion of GSSG to total glutathione remained constant with age. l h e activity of glutathione transferase, an enzyme that catabolizes lipidhydroperoxides, increased by 44 and 205% on a fresh weight and protein basis, respectively, as tubers aged from 6 to 30 months. Glutathione reductase activity also increased with advancing age, by 90% on a fresh weight basis and 305% on a protein basis. Older tubers had more glutathione reductase per unit of soluble and mitochondrial protein. The age-induced increase in cytosolic glutathione transferase activity was likely due to increased availability of lipid-hydroperoxides and/or a positive effector. Synthesis of glutathione requires ATP, and the increased reduction of CSSC resulting from catalysis of lipidhydroperoxides is NADPH-dependent. Thus, increased plasma membrane ATPase and glutathione-mediated free-radical-scavenging activities likely constitute substantial sinks for ATP in older tubers prior to and during sprouting. lncreased oxidative stress and loss in membrane integrity are central features of aging that undoubtedly contribute to the enhanced respiration of sprouting older tubers.Potato (Solanum tuberosum L.) seed-tubers provide a model system for studies of the metabolic processes associated with aging of plant tissues in general and vegetative propagules in particular. Russet Burbank seed-tubers maintain their viability for about 3 years in cold storage (4"C, 95% RH); however, vigor and growth potential of tuber meristems depend on age. As tubers advance in age beyond about 7 months, apical dominance and plant vigor gradually decline, resulting in the production of many shoots per tuber, each with low specific leaf area and sparse root systems (Mikitzel and Knowles, 1990; Kumar and Knowles, 1993a). Paradoxically, older tubers have substantially higher rates of fully coupled, Cyt-mediated respiration than younger tubers at similar stages of sprout development (Kumar and Knowles, 1996). This greater ATP production by sprouting older tubers is required to '

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A 2,4-D-inducible glutathione S-transferase from soybean (Glycine max). Purification, characterisation and induction

Cited by 10 publications

References 0 publications

Isolation and Characterization of GlutathioneS-Transferase Isozymes from Sorghum1

Isolation and Characterization of GlutathioneS-Transferase Isozymes from Sorghum1

An Inducible Glutathione S-Transferase in Soybean Hypocotyl Is Localized in the Apoplast

Oxidative Stress Results in Increased Sinks for Metabolic Energy during Aging and Sprouting of Potato Seed-Tubers

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