Inger Carlberg

[59] Glutathione reductase

Publisher Summary Glutathione reductase is a flavoprotein catalyzing the NADPH-dependent reduction of glutathione disulfide (GSSG) to glutathione (GSH). The reaction is essential for the maintenance of glutathione levels. Glutathione has a major role as a reductant in oxidation–reduction processes, and serves in detoxication and several other cellular functions of great importance. A purification method of this enzyme from calf liver and rat liver is described in this chapter. Similar methods are used for the purification of the enzyme from yeast, porcine, and human erythrocytes. All the steps are carried out at about 5 ° . The purification method from calf liver consists of various steps including preparation of cytosol fraction, chromatography on DEAE-sephadex, precipitation with ammonium sulfate, and chromatography on hydroxyapatite. The purification of glutathione reductase from rat liver is usually combined with the preparation of glutathione transferases, thioltransferase, and glyoxalase I.

Phosphorylation of Light-harvesting Complex II and Photosystem II Core Proteins Shows Different Irradiance-dependent Regulation in Vivo APPLICATION OF PHOSPHOTHREONINE ANTIBODIES TO ANALYSIS OF THYLAKOID PHOSPHOPROTEINS

An immunological approach using a polyclonal phosphothreonine antibody is introduced for the analysis of thylakoid protein phosphorylation in vivo. Virtually the same photosystem II (PSII) core phosphoproteins (D1, D2, CP43, and thepsbH gene product) and the light-harvesting chlorophylla/b complex II (LHCII) phosphopolypeptides (LHCB1 and LHCB2), as earlier identified by radiolabeling experiments, were recognized in both pumpkin and spinach leaves. Notably, the PSII core proteins and LHCII polypeptides were found to have a different phosphorylation pattern in vivo with respect to increasing irradiance. Phosphorylation of the PSII core proteins in leaf discs attained the saturation level at the growth light intensity, and this level was also maintained at high irradiances. Maximal phosphorylation of LHCII polypeptides only occurred at low light intensities, far below the growth irradiance, and then drastically decreased at higher irradiances. These observations are at variance with traditional studies in vitro, where LHCII shows a light-dependent increase in phosphorylation, which is maintained even at high irradiances. Only a slow restoration of the phosphorylation capacity for LHCII polypeptides at the low light conditions occurred in vivo after the high light-induced inactivation. Furthermore, if thylakoid membranes were isolated from the high light-inactivated leaves, no restoration of LHCII phosphorylation took place in vitro. However, both the high light-induced inactivation and low light-induced restoration of LHCII phosphorylation seen in vivo could be mimicked in isolated thylakoid membranes by incubating with reduced and oxidized dithiothreitol, respectively. We propose that stromal components are involved in the regulation of LHCII phosphorylation in vivo, and inhibition of LHCII phosphorylation under increasing irradiance results from reduction of the thiol groups in the LHCII kinase.

In vitro studies on light-induced inhibition of Photosystem II and D1-protein degradation at low temperatures

Abstract In order to get information on the molecular background behind the aggrevated photodamage to photosynthesis at low temperatures and to investigate the general mechanism of D1-protein degradation, isolated spinach thylakoids were subjected to photoinhibitory treatment at various temperatures. The results reveal that: (i) the Photosystem II electron transport per se is less sensitive to high light at low temperatures in contrast to the overall photosynthetic process; (ii) the degradation of D1-protein is severely retarded below 7°C; (iii) inhibition of Photosystem II electron transport and D1-protein degradation are separate events the two reactions could be completely separated in time; (iv) D1-protein is degraded by enzymatic proteolysis and not by a direct photocleavage reaction; (v) degradation of the D1-protein readily proceeds in the dark but its triggering for the proteolytic attack requires light; (vi) strong illumination at low temperature does not induce any lateral rearrangement in the location of Photosystem II; and (vii) D1-protein fragments can be identified in vitro and be used to verify the specificity of D1-protein degradation under various experimental conditions.

A novel plant protein undergoing light-induced phosphorylation and release from the photosynthetic thylakoid membranes

The characteristics of a phosphoprotein with a relative electrophoretic mobility of 12 kDa have been unknown during two decades of studies on redox-dependent protein phosphorylation in plant photosynthetic membranes. Digestion of this protein from spinach thylakoid membranes with trypsin and subsequent tandem nanospray-quadrupole-time-of-flight mass spectrometry of the peptides revealed a protein sequence that did not correspond to any previously known protein. Sequencing of the corresponding cDNA uncovered a gene for a precursor protein with a transit peptide followed by a strongly basic mature protein with a molecular mass of 8,640 Da. Genes encoding homologous proteins were found on chromosome 3 of Arabidopsis and rice as well as in ESTs from 20 different plant species, but not from any other organisms. The protein can be released from the membrane with high salt and is also partially released in response to light-induced phosphorylation of thylakoids, in contrast to all other known thylakoid phosphoproteins, which are integral to the membrane. On the basis of its properties, this plant-specific protein is named thylakoid soluble phosphoprotein of 9 kDa (TSP9). Mass spectrometric analyses revealed the existence of non-, mono-, di-, and triphosphorylated forms of TSP9 and phosphorylation of three distinct threonine residues in the central part of the protein. The phosphorylation and release of TSP9 from the photosynthetic membrane on illumination favor participation of this basic protein in cell signaling and regulation of plant gene expression in response to changing light conditions.

Restoration of light induced photosystem II inhibition without de novo protein synthesis

Illumination of isolated spinach thylakoid membranes under anaerobic conditions gave rise to severe inhibition of photosystem II electron transport but did not result in D1‐protein degradation. When these photoinhibited thylakoids were incubated in total darkness the photosystem II activity could be fully restored in vitro in a process that required 1–2 h for completion.

Purification and characterization of glutathione reductase from calf liver. An improved procedure for affinity chromatography on 2',5'-ADP-Sepharose 4B.

Abstract Glutathione reductase has been purified to at least 98% homogeneity from calf liver. An essential part in the procedure involves affinity chromatography on 2′,5′-ADP-Sepharose 4B to which the enzyme remains bound in the presence of 0.4 m phosphate. This step separates glutathione reductase from the closely related thioredoxin reductase. Some of the physical and catalytic properties as well as the amino acid composition of the enzyme are reported.

Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c-Fo subunit P1 isoform

Despite the significance of mitochondrial ATP synthase for mammalian metabolism, the regulation of the amount of ATP synthase in mammalian systems is not understood. As brown adipose tissue mitochondria contain very low amounts of ATP syn‐thase, relative to respiratory chain components, they constitute a physiological system that allows for examination of the control of ATP synthase assembly. To examine the role of the expression of the P1‐isoform of the c‐Fo subunit in the biogenesis of ATP synthase, we made transgenic mice that express the P1‐c subunit isoform under the promoter of the brown adipose tissue‐specific protein UCP1. In the resulting UCPlpl transgenic mice, total P1‐c subunit mRNA levels were increased;mRNA levels of other F1Fo‐ATPase subunits were unchanged. In isolated brown‐fat mitochondria, protein levels of the total c‐Fo subunit were increased. Remarkably, protein levels of ATP synthase subunits that are part of the F1‐ATPase complex were also increased, as was the entire Complex V. Increased ATPase and ATP synthase activities demonstrated an increased functional activity of the F1Fo‐ATPase. Thus, the levels of the c‐Fo subunit P1‐isoform are crucial for defining the final content of the ATP synthase in brown adipose tissue. The level of c‐Fo subunit may be a determining factor for F1Fo‐ATPase assembly in all higher eukaryotes.— Kramarova T. V., Shabalina, I. G., Andersson, U., Westerberg, R., Carlberg, I., Houstek, J., Nedergaard, J., Cannon B. Mitochondrial ATP synthase levels in brown adipose tissue are governed by the c‐Fo subunit P1 isoform. FASEB J. 22, 55–63 (2008)

Phosphatase activities in spinach thylakoid membranes-effectors, regulation and location.

The dephosphorylation of seven phosphoproteins associated with Photosystem II or its chlorophyll a/b antenna in spinach thylakoids, was characterised. The rates were found to fall into two distinct groups. One, rapidly dephosphorylated, consisted of the two subunits (25 and 27 kD) of the major light harvesting complex of Photosystem II (LHC II) and a 12 kD polypeptide of unknown identity. A marked correlation between the dephosphorylation of these three phosphoproteins, strongly suggested that they were all dephosphorylated by the same enzyme. Within this group, the 25 kD subunit was consistently dephosphorylated most rapidly, probably reflecting its exclusive location in the peripheral pool of LHC II. The other group, only slowly dephosphorylated, included several PS II proteins such as the D1 and D2 reaction centre proteins, the chlorophyll-a binding protein CP43 and the 9 kD PS II-H phosphoprotein. No dephosphorylation was observed in either of the two groups in the absence of Mg2+-ions. Dephosphorylation of the two LHC II subunits took place in both grana and stroma-exposed regions of the thylakoid membrane. However, deposphorylation in the latter region was significantly more rapid, indicating a preferential dephosphorylation of the peripheral (or ‘mobile’) LHC II. Dephosphorylation of LHC II was found to be markedly affected by the redox state of thiol-groups, which may suggest a possible regulation of LHC II dephosphorylation involving the ferredoxin-thioredoxin system.

Intrinsically unstructured phosphoprotein TSP9 regulates light harvesting in Arabidopsis thaliana.

Thylakoid-soluble phosphoprotein of 9 kDa, TSP9, is an intrinsically unstructured plant-specific protein [Song, J., et al. (2006) Biochemistry 45, 15633-15643] with unknown function but established associations with light-harvesting proteins and peripheries of both photosystems [Hansson, M., et al. (2007) J. Biol. Chem. 282, 16214-16222]. To investigate the function of this protein, we used a combination of reverse genetics and biochemical and fluorescence measurement methods in Arabidopsis thaliana. Differential gene expression analysis of plants with a T-DNA insertion in the TSP9 gene using an array of 24000 Arabidopsis genes revealed disappearance of high light-dependent induction of a specific set of mostly signaling and unknown proteins. TSP9-deficient plants had reduced levels of in vivo phosphorylation of light-harvesting complex II polypeptides. Recombinant TSP9 was phosphorylated in light by thylakoid membranes isolated from the wild-type and mutant plants lacking STN8 protein kinase but not by the thylakoids deficient in STN7 kinase, essential for photosynthetic state transitions. TSP9-lacking mutant and RNAi plants with downregulation of TSP9 showed reduced ability to perform state transitions. The nonphotochemical quenching of chlorophyll fluorescence at high light intensities was also less efficient in the mutant compared to wild-type plants. Blue native electrophoresis of thylakoid membrane protein complexes revealed that TSP9 deficiency increased relative stability of photosystem II dimers and supercomplexes. It is concluded that TSP9 regulates plant light harvesting acting as a membrane-binding protein facilitating dissociation of light-harvesting proteins from photosystem II.

Effect of inducers of drug-metabolizing enzymes on glutathione reductase and glutathione peroxidase in rat liver

Cytosolic glutathione reductase activity of rat liver was shown to increase to about 250% of control values after treatment of the animals by intraperitoneal injections of trans-stilbene oxide. The time course and dose-response relationship of the induction brought about by trans-stilbene oxide were determined. The increase of activity was accompanied by a similar increase of protein precipitable by antibodies to rat liver glutathione reductase. These results strongly indicate that increase of glutathione reductase activity in response to treatment with this inducer is the result of true induction. Phenobarbital and 3-methylcholanthrene increased the activities per mg protein in the cytosol fraction by 80 and 24%, respectively. Glutathione reductase was purified to homogeneity from rats treated with trans-stilbene oxide. The molecular and kinetic properties investigated were not significantly different from those of the enzyme from control animals. Selenium-dependent glutathione peroxidase was not induced in the hepatic cytosol of animals treated with trans-stilbene oxide.