Microsomal glutathione transferase : catalysis, in vitro mutagenesis and heterologous expression

Microsomal glutathione transferase is a membrane-bound member of the glutathione transferases, a family of multifunctional enzymes involved in the cellular detoxification of xenobiotics and reactive endogenous compounds formed during oxidative stress. Rat liver microsomal glutathione transferase is a trimeric protein with a molecular mass of 17.3 kDa per subunit which displays unique features regarding DNA and amino acid sequence, molecular weight, enzymatic properties and ability of activation by various agents as compared to its cytosolic counterparts. At the outset of this work we were interested in obtaining: specific substrates and expression systems for the enzyme. These experimental tools combined with in vitro mutagenesis were used to obtain information on the functional role of individual amino acids as well as membrane topology. N-acetyl-L-cysteine was found to serve as a substrate for the microsomal glutathione transferase (with l-chloro-2,4-dinitrobenzene (CDNB) as second substrate). In examining the activity of liver subcellular fractions, no activity with N-acetyl-L-cysteine could be detected in cytosols devoid of endogenous glutathione. Thus, N-acetyl-L-cysteine is a specific substrate for microsomal glutathione transferase. The pH dependence of kCat/Km(CDNB) for the microsomal glutathione transferase with different thiol substrates indicates that the enzyme has the ability to lower the pKa of bound glutathione by 3 orders of magnitude. The microsomal glutathione transferase stabilizes Meisenheimer complex formation between 1,3,5-trinitrobenzene and various glutathione analogues, including some non-substrate thiols, thus offering new possibilities for examining ligand interactions of glutathione transferases. Rat liver microsomal glutathione transferase was successfully expressed both in mammalian COS- cells and in E. Coli BL 21 (DE3). Significant amounts of enzymatically active protein was expressed in the inner membrane of this E. Coli strain. Recombinant rat microsomal glutathione transferase was purified from bacterial membranes and was found to be indistinguishable from the liver enzyme with regard to enzymatic activity, molecular mass, immunoreactivity and N-terminal amino acid sequence. Chemical modification of rat liver microsomal glutathione transferase indicated that arginine 107 and Iysine 67 are essential for enzyme activity and may thus reside in the active site. A set of mutant forms of the rat enzyme were constructed by site-directed mutagenesis and heterologously expressed in E. coli BL21(DE3). Arginine 107 was exchanged for alanine and Iysine residues. The alanine mutant (R107A) displayed a decreased thermostability and an important structural role is suggested for this residue. Neither mutation of Iysine 67 to alanine and arginine nor replacement of the three histidines by glutamines yielded any drastic changes of activity in contrast to the chemical modification experiments. All tyrosine to phenylalanine substitutions resulted in mutants with activities similar to the wild type. Thus, the microsomal glutathione transferase must perform an alternate stabilization of the thiolate anion of glutathione than through interaction with a phenolic hydroxyl group of a tyrosine. Substitution of cysteine 49 with alanine resulted in a semi-activated mutant enzyme which was not affected by N- ethylmaleimide. Cysteine 49 is therefore unambiguously demonstrated as the site of modification that results in activation of microsomal glutathione transferase. The membrane topology of rat liver microsomal glutathione transferase was investigated by in situ proteolysis of intact and permeabilized rat liver microsomes. Lysine 4 is accessible at the luminal surface of the endoplasmic reticulum, whereas Iysine 41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids which comprises a likely membrane-spanning region. The cytosolic location of the active site was demonstrated using radiolabeled glutathione. Additional membrane anchoring(s) are indicated since the C-terminal part of the trypsin-cleaved protein was not separated from the membrane fraction by intensive salt washing or phase separation in Triton X-l 14. Cleavage of the purified protein at Iysine 41 and subsequent separation of the fragments yielded a catalytically competent C-terminal polypeptide. The topology of recombinant rat microsomal glutathione transferase expressed in E. coli was investigated by comparing the proteolytic cleavage products from intact and permeabilized spheroplasts. Tryptic cleavage at Iysine 4 in intact spheroplasts shows that this residue is directed towards the periplasmic side, whereas Iysine 41 faces the inside of the E. Coli inner membrane. Intact spheroplasts treated with pronase yielded a cleavage pattern consistent with two additional C-terminal sites exposed to the periplasmic side indicating a polytopic membrane association of microsomal glutathione transferase . Doctoral Thesis c 1996 Rolf Weinander ISBN 91-628-2217-

Språkutvecklande arbetssätt i SO- och NO-undervisning i grundskolans mellanår

I många av skolans ämnen ställs stora krav på elevernas språkliga förmågor gällande läsning, skrivande och begreppsförståelse. Elever i språklig sårbarhet kan möta stora utmaningar i SO- och NO-ämnena där explicit undervisning och scaffolding behövs för att stödja eleverna i ämnesspecifika läs- och skrivaktiviteter. Om undervisningen inte kan möta elevernas språkliga behov och erbjuda adekvat stöttning kan det få stora konsekvenser för elevernas kunskapsutveckling och måluppfyllelse. Därför är ett språkutvecklande förhållningssätt en nyckel till en mer tillgänglig undervisning. Syftet med arbetet är att undersöka hur SO- och NO-lärare på mellanstadiet beskriver att de arbetar språkutvecklande i sina ämnen. I arbetet har en kvalitativ metod använts där datainsamling skett genom fokusgruppssamtal och enskilda intervjuer med 12 mellanstadielärare, varav hälften undervisar i SO och hälften i NO. Resultatet visar att lärarna överlag har en medvetenhet kring språkets betydelse i sina ämnen och att de anser sig ha en roll både som ämneslärare och språklärare. Några slutsatser som kan dras är att det, trots en samstämmig syn, finns stor variation i lärarnas sätt att stötta elever i språklig sårbarhet samt att begrepp som stöttning kan ha olika innebörd för olika lärare. En mängd faktorer kan också påverka hur den språkutvecklande undervisningen gestaltar sig, och de språkliga utmaningar som finns i elevgruppen får konsekvenser för vad man prioriterar att arbeta med. Generellt läggs dock stort fokus på begreppsinlärning och ämnesspråk samt gemensam textläsning

Glutathione transferase mimics: micellar catalysis of an enzymic reaction

Substances that mimic the enzyme action of glutathione transferases (which serve in detoxification) are described. These micellar catalysts enhance the reaction rate between thiols and activated halogenated nitroarenes as well as α,β-unsaturated carbonyls. The nucleophilic aromatic substitution reaction is enhanced by the following surfactants in descending order: poly(dimethyldiallylammonium-co-dodecylmethyldiallylammonium) bromide (86/14) » cetyltrimethylammonium bromide > zwittergent 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate) > zwittergent 3-14 (n-tetradecyl-N,N-dimethyl- 3-ammonio-1-propanesulphonate) ≈ N,N-dimethyl-laurylamine N-oxide > N,N-dimethyloctylamine N-oxide. The most efficient catalyst studied is a polymeric material that incorporates surfactant properties (n-dodecylmethyldiallylammonium bromide) and opens up possibilities for engineering sequences of reactions on a polymeric support. Michael addition to α,β-unsaturated carbonyls is exemplified by a model substance, trans-4-phenylbut-3-en-2-one, and a toxic compound that is formed during oxidative stress, 4-hydroxy-2-undecenal. The latter compound is conjugated with the highest efficiency of those tested. Micellar catalysts can thus be viewed as simple models for the glutathione transferases highlighting the influence of a positive electrostatic field and a non-specific hydrophobic binding site, pertaining to two catalytic aspects, namely thiolate anion stabilization and solvent shielding.

Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination.

Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathione-dependent conversion of PGH2 to PGE2 (Vmax; 170 µmol min–1 mg–1) and high kcat/Km (310 mM–1 s–1). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG2 to 15-hydroperoxy-PGE2 (Vmax; 250 µmol min–1 mg–1). The formation of 15-hydroperoxy-PGE2 represents an alternative pathway for the synthesis of PGE2, which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 µmol min–1 mg–1), 5-hydroperoxyeicosatetraenoic acid (0.043 µmol min–1 mg–1), and 15-hydroperoxy-PGE2 (0.04 µmol min–1 mg–1). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 µmol min–1 mg–1). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm3/g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1