Isolation and immunological characterization of theta class glutathione-s-transferase gstt2-2 from bovine liver

Download

index.pdf

Date

2004

Author

İşgör, Sultan Belgin

Metadata

Show full item record

Item Usage Stats

202
views

75
downloads

The glutathione-S-transferases (GSTs) (EC.2.5.1.18) are enzymes that participate in cellular detoxification of endogenous as well as foreign electrophilic compounds, function in the cellular detoxification systems and are evolved to protect cells against reactive oxygen metabolites by conjugating the reactive molecules to the nucleophile scavenging tripeptide glutathione (GSH, ?-glu-cys-gly). The GSTs are found in all eukaryotes and prokaryotic systems, in the cytoplasm, on the microsomes, and in the mitochondria. Cytosolic GSTs have been grouped into seven distinct classes as: alpha (?), mu (?), pi (?), sigma (?), omega, theta (?) and zeta (?). In comparison with other GSTs, class theta enzymes have proven difficult to isolate and characterize. Two distinct theta GSTs have been identified in man, GSTT1-1 and GSTT2-2 three in the rat rGST1-1, rGSTT2-2 and 13-13 and one in the mouse. this study, a class theta GST (GSTT2-2), with high activity towards 1-MS was isolated and purified from bovine liver in 3% yield with a purification factor of 3-fold. The purification protocol included a sequential DEAE cellulose anion exchanger liquid chromatography column, S-hexylglutathione agarose affinity column, dye binding orange A and chromatofocusing columns. The enzyme activity and protein content decreased rapidly after the last step of purification. The purified GSTT2-2 showed significant activity only towards 1-MS as 77 nmole/min/mg. The GSTT2-2 purified from bovine liver had a molecular weigth (Mr) value of about 28,200 which was also confirmed by Western Blott Analysis. The purified farctions of GSTT2-2 with other kolon farctions were tested with anti GSTT2-2, antiGST alfa, antiGST mu and antiGST pi antibodies. The enzyme activities towards CDNB, 4-nitrobenzylchloride (NBC) and 1-menapthyl sulfate were measured as described by Habig and Jacoby.

Subject Keywords

Biochemistry.

URI

http://etd.lib.metu.edu.tr/upload/3/12604851/index.pdf
https://hdl.handle.net/11511/14104

Collections

Graduate School of Natural and Applied Sciences, Thesis

Suggestions

OpenMETU
Core

Investigation for natural extract inhibitors of bovine lens aldose reductase responsible for the formation of diabetis dependent cataract Onay, Melih; Çoruh, Nursen; Department of Biochemistry (2008) In the polyol pathway, Aldose reductase (AR) is an important enzyme in reduction of aldehydes and aldosugars to their suitable alcohols. AR, using NADPH as a coenzyme, has a molecular weight of 37 000 dalton. AR in its activated form, known to increase the sorbitol accumulation in lens, is responsible for the cataract formation in diabetis diseases. Therefore, the inhibition of aldose reductase is important to prevent the incedence of cataract formation in diabetus mellitus. In the treatment of diabetis dep...
Sequences in the intracellular loops of the yeast pheromone receptor Ste2p required for G protein activation. Celić, A; Martin, NP; Son, Çağdaş Devrim; Becker, JM; Naider, F; Dumont, ME (American Chemical Society (ACS), 2003-03-18) The α-factor receptor of the yeast Saccharomyces cerevisiae encoded by the STE2 gene is a member of the large family of G protein-coupled receptors (GPCRs) that mediate multiple signal transduction pathways. The third intracellular loop of GPCRs has been identified as a likely site of interaction with G proteins. To determine the extent of allowed substitutions within this loop, we subjected a stretch of 21 amino acids (Leu228−Leu248) to intensive random mutagenesis and screened multiply substituted alleles...
KINETIC-PROPERTIES OF PURIFIED SHEEP LUNG MICROSOMAL NADH-CYTOCHROME B5 REDUCTASE Güray, Nülüfer Tülün (Elsevier BV, 1991-01-01) 1. Lung NADH-cytochrome b5 reductase was saturated with its artificial substrate, potassium ferricyanide at approximately 0.1 mM ferricyanide concentration, and the activity of the lung enzyme was inhibited by the higher concentrations of potassium ferricyanide. Ferricyanide at 0.5 and 1.0 mM inhibited the activity of the enzyme by about 20 and 61% respectively. The apparent K(m) value was calculated as 13.7-mu-M potassium ferricyanide and 4.3-mu-M NADH.
AMINO-ACID SUBSTITUTIONS WITHIN THE ANALOGOUS NUCLEOTIDE-BINDING LOOP (P-LOOP) OF AMINOGLYCOSIDE 3'-PHOSPHOTRANSFERASE-II KOCABIVIK, S; PERLIN, MH (Elsevier BV, 1994-01-01) 1. Oligonucleotide-directed mutagenesis of APH(3')-II was used to investigate the functions of key amino acids in the P-loop analogous motif of the enzyme. 2. The mutations of Gly205 --> GIu, Gly210 --> Ala and Arg211 --> Pro considerably reduced the resistance of the resulting strains to KM and to related drugs, e.g. G418. 3. Similarly, enzyme activity in the crude extracts of these mutants was substantially reduced as well as the enzyme's affinity for Mg2+ ATP. 4. Alternatively substitutions at a highly c...
Aromatic amino acid synthesis performance of bacillus acidocaldarius Kocabaş, Pınar; Çalık, Pınar; Department of Chemical Engineering (2004) In this study, the effects of bioprocess operation parameters on aromatic amino acid synthesis performance of Bacillus acidocaldarius were investigated. Firstly, in laboratory scale shake-bioreactors, a defined medium was designed in terms of its carbon and nitrogen sources, to achieve the highest cell concentration. Thereafter, the effects of bioprocess operation parameters, i.e., pH and temperature were investigated; and the optimum medium contained (kg m-3): fructose, 8; (NH4)2HPO4, 5; CaCl2, 0.2; KH2PO4...

Citation Formats

S. B. İşgör, “Isolation and immunological characterization of theta class glutathione-s-transferase gstt2-2 from bovine liver,” Ph.D. - Doctoral Program, Middle East Technical University, 2004.