Purification and Characterization of Two Isoforms of Glucose 6-Phosphate Dehydrogenase (G6PDH) fromChlorella vulgarisC-27

Abstract

Two kinds of isoforms of glucose 6-phosphate dehydrogenase (G6PDH) were purified from cells of a freezing-tolerant strain, Chlorella vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a HiTrap Blue column which showed excellent separation of the isoforms from each other. The two isoforms (G6PDH1 and G6PDH2) were purified up to 109-fold and 197-fold with specific activity of 14.4 and 26.0 U/mg-protein, respectively. G6PDH1 showed an apparent Mr of 200,000 with a subunit Mr of about 58,000, whereas G6PDH2 showed an apparent Mr of 450,000 with a subunit Mr of about 52,000. The kinetic parameters were measured and several enzymatic features of the isoforms, such as effects of metal ions on the enzyme activity, were clarified, which showed that the two isoforms were different from each other in many respects. Among the effective ions, Cd2+ showed marked stimulating effects on both isoforms. G6PDH1 and G6PDH2 seem to be a cytosolic and a chloroplastic type, respectively, as judged by their sensitivity to DTT, and also from the results of sequence similarity searches using their N-terminal and internal amino acid sequences.

Regulation and properties of glucose-6-phosphate dehydrogenase: A review

“…Studies about regulation of G6PD has been carried out in plants, eukaryotic algae and cyanobacteria but glucose-6phophate dehydrogenase encoding gene has been isolated from only few of these sources (Srivastava and Anderson, 1983;Fickenscher and Scheibe, 1986;Graeve, Schaewen and Scheibe, 1994;Gleason, 1996). Two isoforms (cytosolic and chloroplastic isoforms) of G6PD has been reported in green algae Chlorella vulgaris C-27 (Honjoh et al, 2003). Several isoforms of G6PDH have been reported in cytosol, plastidic stroma, and peroxisomes of higher plants (Corpas et al, 1998).…”

“…2",5"-ADP-Sepharose 4B affinity gel chromatography 650/31% Ciftci et al(2003) Goose erythrocyte Ammonium sulphate precipitation and 2",5"-ADP-Sepharose 4B affinity gel chromatography 3892/68.79% Beydemir et al(2003) Chlorella vulgaris C-27(Two isoforms) Hi Trap blue Column 109 and 197/- Honjoh et al(2003) Coriandrum sativum Ammonium sulphate fractionation and DEAE-Sephadex A50 ion exchange chromatography -/26.4% Demir et al 2004Rainbow trout Ammonium sulphate precipitation and 2",5"-ADP-Sepharose 4B affinity gel chromatography 1271.19/70.40% Ciftci et al 2004Goat Erythrocyte Warburg and Christian, 1937) found that the active form of protein can be obtained from Lebdev juice by acidification with carbon dioxide. Glaser and Brown made attempts for extraction and purification of brewers" yeast enzyme by ammonium sulphate precipitation, calcium phosphate gel adsorption and ethyl alcohol fractionation (Glaser and Brown, 1955).…”

“…Goose erythrocyte G6PD has been purified to 3892 fold with a specific activity of 36.2 EU/mg protein and 68.79% yield by using ammonium sulphate precipitation and 2", 5"-ADP Sepharose 4B affinity gel chromatography (Beydemir et al, 2003). Two isoforms of G6PD from cells of a freezing tolerant strain of C. vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a Hi Trap Blue column which showed excellent separation of the isoforms from each other (Honjoh et al, 2003). The two isoforms were purified up to 109 and 197 fold respectively with specific activity of 14.4 and 26 U/mg proteins, respectively (Honjoh et al, 2003).…”

“…Two isoforms of G6PD from cells of a freezing tolerant strain of C. vulgaris C-27, by sequential steps of chromatography on five kinds of columns, including a Hi Trap Blue column which showed excellent separation of the isoforms from each other (Honjoh et al, 2003). The two isoforms were purified up to 109 and 197 fold respectively with specific activity of 14.4 and 26 U/mg proteins, respectively (Honjoh et al, 2003). Rat small intestine G6PD was purified with a yield of 19.2% and specific activity of 53.8 U/mg proteins (Danisan et al, 2004).…”

“…Monomeric enzyme has been reported from dog liver with a molecular weight of 52.5 KDa (Ozer et al, 2002). Two isoforms of G6PD were reported from a free tolerant strain of C. vulgaris C-27 with an apparent molecular weight of 200 KDa a with a subunit molecular weight of about 58 KDa (Honjoh et al, 2003). The apparently homogeneous enzyme from A. aculeatus had a molecular weight of 105± 5 KDa with a subunit molecular weight of 52±1.1 KDa (Ibraheem et al, 2005), whereas the enzyme from Aspergillus parasiticus had a molecular weight of 180 KDa and was composed of four subunits of apparently equal size (Niehaus and Dilts, 1984).…”