Purification and characterization of two isoforms of glucose 6-phosphate dehydrogenase (G6PDH) from Chlorella vulgaris C-27.
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.
Honjoh
Ken-ichi
K
Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan. honjoh@agr.kyushu-u.ac.jp
Mimura
Ayano
A
Kuroiwa
Eiko
E
Hagisako
Takahiro
T
Suga
Koushirou
K
Shimizu
Hideyuki
H
Dubey
Rama Shanker
RS
Miyamoto
Takahisa
T
Hatano
Shoji
S
Iio
Masayoshi
M
D000595:Amino Acid Sequence; D002708:Chlorella / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002845:Chromatography / Q000379:methods; D004229:Dithiothreitol / Q000737:chemistry; D004795:Enzyme Stability; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics; D016415:Sequence Alignment; D013696:Temperature
D007477:Ions; D007527:Isoenzymes; D008670:Metals; D010446:Peptide Fragments; D005954:Glucosephosphate Dehydrogenase; D004229:Dithiothreitol