BBB : Vol. 67 (2003) , No. 9 pp.1888-1896

Purification and Characterization of Two Isoforms of Glucose 6-Phosphate Dehydrogenase (G6PDH) from Chlorella vulgaris C-27

Ken-ichi HONJOH¹⁾, Ayano MIMURA²⁾, Eiko KUROIWA²⁾, Takahiro HAGISAKO²⁾, Koushirou SUGA²⁾³⁾, Hideyuki SHIMIZU²⁾, Rama Shanker DUBEY⁴⁾, Takahisa MIYAMOTO¹⁾, Shoji HATANO⁵⁾ and Masayoshi IIO¹⁾

1) Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University
2) Department of Bioscience and Biotechnology, Graduate School of Bioresource and Environmental Sciences, Kyushu University
3) Chlorella Industries Co., Ltd.
4) Department of Biochemistry, Faculty of Science, Banaras Hindu University
5) Graduate School of Health and Social Welfare Science, Nishikyushu University

(Received February 24, 2003)
(Accepted June 18, 2003)

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 M_r of 200,000 with a subunit M_r of about 58,000, whereas G6PDH₂ showed an apparent M_r of 450,000 with a subunit M_r 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.

Key words:

Chlorella vulgaris C-27; freezing tolerance; glucose 6-phosphate dehydrogenase (G6PDH); purification

	To cite this article:
	Ken-ichi HONJOH, Ayano MIMURA, Eiko KUROIWA, Takahiro HAGISAKO, Koushirou SUGA, Hideyuki SHIMIZU, Rama Shanker DUBEY, Takahisa MIYAMOTO, Shoji HATANO and Masayoshi IIO, “Purification and Characterization of Two Isoforms of Glucose 6-Phosphate Dehydrogenase (G6PDH) from Chlorella vulgaris C-27”, Biosci. Biotechnol. Biochem., Vol. 67, 1888-1896 (2003) .

	doi:10.1271/bbb.67.1888
	JOI JST.JSTAGE/bbb/67.1888