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

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