6-Phosphogluconate dehydrogenase (6PGDH) is the third enzyme of the oxidative phase of the pentose phosphate pathway. The cytosolic form of this pathway is well characterised, but the details and significance of the endoplasmic reticulum (ER) version are only beginning to be understood. We have previously identified hexose-6-phosphate dehydrogenase (H6PDH), which catalyses the first two steps of this pathway within the lumen of the ER, as a pivotal regulator of ER reductases such as 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). The cortisone-reductase activity of 11β-HSD1 is dependent on a high NADPH/NADP+ ratio, which in turn is dependent on the NADPH-generating activity of H6PDH. Activity of 6PGDH also generates NADPH, and hence its presence within the ER would be of potential significance. 6PGDH activity has previously been reported in microsomes, but neither the protein nor the corresponding gene has been characterised.
Significant 6PGDH activity was observed in microsomes isolated from mouse liver. This activity was lower than the cytosolic activity (44.6±10.6 vs 300.1±15.4 mmol NADPH/min per mg protein, respectively), and was only measurable upon solubilisation indicative of a true microsomal form. There was no detectable cytosolic contamination of the microsomes, as indicated by either western immunodetection or activity measurements, using the known cytosolic marker, lactate dehydrogenase. On gel filtration and ion-exchange chromatography, the microsomal activity behaved identically to the cytosolic activity. FT-ICR mass spectrometry of trypsin digests of the chromatographically and electrophoretically-separated microsomal form, indicated a protein sequence identical to the cytosolic form. In addition, in western blot analyses, antibody against the cytosolic form of 6PGDH cross-reacted with the active microsomal fractions. These data indicate that microsomal 6PGDH is likely to be derived from its cytosolic counterpart, and suggest the cytosolic 6PGDH can be shuttled into the ER where it can additionally contribute to the NADPH pool for lumenal reductases such as 11β-HSD1.