In situ kinetic parameters of glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase in different areas of the rat liver acinus

Publisher Website
Google Scholar
Abstract
Summary The reaction velocity of glucose-6-phosphate dehydrogenase (G6PDH) and phosphogluconate dehydrogenase (PGDH) was quantified with a cytophotometer by continuous monitoring of the reaction product as it was formed in liver cryostat sections from normal, young mature female rats at 37°C. Control incubations were performed in media lacking both substrate and coenzyme for G6PDH activity and lacking substrate for PGDH activity. All reaction rates were non-linear but test minus control reactions showed linearity with incubation time up to 5 min using Nitro BT as final electron acceptor. End point measurements after incubation for 5 min at 37°C revealed that the highest specific activity of G6PDH was present in the intermediate area (V max=7.79±1.76 µmol H2 cm−3min−1) and of PGDH in the pericentral and intermediate areas (V max=17.19±1.73 µmol H2 cm−3 min−1). In periportal and pericentral areas,V max values for G6PDH activity were 4.48±1.03 µmol H2 cm−3 min−1 and 3.47±0.78 µmol H2 cm−3 min−1, respectively. PGDH activity in periportal areas showed aV max of 10.84±0.33 µmol H2 cm−3 min−1. Variation of the substrate concentration for G6PDH activity yielded similarK m values of 0.17±0.07mm, 0.15±0.13mm and 0.22±0.11mm in periportal, pericentral and intermediate areas, respectively.K M values of 0.87±0.12mm in periportal and of 1.36±0.10mm in pericentral and intermediate areas were found for PGDH activity. The significant difference betweenK M values for PGDH in areas within the acinus support the hypothesis that PGDH is present in the cytoplasmic matrix and in the microsomes. A discrepancy existed betweenK M andV max values determined in cytochemical assays using cryostat sections and values calculated from biochemical assays using diluted homogenates. In cytochemical assays, the natural microenvironment for enzymes is kept for the demonstration of their activity and thus may give more accurate information on enzyme reactions as they take placein vivo.