Stéphanie Mörikofer-Zwez

Fructose 1,6-bisphosphatase in rat liver cytosol: Activation after glucagon treatment in vivo and inhibition by fructose 2,6-bisphosphate in vitro

Summary 1. After intraperitoneal injection of glucagon, the activity of fructose 1,6-bisphosphatase is increased in subsequently isolated liver cytosol from fed rats. The glucagon effect is time-dependent and is maximal 15–20 minutes after injection. At 20 minutes the activation is 44 ± 6% as compared to the controls (mean ± S.E.M., n = 16). 2. Fructose 1,6-bisphosphatase is shown to be inhibited by fructose 2,6-bisphosphate prepared according to Van Schaftingen and Hers ((1980) Biochem. Biophys. Res. Commun. 96, 1524–1531). The K i of the enzyme for fructose 2,6-bisphosphate is about 0.1 μM in control animals and is approximately doubled after injection of glucagon.

Fructose 1,6-bisphosphatase in rat liver cytosol: interactions between the effects of K+, Zn2+, Mn2+, and fructose 2,6-bisphosphate as measured in a steady-state assay.

Fructose 1,6-bisphosphatase activity was determined in rat liver cytosols using glyceraldehyde 3-phosphate as primary substrate. Fructose 1,6-bisphosphate was formed in situ and steady-state concentrations ranging from 1 to 30 microM were observed depending on the activity of fructose 1,6-bisphosphatase and the concentration of added glyceraldehyde 3-phosphate. The system was free of contaminating low-molecular-weight compounds, divalent cations, and chelators. Under these conditions, fructose 1,6-bisphosphatase was inhibited by K+ (less than or equal to 200 mM). This inhibition was due to a reduction of V and was observed in presence of low (0.4 mM) and high (5 mM) concentrations of Mg2+. In presence of 0.4 mM Mg2+, 1 microM Zn2+ inhibited fructose 1,6-bisphosphatase by 50%; the same effect was obtained with 0.3 microM Zn2+ when the system was supplemented with 100 mM KCl. On the other hand, 0.2 microM Zn2+ enhanced the inhibitory effect of K+ and decreased the concentration of K+ yielding half-maximal inhibition from 175 to 100 mM when measured at 0.4 mM Mg2+. The effect of Zn2+ on the inhibition by K+ could be abolished by Mn2+ (less than 5 microM) or by 5 mM Mg2+. One hundred millimolar K+ enhanced the inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate and changed the type of inhibition from mainly competitive to a mixed-type inhibition (increase of Km, decrease of V). Mn2+ (less than 10 microM) reduced the effect of fructose 2,6-bisphosphate, especially in the presence of K+. It is proposed that K+ and Mn2+ may play a role in the regulation of gluconeogenesis.

In vitro formation of glucose-6-phosphate from glyceraldehyde-3-phosphate by liver cytosol from fed and starved rats. Effect of divalent cations on the conversion rate.

Abstract Liver cytosol preparations from fed rats are shown to form glucose-6-phosphate from glyceraldehyde-3-phosphate at a rate of 1.6 μmoles·min −1 ·g liver wet weight −1 in presence of 0.4 mM Mg 2+ . This rate is more than doubled by 30 μM EGTA and/or Mg 2+ -concentrations ≥2 mM. 48 hours starvation increases the rate of glucose-6-phosphate formation at 0.4 mM Mg 2+ to 3.0 μmoles·min −1 ·g liver wet weight −1 and greatly diminishes the effect of EGTA and of higher Mg 2+ -concentrations. Inhibition of glucose-6-phosphate formation by Ca 2+ and Zn 2+ is shown to be more pronounced with cytosol from fed than from 48 hours starved rats.