Abstract
NH+4NH4+<math> <msubsup><mrow><mtext>NH</mtext></mrow> <mrow><mn>4</mn></mrow> <mrow><mo>+</mo></mrow> </msubsup> </math> increased growth rates and final densities of several human metastatic cancer cells. To assess whether glutamate dehydrogenase (GDH) in cancer cells may catalyze the reverse reaction of NH+4NH4+<math> <msubsup><mrow><mtext>NH</mtext></mrow> <mrow><mn>4</mn></mrow> <mrow><mo>+</mo></mrow> </msubsup> </math> fixation, its covalent regulation and kinetic parameters were determined under near-physiological conditions. Increased total protein and phosphorylation were attained in NH+4NH4+<math> <msubsup><mrow><mtext>NH</mtext></mrow> <mrow><mn>4</mn></mrow> <mrow><mo>+</mo></mrow> </msubsup> </math> -supplemented metastatic cells, but total cell GDH activity was unchanged. Higher V max values for the GDH reverse reaction vs. forward reaction in both isolated hepatoma (HepM) and liver mitochondria [rat liver mitochondria (RLM)] favored an NH+4NH4+<math> <msubsup><mrow><mtext>NH</mtext></mrow> <mrow><mn>4</mn></mrow> <mrow><mo>+</mo></mrow> </msubsup> </math> -fixing role. GDH sigmoidal kinetics with NH+4NH4+<math> <msubsup><mrow><mtext>NH</mtext></mrow> <mrow><mn>4</mn></mrow> <mrow><mo>+</mo></mrow> </msubsup> </math> , ADP, and leucine fitted to Hill equation showed n H values of 2 to 3. However, the K 0.5 values for NH+4NH4+<math> <msubsup><mrow><mtext>NH</mtext></mrow> <mrow><mn>4</mn></mrow> <mrow><mo>+</mo></mrow> </msubsup> </math> were over 20 mM, questioning the physiological relevance of the GDH reverse reaction, because intracellular NH+4NH4+<math> <msubsup><mrow><mtext>NH</mtext></mrow> <mrow><mn>4</mn></mrow> <mrow><mo>+</mo></mrow> </msubsup> </math> in tumors is 1 to 5 mM. In contrast, data fitting to the Monod-Wyman-Changeux (MWC) model revealed lower K m values for NH+4NH4+<math> <msubsup><mrow><mtext>NH</mtext></mrow> <mrow><mn>4</mn></mrow> <mrow><mo>+</mo></mrow> </msubsup> </math> , of 6 to 12 mM. In silico analysis made with MWC equation, and using physiological concentrations of substrates and modulators, predicted GDH N-fixing activity in cancer cells. Therefore, together with its thermodynamic feasibility, GDH may reach rates for its reverse, NH+4NH4+<math> <msubsup><mrow><mtext>NH</mtext></mrow> <mrow><mn>4</mn></mrow> <mrow><mo>+</mo></mrow> </msubsup> </math> -fixing reaction that are compatible with an anabolic role for supporting growth of cancer cells.