Intracellular enzyme kinetics in triiodothyronine-grown ascites cell cultures

Abstract

The transient changes in pyridine nucleotide reduction (fluorescence pulse) which result from the microelectrophoretic addition of glycolytic substrate (e.g. Glc-6-P) in gradually increasing amounts can be followed in single living triiodothyronine (T 3)-grown EL2 cells and controls by microfluorimetry. With Glc-6-P each fluorescence pulse represents the summated contributions of glyceraldehyde phosphate dehydrogenase (GAPDH, NAD-reducing) and lactic dehydrogenase (LDH, NAD-reoxidizing) under the most simplified assumptions (disregarding the NADP-linked hexose monophosphate shunt, the alpha-glycerophosphate “shuttle”, etc.) So far the most consistent changes are observed in EL2 cells maintained for at least 2 weeks in presence of T3. While the maximal levels of NAD reduction are quite comparable in the control and T3-grown, the required Glc-6-P concentration and the rates of Glc-6-P utilization are about ten times smaller in the T3-grown. Also EL2 controls are quite self-sufficient in terms of endogenous activators of the glycolytic chain and ATP acts as an inhibitor, while in the T3-grown the rate is considerably enhanced by ATP, the simultaneous addition of ADP and P 1, or inhibitors which block the mitochondrial-extramitochondrial exchange of adenine nucleotides (e.g. atractylate). When Glc-6-P is increased gradually (dose response curve) the plot of substrate against the area of the fluorescence pulse reveals sigmoidal kinetics which are altered by adenine nucleotides, LDH inhibitor and substrates, etc. In the EL2 control the ascending branch of the sigmoid is displaced to the left (lower substrate levels) in presence of adenine nucleotides, but it moves to the right in similarly treated T3-grown cells. The rate of Glc-6-P utilization is considerably decreased in controls treated with oxamate inhibitors, but it remains little affected in the T3-grown. The observed changes in the T3-grown cells and their coupling with a high level of pyridine nucleotide reduction might be due to shifts in the balance between various steps along glycolytic pathways as well as to alterations in intracytoplasmic organelles affecting the compartmentalization of metabolites or mitochondrial-extramitochondrial competition for such metabolites.