Studies on glycolysis in vitro: role of glucose phosphorylation and phosphofructokinase activity on total velocity

An in vitro glycolysis system has been developed to study the regulation of glycolysis on kinetic structure basis, in order to determine the extent of regulatory effects on the whole system of individual enzymes according to their kinetic data, in rat liver and muscle. Hexokinase or glucose-6-phosphate addition to the system with glucose as substrate increases lactate production rate by 2.5 in liver and by 10 in muscle, which suggest glucose phosphorylation step is a limiting step in this system. Fructose 2,6-bisphosphate addition to the system increases lactate production rate in liver only when glucose is the substrate, but not with glucose-6-phosphate as substrate. There is a linear relationship between glycolytic activity, as lactate produced per min and protein quantity, which suggests that this system can also be used to assay glycolytic activity in tissue extracts. Specific glycolytic activity found, as mumol of L-lactate produced per min, per protein mg was 0.1 for muscle and 0.01 for liver.