The feasibility of using an in vitro-based kinetic model for understanding the regulatory properties of glycogenolytic flux in skeletal muscle has been investigated by exploring its property at a number of levels. Through a theoretical analysis, the range of known in vivo fluxes were tested and control properties established; by these methods, the dominance of the ATPase as the driving force of the pathway emerged. The model with the addition of pH and hexokinase offers insight in a comparison between glycogen and glucose metabolism and the ability to calculate directly the acidification of muscle due to glycolysis. The model was fitted to a variety of rich data sets—in vitro reconstituted multi-enzyme systems, in vivo NMR dynamics of intact mouse muscle, and isolated mouse muscles of varying fiber types. The ability of the model to account for such diverse experimental conditions calls into question the functional role of enzyme localization and its influence on kinetics. Finally, a novel method of metabolite quantification via ion chromatography was developed in order to acquire dynamic information by which the model could be compared and refined.
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