Efflux Time Courses of Cytosolic Proteins from Rabbit Skinned Muscle Fibers Reflect Dissociation of Enzyme Complexes

Abstract

In many multi-system metabolic diseases characterized by mitochondrial dysfunction (e.g. Myalgic Encephalomyelitis), glycolysis appears to be an important compensatory pathway for generating ATP. It is postulated that glycolytic enzymes form a complex that enhances the rate of ATP production through substrate channeling of metabolic product intermediates. To search for evidence of a reduced diffusion coefficient indicative of supramolecular complexes, we examined the efflux of endogenous glycolytic enzymes from rabbit psoas muscle fibers. Single fiber segments were skinned in oil and transferred to physiological salt solution. Cytosolic proteins that diffused into the solution were separated by gel electrophoresis and compared to load-matched standards for quantitative analysis. A radial diffusion model incorporating the dissociation and dissipation of supramolecular complexes accounts for an initial lag and subsequent efflux of glycolytic enzymes. The model includes terms representing protein crowding, myofilament lattice hindrance, and cytomatrix binding. Optimization of model to data returned estimates of apparent diffusion coefficients that were very low at the onset of diffusion (∼10−10 cm2 s−1) but increased with time as cytosolic protein density decreased. The initial values are consistent with the presence of complexes in situ; higher later values (e.g., 0.2 × 10−7 cm2 s−1 for phosphofructose kinase), with molecular sieving and transient binding of dissociated proteins. Channeling of metabolic intermediates via enzyme complexes may enhance production of ATP at rates beyond that possible with randomly distributed enzymes, thereby matching supply with demand. Metabolic channeling may allow glycolysis to better compensate for reduced ATP production in aerobic metabolic diseases.