Binding of Glycolytic Enzymes to Structure Proteins of the Muscle

Abstract

Only 60–70% of the total activity of aldolase can be extracted from rat or rabbit muscle homogenates with aqueous solutions of relatively low ionic strength. The extraction of aldolase from muscle tissue is only complete in aqueous solutions with an ionic strength greater than 0.2. The fraction of aldolase which is set free at high ionic strength is not located within a special cellular compartment but is present in a bound form and is desorbed in dependence of the ionic strength of the extraction medium. F‐actin, myosin, acto‐myosin and stroma‐protein were prepared from rabbit muscle and the binding of aldolase to each of these structure proteins was studied in vitro. A completely reversible binding of aldolase to F‐actin, actomyosin, myosin and stroma protein was found. F‐actin possesses by far the highest binding capacity. 100% of the enzyme is bound when 1 mg of aldolase is added to 1 mg of highly purified F‐actin. Under identical experimental conditions acto‐myosin binds 40%, myosin 25%, and stroma protein only 15% of the aldolase activity. A modified Langmuir isotherm is derived, and the analytical evaluation supports the assumption that the binding of aldolase to F‐actin occurs at two different binding sites. The binding of aldolase to F‐actin and other structure proteins depends on the ionic strength. At 150 mM KCl, a complete desorption occurs, and 50% of the actin‐bound aldolase is set free at a concentration of 80 mM KCl. The sigmoid shape of the desorption curve indicates a cooperative mechanism of the binding phenomenon. Within the physiological range, the pH does not influence the binding of aldolase to F‐actin. Similar to aldolase, glyceraldehydephosphate dehydrogenase is bound to F‐actin, and under the experimental conditions, 1 mg of F‐actin binds up to 1.2 mg of glyceraldehydephosphate dehydrogenase. Studies with a purified preparation of myogen reveal that also fructose‐6‐phosphate kinase, and in a lower degree phosphoglycerate kinase, pyruvate kinase and lactate dehydrogenase can be adsorbed to F‐actin. No binding occurs in the case of creatine kinase. The possible significance of the binding phenomenon is discussed with respect to the location of the Embden‐Meyerhof system at the site of the actin filaments within the isotropic zones of the cross‐striated muscle fiber.