1. 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 a t 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.2. 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. 1000/, 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 250/,, and stroma protein only 1501, of the aldolase activity. 3. A modified Langmuir isotherm is derived, and the analytical evaluation supports the assumption that the binding of aldolase to F-actin occurs a t two different binding sites. 4.The binding of aldolase to F-actin and other structure proteins depends on the ionic strength. At 150 mM KC1, a complete desorption occurs, and 5001, of the actin-bound aldolase is set free a t a concentration of 80 mM KC1. The sigmoid shape of the desorption curve indicates a cooperative mechanism of the binding phenomenon.5. Within the physiological range, the pH does not influence the binding of aldolase to F-actin. 6. 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.7. 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.8. 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.

The binding of aldolase and triosephosphate dehydrogenase to F-actin was studied in vitro under various conditions. Several monovalent and bivalent cations and anions, most of the glycolytic metabolites, and some nucleotides were examined with respect to their influence on the binding. Desorption of the bound enzymes as well as adsorption hindrance may be due to relatively specifk factors (metabolites, ions) and relatively non-specific factors (pH, ionic strength). I n the case of aldolase it was found that fructose-l,6-diphosphate, dihydroxyacetone phosphate, ATP, ADP, and inorganic phosphate cause a marked adsorption hindrance within their physiological concentration ranges. I n analogy with the effect of fructose-l,6-diphosphate on aldolase, the binding of triosephosphate dehydrogenase to F-actin is also inhibited most strongly by its substrate glyceraldehydephosphate. However, adsorption hindrance of triosephosphate dehydrogenase is also caused by fructose-1 ,Sdiphosphate, ATP, and inorganic phosphate, although higher concentrations of these compounds are needed in comparison with aldolase. Kinetic studies of F-actin-bound aldolase revealed that the properties of the enzyme are modified by the binding. Under the chosen experimental conditions, binding of aldolase to F-actin doubles Vmax and increases K m for fructose-1,6-diphosphate by almost one order of magnitude. K , determinations were performed in the absence of ammonium sulfate. Sulfate and also phosphate anions are strong inhibitors of muscle aldolase. I n both cases the inhibition type appears to be competitive. Determinations of K , for fructoseie-1,6-diphosphate of native muscle aldolase gave values as low as 0.4 @.As has been shown in previous communications, certain glycolytic enzymes of the muscle can be bound reversibly in vitro to structure proteins of the contractile apparatus [l -31. A comparison of the main structure proteins revealed that F-actin possesses by far the lughest binding capacity. The affinity of these enzymes to F-actin is also documented by histochemical findings. It has been shown that glycogenolytic and glycolytic enzymes are located almost exclusively within the isotropic zones of the myofibrils [4-71. This distribution corresponds to the site of the actin filaments in the relaxed myofibril [S,9].Enzymes.

The binding to F-actin of several crystalline rabbit muscle enzymes of glycogenolysis and glycolysis, was investigated in vitro. Under the conditions chosen, no binding occurs in the case of glycogen phosphorylase, phosphoglucomutase, glycerate phosphomutase and enolase. Aldolase, pyruvate kinase and triosephosphate dehydrogenase are strongly bound to F-actin, whereas lactate dehydrogenase and phosphoglycerate kinase are bound to a lesser degree. The quantitative differences in the affinity to F-actin are expressed by differences in the determined binding constants. Analytical evaluation of the binding characteristics suggests two binding sites in the case of aldolase and pyruvate kinase. Analytical ultracentrifugation studies and density gradient centrifugation in sucrose revealed a complex formation between aldolase and G-actin monomer. An average ~2 0 ,~ value of 9.2 S was obtained for the complex. A t higher aldolase concentration, a gelation of the G-actin was observed which resembles the action of a-actinin.Several glycolytic enzymes, especially fructosediphosphate aldolase and triosephosphate dehydrogenase have been shown to be bound reversibly to structure proteins of muscle.

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