Binding of Glycolytic Enzymes to Structure Proteins of the Muscle

Abstract

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.

Effects of Tissue Protectants on the Kinetics of Lactate Dehydrogenase in Cells

“…Many reports of the interactions of the enzyme with F-actin in vitro have been published since Arnold and Pette (1968) first suggested it. The dissociation constants (K d ) of the LDH-F-actin complex are very low, 8 nM (Poglazov and Livanova 1986) to 2.1 M (Arnold et al 1971).…”

“…Previous histochemical studies, for example, have shown that muscle LDH, as well as other glycolytic enzymes, is predominantly localized within the isotropic zones of myofibrils (e.g., Dölken et al 1975, and references cited therein). Many biochemical studies have reported that some glycolytic enzymes, including LDH, interact with cytoskeletal proteins such as actin filaments (F-actin), tropomyosin, and troponin (e.g., Arnold and Pette 1968;Arnold et al 1971;Clarke et al 1985;Poglazov and Livanova 1986;Walsh and Knull 1987;Yasykova et al 1990), microtubules, and tubulin (Karkhoff-Schweizer and Knull 1987;Walsh et al 1989;Marmillot et al 1994). This evidence suggests that some glycolytic enzymes exist in both the fluid phase and the solid phase of the cytoplasm and that changes in the equilibrium between the two phases may play a role in the regulation of glycolytic metabolism within cells (Wilson 1978).…”

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

“…The interaction of aldolase with actin has been demonstrated histochemically (8,61), biochemically (5,6,8,69), ultrastructurally (16,52), and with immunofluorescence techniques (27). In fact, aldolase was among the first actinbinding proteins to be identified (10), although this interaction was not considered specific at the time.…”

Rabbit muscle myogen. Interactions with phosphate as the source of non-enantiography in moving-boundary electrophoresis

“…Myogen preparations were obtained from freshly excised white muscle of adult rabbits, essentially by procedures described previously (Arnold & Pette, 1968;Clarke & Masters, 1973, 1974, except that 0.3 M-sucrose / l 0mM-triethanolamine /3 mm -EDTA (pH 7.2 at 40C) (Czok & Bticher, 1960) was used as the extracting medium. This change of procedure proved effective in eliminating the traces of myosin that were present in earlier myogen preparations made in this laboratory (Clarke & Masters, 1973, 1974.…”

“…From the biological viewpoint this concept of myogen itself as a multi-enzyme complex has been made largely redundant by the detection of interactions between most of the individual glycolytic enzymes and the fibrous components of muscle (Arnold & Pette, 1968;Amberson & Bauer, 1971; Clarke & Masters, 1975). Indeed, Clarke & Masters (1974) have attributed their earlier ultracentrifugal evidence of complex-formation in myogen (Clarke & Masters, 1973) to the presence of myosin in the preparation.…”