Binding Of Glycolytic Enzymes Research Articles

Tyrosine phosphorylation of band 3 inhibits peripheral protein binding.

The cytoplasmic domain of band 3 (cdb3) of the human erythrocyte membrane is a good substrate of endogenous and exogenous protein-tyrosine kinases. Because one site of tyrosine phosphorylation is within the glycolytic enzyme/hemoglobin-binding region at the N terminus of the polypeptide, we have investigated whether tyrosine phosphorylation of cdb3 might influence its interaction with the above peripheral proteins. Using p40, a protein-tyrosine kinase isolated from bovine thymus, we demonstrate that aldolase binding to cdb3 linked to Affi-Gel 15 is significantly inhibited by phosphorylation of the immobilized band 3. Importantly, upon dephosphorylation of the gel with acid phosphatase, aldolase binding returns to prephosphorylated values. Similarly, cdb3 phosphorylation was found to inhibit glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, and hemoglobin binding to immobilized cdb3. In the converse experiment, untreated soluble cdb3 was shown to bind to immobilized aldolase, whereas phosphorylated cdb3 (approximately equal to 1.8 mol of Pi/mol of cdb3) did not. Furthermore, phosphorylated cdb3 was unable to inhibit aldolase catalysis, whereas untreated cdb3, as shown previously by others, was a potent inhibitor. Taken together, these results demonstrate that phosphorylation of cdb3 on tyrosine residues inhibits peripheral protein binding at the polypeptide's N terminus. In view of the known effect of glycolytic enzyme binding to band 3 on catalytic activity, tyrosine phosphorylation of band 3 may modulate glycolysis in vivo.

Glycolytic enzyme binding and metabolic control in anaerobiosis

Metabolic rate depression is a key survival strategy used by facultative anaerobes for enduring periods of environmental anoxia. In determining the molecular mechanisms of this phenomenon the role of enzyme binding to the subcellular particulate fraction was assessed in muscle tissues (ventricle and foot) of the anoxia tolerant marine gastropod,Busycotypus canaliculatum. Using two different methodologies for preparation, soluble versus particulate fractions of muscle were separated and assayed for their contents of eight glycolytic enzymes. Preparations from anoxic animals showed decreased percentages of enzymes associated with the particulate fraction as compared to controls; this was particularly pronounced for hexokinase and aldolase. A return to aerated seawater reversed this effect, and increased enzyme binding to the particulate fraction. The absence of a Pasteur effect in animal facultative anaerobes may be due, in part, to an anoxia-induced dissociation of enzymes from the particulate fraction of the cell promoting a decrease in glycolytic rate.

Binding of glycolytic enzymes to cardiac sarcolemmal and sarcoplasmic reticular membranes.

Glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase activities were associated with a particulate fraction in a cardiac homogenate preparation. NaCl treatment displaced these activities from the particulate fraction. These membrane-associated glycolytic enzymes were gradually lost during the rigorous isolation procedures needed to purify sarcolemmal and sarcoplasmic reticular vesicles. However, exogenously added enzymes did bind to these purified membranes in relatively large quantities (glyceraldehyde-phosphate dehydrogenase: 1.14 mg of enzyme/mg of sarcolemma, 0.30 mg of enzyme/mg of sarcoplasmic reticulum; phosphoglycerate kinase: 1.02 mg of enzyme/mg of sarcolemma, 0.12 mg of enzyme/mg of sarcoplasmic reticulum), which suggests a nonspecific type of binding interaction. Approximately 50% of the glyceraldehyde-phosphate dehydrogenase and 90% of the phosphoglycerate kinase were inactivated upon binding. NaCl removed the enzyme from the membranes in a concentration-dependent manner similar to its effects in the particulate fraction. Dimethonium (5 mM), an organic divalent cation which screens membrane-surface charge, removed greater than 90% glyceraldehyde-phosphate dehydrogenase from the sarcolemmal membrane. These results, and those with NaCl, suggest that the binding of the enzyme to the membrane is charge-related. Phospholipids extracted from the sarcolemmal membrane were capable of binding and inactivating both glycolytic enzymes. Purified anionic phospholipids were more potent at binding and inactivating both enzymes than were neutral phospholipids. These data show that glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase can bind to sarcolemmal and sarcoplasmic reticular membranes. This binding is reversible, charge-dependent, and inhibitory. This binding is likely to involve anionic membrane phospholipids.

Metabolic dependence of glycolytic enzyme binding in rat and sheep heart.

Perfused rat hearts show a markedly increased binding of phosphofructokinase and fructose-bisphosphate aldolase as a consequence of ischaemia, but little change in binding of pyruvate kinase, lactate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase. After 10 min ischaemia over one quarter of the phosphofructokinase and three quarters of the aldolase are bound. The effect of anoxia is less well marked in its influence on binding with only aldolase showing a significant increase in binding. These results suggest that one factor involved in the increased binding during ischaemia is the fall in pH of the heart. Binding studies with isolated myofibrils confirm that the affinity and stoichiometry of aldolase binding are considerably increased as the pH is lowered over a range comparable to that which occurs in ischaemic heart. The low level of binding of glyceraldehyde-3-phosphate dehydrogenase in perfused rat hearts correlates with the relatively low affinity of this enzyme for binding to rat or rabbit cardiac myofibrils. There are species differences in the enzyme binding response to ischaemia. Sheep hearts show rapid and large increases in the binding of glyceraldehyde-3-phosphate dehydrogenase in addition to changes in aldolase and phosphofructokinase binding. The greater binding of glyceraldehyde-3-phosphate dehydrogenase reflects the greater affinity of sheep cardiac myofibrils. It is suggested that the altered metabolic demands of ischaemia are satisfied by changes in glycolytic enzyme organisation as the enzymes shift from the soluble to the particulate phase of cardiac muscle.

Hemoglobin binds to the amino-terminal 23-residue fragment of human erythrocyte band 3 protein.

Hemoglobin, aldolase and glyceraldehyde 3-phosphate dehydrogenase are known to bind to the cytoplasmic domain of band 3 protein. Binding of glycolytic enzymes to band 3 protein is inhibited by its amino-terminal fragments. To precisely localize the sequence portion of band 3 protein to which hemoglobin binds and to see whether the same region of amino-acid sequence binds both hemoglobin and glycolytic enzymes, a simple, direct solid-phase binding assay was developed. Peptides generated from the 23-kDa fragment by trypsin, cyanogen bromide and mild acid hydrolysis were used as inhibitors to determine the minimal sequence structure involved in the binding of the 23-kDa fragment to hemoglobin. The shortest peptide which inhibits the binding of the 23-kDa fragment is an acid cleavage peptide containing the sequence positions 1 to 23. This sequence is unusual as 14 of its residues are negatively charged, it contains no basic residues and has its amino terminus blocked. Using aldolase, glyceraldehyde-3-phosphate dehydrogenase and hemoglobin as competitive inhibitors in the binding of 23-kDa fragment, the affinity of hemoglobin to this fragment appears several-fold weaker than that of both the enzymes. These findings demonstrate that glycolytic enzymes and hemoglobin bind competitively to the same polyanionic sequence region of band 3 protein.

Glycolytic enzyme binding in fetal brain — The role of actin

The binding of aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase in fetal calf brain homogenates and extracts has been investigated at both 0° and 37°C under high ionic strength conditions. The results demonstrate far greater enzyme binding at 37°C than at 0°C, which correlates with an increased sedimentation of cytoskeletal actin at the higher temperature. A dependence of enzyme sedimentation on the presence of polymerised actin was also demonstrated, and this indicates that cytoskeletal actin is a major adsorbent of glycolytic enzymes in this non-muscle tissue.

The reversible binding of glycolytic enzymes in ovine skeletal muscle in response to tetanic stimulation

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in sheep hind muscles after electrical stimulation. As compared to the control muscles, stimulation led to significant increases in the amount of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase bound to the particulate fraction. The bindng of other glycolytic enzymes was not significantly altered. A servey of different hind limb muscles at variable rates of stimulation revealed that each muscle exhibited its own characteristic response pattern in terms of the level of increased enzyme binding. Generally, an increased stimulation rate led to greater enzyme adsorption. The increase in enzyme binding was rapidly reversible for it was shown that the amount of enzyme bound quickly returned to control values when the muscles were allowed to recover in the live anaesthetised animal following cessation of stimulation. Those muscles which exhibited increased enzyme binding were characterised by a marked loss of glycogen and accumulation of lactate suggesting that accelerated glycolytic flux was a necessary condition for the observation of increased enzyme binding. In support of this, enzyme adsorption was observed to be greatest on stimulation of ischemic muscles, whereas in trained muscles, or muscles with depleted glycogen stores induced by prior adrenalin treatment, the increased enzyme binding response was greatly diminished. It is concluded that the variable binding of key glycolytic enzymes has a role to play in the regulation of glycolytic behaviour in skeletal muscle.

Effect of electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Functional and structural implications

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in bovine psoas muscle before and after electrical stimulation. In association with an accelerated glycolytic rate on stimulation, there was a significant increase in the binding of certain glycolytic enzymes, the most notable of which were phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase. From the known association of glycolytic enzymes with the I-band of muscle it is proposed that electrical stimulation of anaerobic muscle increases enzyme binding to actin filaments. Calculations of the extent of enzyme binding suggest that significant amounts of enzyme protein, particularly aldolase and glyceraldehyde 3-phosphate dehydrogenase, are associated with the actin filaments. The results also imply that kinetic parameters derived from considerations of the enzyme activity in the soluble state may not have direct application to the situation in the muscle fibre, particularly during accelerated glycolysis.

Changes associated with glycolytic-enzyme binding in the equatorial X-ray-diffraction pattern of glycerinated rabbit psoas muscle.

The binding of fructose biphosphate aldolase to the thin filaments of glycerinated rabbit psoas muscle produces a significant change in its low-angle X-ray-diffraction pattern. The intensity of the (11) reflection relative to that of the (10) reflection increases by 26 +/- 3% (mean +/- S.E.M.), which is consistent with the increase in the mass of the thin filaments produced by enzyme binding. A similar effect is found with a mixture of aldolase and glyceraldehyde 3-phosphate dehydrogenase. The significance of the change in intensity is considered with reference to the interpretation of the equatorial patterns obtained from muscles in different physiological states. The magnitude of the increase in the relative intensity of the (11) reflection is lower than that observed between relaxed and contracting muscle and does not bring into question the interpretation linking changes in these patterns to cross-bridge movement. However, the effect due to enzyme binding may be important when making detailed interpretations of these changes. It may also be related to an unusual pattern sometimes observed in cardiac muscle.

Quantitative comparison of the binding of various glycolytic enzymes to F-actin and the interaction of aldolase with G-actin.

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 s20, w value of 9.2 S was obtained for the complex. At higher aldolase concentration, a gelation of the G‐actin was observed which resembles the action of α‐actinin.

Binding of glycolytic enzymes to structure proteins of the muscle.

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.

A histochemical-physiological correlation of frog skeletal muscle fibers.

A histochemical-physiological correlation of frog skeletal muscle fibers.