Quantitative Comparison of the Binding of Various Glycolytic Enzymes to F‐Actin and the Interaction of Aldolase with G‐Actin

Arnold, H.; Henning, Roland; Pette, Dirk

doi:10.1111/j.1432-1033.1971.tb01522.x

Cited by 128 publications

(51 citation statements)

References 24 publications

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“…A general effect of decreasing pH on increasing enzyme binding to actin was noted by Pette [6] for several enzymes other than aldolase. However, during the studies with perfused rat hearts we have not observed any marked increases in the binding of pyruvate kinase, lactate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase although such increases might have been expected on the basis of the known pH dependency of their absorption to actin filaments in vitro.…”

Section: Discussionmentioning

confidence: 76%

Metabolic dependence of glycolytic enzyme binding in rat and sheep heart

Clarke

Stephan

Huxham

et al. 1984

European Journal of Biochemistry

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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 p H 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.Pette and co-workers first demonstrated an association between glycolytic enzymes and the I-band of cardiac muscle [I] and this localisation was shown to be general [l -31 for striated muscle. F-actin, the main structural protein of the I-band, was shown to bind phosphofructokinase, fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, lactate dehydrogenase and a number of other glycolytic enzymes 14-61. Subsequently we have shown that the accessory I-band proteins, tropomyosin and troponin, are also sites for enzyme binding and that this binding can be observed at physiologically relevant ionic strengths [7 -121.It has been suggested [5, 71 that a reversible interaction of glycolytic enzymes with structural proteins could constitute an effective mechanism for metabolic control as the kinetic parameters of some enzymes are known to alter on binding. If such mechanisms are operative, then the degree of enzyme association with structural components might be expected to vary depending on the metabolic state of the muscle. Earlier reports [13,14] from these laboratories have shown that in both ovine and bovine skeletal muscle there is an appreciable binding of a range of glycolytic enzymes notably phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase and this binding is markedly increased upon electrical stimulation. In the live animal the increased binding is rapidly rev...

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Section: Discussionmentioning

confidence: 76%

Metabolic dependence of glycolytic enzyme binding in rat and sheep heart

Clarke

Stephan

Huxham

et al. 1984

European Journal of Biochemistry

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“…The uniform distribution of Rh-aldolase we observed in vivo initally seemed paradoxical, in light of data demonstrating that aldolase hinds to F-actin (7,15,16), skeletal muscle homogenates (19), and fetal brain homogehates (17) with affinities greater than or equal to those of other glycolytic enzymes. However, the spatial differences we measured in the mobile fraction and diffusion coefficient of Rh-aldolase indicate that a sensitive equilibrium may regulate the actin-binding activity of aldolase in vivo.…”

Section: Discussionmentioning

confidence: 99%

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]

Pagliaro

Taylor

1988

The Journal of Cell Biology

115

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Abstract. We have prepared a functional fluorescent analogue of the glycolytic enzyme aldolase (rhodamine [Rh]-aldolase), using the succinimidyl ester of carboxytetramethyl-rhodamine. Fluorescence redistribution after photobleaching measurements of the diffusion coefficient of Rh-aldolase in aqueous solutions gave a value of 4.7 × 10 -7 cm2/s, and no immobile fraction. In the presence of filamentous actin, there was a 4.5-fold reduction in diffusion coefficient, as well as a 36% immobile fraction, demonstrating binding of Rh-aldolase to actin. However, in the presence of a 100-fold molar excess of its substrate, fructose 1,6-diphosphate, both the mobile fraction and diffusion coefficient of Rh-aldolase returned to control levels, indicating competition between substrate binding and actin cross-linking. When Rh-aldolase was microinjected into Swiss 3T3 cells, a relatively uniform intracellular distribution of fluorescence was observed. However, there were significant spatial differences in the in vivo diffusion coefficient and mobile fraction of Rh-aldolase measured with fluorescence redistribution after photobleaching. In the perinuclear region, we measured an apparent cytoplasmic diffusion coefficient of 1.1 × 10 -7 cm2/s with a 23% immobile fraction; while measurements in the cell periphery gave a value of 5.7 × 10 -g cm2/s, with no immobile fraction. Ratio imaging of Rh-aldolase and FITCdextran indicated that FITC-dextran was relatively excluded from stress fiber domains. We interpret these data as evidence for the partitioning of aldolase between a soluble fraction in the fluid phase and a fraction associated with the solid phase of cytoplasm. The partitioning of aldolase and other glycolytic enzymes between the fluid and solid phases of cytoplasm could play a fundamental role in the control of glycolysis, the organization of cytoplasm, and cell motility. The concepts and experimental approaches described in this study can be applied to other cellular biochemical processes.TIaOUGH glycolytic metabolism is well-defined at the biochemical level, the dynamic and spatial organization of glycolysis in living cells has not been characterized to date. After the elucidation of mitochondrial structure and function 35 years ago (28) it was generally assumed that a corresponding organelle for glycolysis must exist. Efforts to isolate and to characterize such an organelle were unsuccessful, leading de Duve (26) to conclude that the longsought"glycolytic particle" did not exist. As a result, the current concept of in vivo glycolytic metabolism is based on an assumption of dilute solution chemistry, due largely to the absence of evidence for a discrete glycolytic organelle.There has been evidence for over 20 years that some glycoiytic enzymes bind to structural proteins, particularly actin.

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“…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). It is also known that purified LDH binds to intracellular proteins such as tubulin (K d ϭ 1.0 M), microtubules (K d ϭ 3.5 M) (Walsh et al 1989), and troponin (K d ϭ 0.73-2.3 M; Yasykova et al 1990).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

Nakae

Stoward

1997

J Histochem Cytochem.

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SUMMARYWe studied the effects of two tissue protectants, polyvinyl alcohol (PVA) and agarose gel, on a kinetic parameter of lactate dehydrogenase LDH that is assumed to be related to the extent of diffusion of the enzyme out of tissue sections during its histochemical assay. The kinetics of the enzyme in mouse gastrocnemius (skeletal) muscle fibers and periportal hepatocytes were determined in unfixed sections incubated either on substrate ( L -lactate)-containing agarose gel films or in aqueous assay media in the presence or absence of 18% PVA. The absorbances of the formazan final reaction products at their isobestic point were measured continuously in the cytoplasm of individual cells as a function of incubation time, using a real-time image analysis system. Whichever incubation medium was used, the absorbances in the two cell types increased nonlinearly during the first minute of incubation but linearly for incubation times between 1 and 3 min. The nonlinearity of the LDH reaction was analyzed using the equation v i Ϫ v ϭ a Њ A , where v i is the observed initial velocity determined from the absorbance changes during the first 10 sec of incubation and v and Њ A are respectively the gradient and intercept on the absorbance axis of the linear regression line of the absorbance on incubation times between 1 and 3 min. The plots of the observed ( v i Ϫ v ) against Њ A were linear. Their gradients a were characteristic for each cell type and tissue protectant. The a values for skeletal muscle fibers were 12-43% lower than those for hepatocytes. The a value for hepatocytes obtained with the PVA method was 32% lower than that determined with the gel film method. For skeletal muscle fibers, the a values determined by the two methods were almost the same. Addition of excess pyruvate to the aqueous assay medium had no effects on a for either muscle fibers or hepatocytes. In contrast, a was zero for sections of polyacrylamide gels containing purified enzyme, whether incubated on agarose films or in PVA media. These data confirmed that the constant a is related to the extent to which the enzyme diffuses out of sections during incubation but not to product inhibition of LDH by pyruvate. PVA was more effective for protecting diffusion of LDH from hepatocytes than from skeletal muscle fibers, possibly because hepatocytes contain a greater proportion of diffusable (unbound) LDH than skeletal muscle fibers.

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Quantitative Comparison of the Binding of Various Glycolytic Enzymes to F‐Actin and the Interaction of Aldolase with G‐Actin

Cited by 128 publications

References 24 publications

Metabolic dependence of glycolytic enzyme binding in rat and sheep heart

Metabolic dependence of glycolytic enzyme binding in rat and sheep heart

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]

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

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