Ionic strength dependence of F-actin and glycolytic enzyme associations: a Brownian dynamics simulations approach

The association of glycolytic enzymes with F-actin is proposed to be one mechanism by which these enzymes are compartmentalized, and, as a result, may possibly play important roles for: regulation of the glycolytic pathway, potential substrate channeling, and increasing glycolytic flux. Historically, in vitro experiments have shown that many enzyme/actin interactions are dependent on ionic strength. Herein, Brownian dynamics (BD) examines how ionic strength impacts the energetics of the association of F-actin with the glycolytic enzymes: lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1,6-bisphosphate aldolase (aldolase), and triose phosphate isomerase (TPI). The BD simulations are steered by electrostatics calculated by Poisson-Boltzmann theory. The BD results confirm experimental observations that the degree of association diminishes as ionic strength increases but also suggest that these interactions are significant, at physiological ionic strengths. Furthermore, BD agrees with experiments that muscle LDH, aldolase, and GAPDH interact significantly with F-actin whereas TPI does not. BD indicates similarities in binding regions for aldolase and LDH among the different species investigated. Furthermore, the residues responsible for salt bridge formation in stable complexes persist as ionic strength increases. This suggests the importance of the residues determined for these binary complexes and specificity of the interactions. That these interactions are conserved across species, and there appears to be a general trend among the enzymes, support the importance of these enzyme-F-actin interactions in creating initial complexes critical for compartmentation.     

Interaction between mammalian glyceraldehyde-3-phosphate dehydrogenase and L-lactate dehydrogenase from heart and muscle

The exceptionally high protein concentration in living cells can favor functional protein-protein interactions that can be difficult to detect with purified proteins. In this study we describe specific interactions between mammalian D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) isozymes from heart and muscle. We use poly(ethylene-glycol) (PEG)-induced coprecipitation and native agarose electrophoresis as two independent methods uniquely suited to mimic some of the conditions that can favor protein-protein interaction in living cells. We found that GAPDH interacts with heart or muscle isozymes of LDH with approximately one-to-one stoichiometry. The interaction is specific; GAPDH shows interaction with two LDH isozymes that have very different net charge and solubility in PEG solution, while no interaction is observed with GAPDH from other species, other NAD(H) dehydrogenases, or other proteins that have very similar net charge and molecular mass. Analytical ultracentrifugation showed that the LDH and GAPDH complex is insoluble in PEG solution. The interaction is abolished by saturation with NADH, but not by saturation with NAD(+) in correlation with GAPDH solubility in PEG solution. The crystal structures show that GAPDH and LDH isozymes share complementary size, shape, and electric potential surrounding the active sites. The presented results suggest that GAPDH and LDH have a functional interaction that can affect NAD(+)/NADH metabolism and glycolysis in living cells.     

Reevaluation of the "glycolytic complex" in muscle: a multitechnique approach using trout white muscle

Preliminary characterization of the "glycolytic complex," formed in trout white muscle, revealed that phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are bound to particulate matter largely by ionic interactions; increasing neutral salt or charged metabolite concentrations released bound PFK and GAPDH. GAPDH was consistently solubilized at lower salt concentrations, indicating that it is not bound as tightly as PFK, but both enzymes were readily solubilized at physiological concentrations of salts and metabolites. pH titrations indicated that PFK binding is dependent on group(s) with a pKa of 7.3 in 30 mM imidazole. PFK binding increased at lower pH values; at 150 mM KCl the apparent pKa value is 6.5. Experiments with polyethylene glycol 8000 (PEG), which is used to mimic the high in vivo protein concentrations under in vitro conditions, showed that the binding of PFK and GAPDH increased with increasing PEG concentrations. Interestingly, at 5% PEG, only the PFK binding response depended on the ionic composition of the medium--with increased binding occurring at the pH of the exhausted muscle and decreased binding at control pH values. These results suggested that only PFK reversibly bound to cellular structures in response to changing conditions and disagrees with previous studies showing binding of several glycolytic enzymes as measured using the dilution method (F. M. Clarke, F.D. Shaw, and D.J. Morton (1980) Biochem. J. 186, 105-109). In order to determine whether artifactual binding was measured by the dilution method, two new methodologies were employed to measure enzyme binding in vivo: (a) whole muscle slices were pressed to quickly extrude cellular juice, and (b) muscle strips were finely minced and centrifuged to liberate cytoplasmic contents. Both methods indicated that, under physiological conditions, up to 70% of the total cellular phosphofructokinase may be bound, but other glycolytic enzymes are bound to a lesser extent (10-30%). This result contrasts those obtained with the dilution method, and suggests that dilution of cellular contents may result in an overestimation of the percentage of enzyme associated with cellular structures; this is dramatically shown for glyceraldehyde-3-phosphate dehydrogenase. The viability of the glycolytic complex in trout white muscle is discussed in light of the decreased binding measured using these new methodologies.     

Effects of ATP on the stability of enzymes against heat treatment in 6-aminonicotinamide treated quail

The stabilities of liver and pectoral muscle enzymes in 6-aminonicotinamide (6-AN) treated quail against heat treatment in the presence and absence of added ATP were investigated. Only ATP level in the brain and pectoral muscle of 6-AN treated group was significantly reduced compared to the control group whereas ADP and AMP levels were not affected. In the thermal stability (55 degrees C) of liver enzymes, the activity of acetylcholinesterase (AChE) was not affected whereas the activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were significantly lowered (P<0.01). The addition of 1mM ATP to liver enzyme extracts of 6-AN group afforded 4- and 1.7-fold more protection for GAPDH and LDH, respectively (P<0.01). In liver, LDH appeared to be more protected by ATP than GAPDH. In muscle, however, GAPDH and AChE activity were significantly affected but not LDH. The addition of 1mM ATP to muscle enzyme extracts of 6-AN group afforded 1.7-fold more protection for GAPDH (P<0.01) but rather inactivated AChE. A marked reduction in ATP levels in muscle did not affect specifically muscle enzyme activities only since liver enzyme activities were also affected to the same degree as muscle.     

Differential activity and synthesis of lactate dehydrogenase isozymes A (muscle), B (heart), and C (testis) in mouse spermatogenic cells

Spermatogenic cells isolated from adult and prepubertal mice by unit gravity sedimentation were used to examine enzyme activities and synthesis of the lactate dehydrogenase (LDH) isozymes during spermatogenesis. The synthesis and activity of LDH-C4, the germ cell-specific isozyme, was detected earliest in isolated preleptotene and leptotene/zygotene spermatocytes prior to the mid-pachytene stage of meiosis reported previously. The LDH-C4 isozyme was prominent in pachytene spermatocytes, round spermatids, and condensing spermatids, whereas spermatozoa contained only the LDH-C4 isozyme. In addition, somatic-type LDH isozymes consisting primarily of LDH-B subunits were present in germ cells throughout spermatogenesis. This is in contrast to a previous report that the LDH-B subunit was not synthesized in germ cells. Sertoli cells were further shown to exhibit comparable amounts of five tetrameric LDH isozymes formed by combination of muscle-type LDH-A and heart-type LDH-B subunits.     

Interaction of lactate dehydrogenase and the sarcoplasmic reticulum membrane]

The binding of lactate dehydrogenase (LDH) to sarcoplasmic reticulum membranes results in a 60-70% decrease of the enzyme specific activity. This binding occurs both in high (Kd = 1 microM) and low affinity sites. Addition of NADH or NAD+ and a increase of ionic strength lead to the solubilization of the bound enzyme. A similar effect is observed after addition of the fluorescent probes--anilinonaphthalene sulfonate (ANS) and auramine O (A0). The effect of ANS consists predominantly in its binding to the membrane, while that of A0 is due to the probe interaction with the enzyme. At low concentrations of toluidinylnaphthalene sulfonate (TNS) under conditions of predominant binding of the probe to the membrane, the LDH binding to microsomes is enhanced. A rise in the TNS concentration leads to the formation of the probe-LDH complex which interaction with membrane is hampered. The sites of the probes binding to the protein are located outside the enzyme active center but are, nevertheless, sensitive to it states. It is assumed that these sites of the LDH molecule are involved in its interaction with the membrane. The decline of activity of the bound enzyme is interpreted in terms of alterations of the physico-chemical properties of the medium during the enzyme transition from the solution to the perimembrane space.     

Heart 6-phosphofructo-1-kinase. Subcellular distribution and binding to myofibrils

6-Phosphofructo-1-kinase (phosphofructokinase) (ATP:D-fructose-6-P 1-phosphotransferase, EC 2.7.1.11) can be identified in sheep heart homogenates in two forms, a soluble form and a form bound to the particulate fraction. Homogenates from immediately-dissected hearts have the enzyme in the soluble form, while those collected after a delay have the enzyme bound to the particulate fraction. Aldolase appears to show the same change in its location. Homogenization in a solution with concentrated macromolecular species (20% albumin) results in a greater association of phosphofructokinase and of aldolase to the particulate fraction in homogenates from immediately dissected hearts. Phosphofructokinase activity can be solubilized by two specific means: by high ionic strength, which is dependent upon specific salts; or by low ionic strength, which is dependent upon the presence of phosphofructokinase substrates or modifier ligands. These two means of solubilization are affected differently upon decreasing the pH below 6.9: the solubilization at low ionic strength is prevented, whereas phosphofructokinase is still solubilized by high ionic strength. Under the latter condition, the enzyme is in the inactive dimeric state, which can be activated at an alkaline pH. Myofibrils present in the particulate fraction can account for the binding of phosphofructokinase in heart homogenates. Purified myofibrils, when added to heart supernatant fluids, can bind phosphofructokinase at a slightly acidic pH. Conditions for phosphofructokinase binding to myofibrils, as well as its dissociation, follow what was observed with the binding of phosphofructokinase to the particulate fraction. At an acidic pH, and in the presence of a high concentration of ATP, phosphofructokinase exhibits low activity. However, if phosphofructokinase is assayed under these conditions while bound to myofibrils, the enzyme is activated.     

Inhibition of rabbit muscle isozymes by vitamin C

The ability of vitamins C, E and K to inhibit enzymes directly has been investigated. It was found that vitamin E and some analogs and menadione (vitamin K3) inhibited several enzymes irreversibility at concentrations below one millimolar. Ascorbate inhibits rabbit muscle 6-phosphofructokinase (MPFK-1; EC 2.7.1.11), muscle type LDH (EC 1.1.1.27), and muscle AK (EC 2.7.4.3) at low concentrations that do not inhibit equivalent liver isozymes. Ascorbate Ki values for muscle-type LDH and heart-type LDH isozymes are 0.007 and 3 mM, respectively. The ascorbate Ki value for rabbit skeletal muscle PFK-1 is 0.16 mM; liver PFK-I is not inhibited by ascorbate. Dehydroascorbate does not inhibit any enzyme at ascorbate concentrations normally found in cells. All ascorbate inhibitions are completely reactivated or nearly so by L-ascorbate oxidase, CYS, GSH, or DTT. We propose a hypothesis that ascorbate facilitates glycogen storage in muscle by inhibiting glycolysis. The relationship between ascorbate metabolism and diabetes is discussed.     

Characteristics of microsome-bound lactate dehydrogenase from skate white muscles]

Microsomes possessing the lactate dehydrogenase (LDH) activity were isolated from white driving muscles of the skate (Raja clavata) using differential centrifugation. It was shown that the increase of the ionic strength after addition of 0.6 M KCl and alkalinization of the medium result in the solubilization of the LDH activity - by 50% and 80%, respectively. The Km values for pyruvate and NADH are 171 microM and 7.5 mM, respectively. Membrane-bound LDH, is not inhibited by pyruvate excess (up to 20 mM); the rate of the enzyme inactivation by trypsin is 3 times as low as that of the solubilized enzyme. The existence of two-membrane-bound LDH pools is postulated. The enzyme from the first pool is bound to the membrane by electrostatic whereas the second pool LDH - by hydrophobic forces.     

Interaction of adenylosuccinate synthetase with F-actin

Both crude and purified preparations of adenylosuccinate synthetase from muscle were found to combine with, and dissociate from, muscle debris precipitated from a homogenate of the muscle with water. The binding and dissociation depended on ionic strength. Further study showed that the muscle enzyme was adsorbed to F-actin, but not to G-actin or myosin. The muscle-type enzyme from the liver also associated with F-actin, but the liver-type enzyme from the liver did not. In the absence of KCl the molar ratio of adenylosuccinate synthetase from skeletal muscle to actin monomer in F-actin in the complex formed was 1 to 4. From a Scatchard plot the dissociation constant was calculated to be 0.72 micrometer. The binding was maximal at pH 5.5-7 in 30 mM potassium phosphate buffer. The complex was completely dissociated in the presence of 0.21 M KCl. The physiological significance of this binding is discussed on the basis of these findings.     

Changes in enzyme binding and activity during aestivation in the frog Neobatrachus pelobatoides

1. The proportion of aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) associated with the particulate fraction of a cell was measured in aestivating and non-aestivating Neobatrachus pelobatoides. 2. Reduced binding of these enzymes was found in the brain, indicating lower glycolytic flux. This was not correlated to metabolic rate suggesting that glycolytic rate was reduced in this tissue in the early stages of aestivation, possibly due to a change in fuel use. 3. Measurement of total enzyme levels showed that the liver of aestivating frogs had less GAPDH and less aldolase than non-aestivating frogs.     

Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids. The contribution of the ATP/ADP binding site to this process can also be considered. In the case of pig skeletal muscle LDH, neither the cofactor binding site nor the subunit interfacing areas seem to be involved in the interaction.     

粘虫飞行肌中与能量代谢有关的酶活性研究

该文报道粘虫Mythimna separata (Walker ) 蛹及不同日龄成虫飞行肌中与3 种代谢途径有关的5 种酶,即3-磷酸甘油醛脱氢酶(GAPDH)、3-磷酸甘油脱氢酶(GDH)、乳酸脱氢酶(LDH)、3-羟酰辅酶A 脱氢酶(HOAD)、柠檬酸合成酶(CS)活性的变化。成虫羽化后,这5 种酶的活性大多数都高于蛹期,表明成虫飞行肌与能量代谢有关的活动比蛹期高。不同日龄成虫飞行肌的能量代谢特点为：成虫羽化后糖酵解循环的活性增加;1 日龄进行糖酵解的能力较强,2 日龄即具备较强的脂肪代谢能力,2～5日龄糖及脂肪代谢的能力基本相当,但7日龄脂肪代谢的能力较强。1～7日龄粘虫蛾飞行肌具有较高的GDH 和LDH活性,这既是粘虫蛾飞行肌能进行高度有氧代谢的重要标志,也是其具有一定无氧代谢能力的最好说明,而飞行肌中较高的CS活性则是粘虫蛾具有较强飞行能力的重要保证。对成虫GAPDH∶HOAD 活性进行分析比较的结果还显示,粘虫蛾持续飞行的能源物质既有脂类也有糖类,而不仅仅只限于脂类。     

HISTOCHEMISTRY OF LACTIC DEHYDROGENASE IN HEART AND PECTORALIS MUSCLES OF RAT

Left-ventricular heart muscle and pectoralis major muscle of the rat were studied to determine the intracellular localization of lactic dehydrogenase (LDH) isoenzymes. Fixation of tissue for 2 hr in 2% buffered formaldehyde provided the best preservation of the ultrastructure and enzyme activity. Total LDH activity was found diffusely in the ground substance of the sarcoplasm and in the mitochondria of the heart muscle. In skeletal muscle a strong reaction was noted in the sarcoplasmic reticulum, and moderate activity was seen in the ground substance of the sarcoplasm and in the mitochondria. Differentiation of the isoenzymes of LDH was accomplished by addition of 4 M urea or application of heat. Heart-type isoenzymes were mainly localized in the mitochondria and sarcoplasm, whereas muscle-type isoenzymes were localized mainly in the sarcoplasmic reticulum of the skeletal muscle. It is speculated that the sarcoplasmic reticulum of the skeletal muscle is the site of anaerobic glycolysis and that the sarcoplasm and mitochondria are involved primarily in aerobic metabolism of pyruvate.     

High performance purification of glycolytic enzymes and creatine kinase from chicken breast muscle and preparation of their specific immunological probes

We developed a novel procedure for isolation of the muscle isozymes of aldolase, triose phosphate isomerase (TPI), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), enolase, pyruvate kinase (PK) and lactic dehydrogenase (LDH), and also creatine kinase (CK), at high purity, specific activity and yield. Protein was extracted from chicken breast muscle and glycolytic enzymes were purified by a three step procedure consisting of: Ammonium sulfate combined with pH fractionation. Phosphocellulose chromatography with performance of high pressure liquid chromatography, exploiting a pH gradient formed by a gradient of the buffering ion for protein elution. Affinity chromatography causing elution by substrate or pH. The enzymes, obtained at over 95% purity as judged by specific activity and silver stained electropherograms, were injected into sheep. Antibody for each enzyme was purified on specific immunosorbant and its specificity was verified by immunotransfer analysis.     

Studies on the nature of the subcellular localization of lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase in chicken skeletal muscle

The elucidation of the subcellular localization of enzymes by the classical technique of homogenization followed by differential centrifugation is limited in that it is difficult to determine the effect of the severe disruptive procedures on the normal relationship of the enzymes to their subcellular environment. Attempts have been made to study this problem under less severe limitations; one of the approaches used has been the use of pressure on whole muscle tissue to extract the cellular fluids. In this report we introduce the concept of “comparative extraction” for evaluation of results obtained by this procedure. By comparing the efflux of enzymes of similar solubility and similar size and shape, it is possible to determine the minimal amount of the less easily extractable enzyme which cannot be removed due to compartmentation or binding to cellular particulate structures. Using this concept of “comparative extraction,” we show in this report that at least 35% of the lactate dehydrogenase of chicken breast muscle is restricted in its removal. The data do not definitely resolve the problem of whether the restriction is due to compartmentation of the enzyme within subcellular organelles or binding to subcellular structures.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values <6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.     

Comparative study of free and bound glycolytic enzymes from sea scorpion brain.

Free and bound forms of hexokinase, pyruvate kinase, and lactate dehydrogenase were prepared from the brain of the sea scorpion (Scorpaena porcus) in a low ionic strength medium. Properties of the free and bound forms were compared to determine whether binding to particulate matter could influence enzyme function or stability in vivo. Changes in pH differently affected the activity of the free and bound forms of all three enzymes. Furthermore, bound forms of hexokinase and pyruvate kinase were more stable than the free enzymes to heating at 45 degrees C. Bound hexokinase showed higher affinity for substrates (ATP, glucose) than the free form and bound lactate dehydrogenase had greater affinity for pyruvate and NADH. Although the affinities of the two forms of pyruvate kinase for substrates were similar, Hill coefficients for phosphoenolpyruvate as well as inhibition by ATP differed between the two enzyme forms. Free and bound lactate dehydrogenase also showed differences in Hill coefficients and bound lactate dehydrogenase was less sensitive to substrate inhibition by high pyruvate concentrations. The possible physiological role of the binding of these glycolytic enzymes to subcellular structures is discussed.     

Lactate dehydrogenase associated with the mitochondrial fraction and with a mitochondrial inhibitor--I. Enzyme binding to the mitochondrial fraction

Rabbit skeletal muscle mitochondrial fraction shows LDH activity (212 +/- 43 U/g pellet). The majority of the mitochondrial enzyme was solubilized by washing with 0.15 M NaCl, pH 6, or by ultrasonic treatment in the same medium. It was also solubilized on increasing the ionic strength and the pH of the medium. Cytosoluble LDH was observed to bind in vitro to the particulate fraction and the enzyme bound was a sigmoidal function of the amount of soluble enzyme added. The bound enzyme is less active than the soluble one. Kinetically, active mitochondrial fraction or in vitro bound enzyme showed non-hyperbolic behavior which is different from the bi-bi sequential-ordered type mechanism of the soluble enzyme.