Affinity chromatography of lactate dehydrogenase on immobilized nucleotides

The interaction of two isoenzymes of lactate dehydrogenase from pig heart muscle (H(4)) and rabbit skeletal muscle (M(4)), with immobilized nucleotides was examined: the effects of pH and temperature on the binding of lactate dehydrogenase were studied with immobilized NAD(+) matrices. The influence of substrate, product and sulphite on the binding of heart muscle lactate dehydrogenase to immobilized NAD(+) was investigated. The interaction of both lactate dehydrogenase isoenzymes with immobilized pyridine and adenine nucleotides and their derivatives were measured. The effects of these parameters on the interaction of lactate dehydrogenase with immobilized nucleotides were correlated with the known kinetic and molecular properties of the enzymes in free solution.     

Kinetic behaviour of soluble and mitochondrial bound lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The kinetic behaviour of mitochondrial bound enzyme fits a bibi sequential type mechanism as well as the cytosolic rabbit liver lactate dehydrogenase. The bound enzyme has greater values of Km(NADH) and Km(pyruvate) than the soluble one, suggesting that binding induces a decrease in the affinity of both substrates. The behaviour of the free and the mitochondrial-bound enzyme is of the Michaelis-Menten type, but the kinetics of a mixture of rabbit liver cytosolic and mitochondrial-bound lactate dehydrogenase is sigmoidal, suggesting that a cooperative phenomenon takes place.     

Thermodynamic studies of the activation of rabbit muscle lactate dehydrogenase by phosphate.

In an attempt to trace the source of phosphate activation of the enzyme-catalysed pyruvate-lactate interconversion by rabbit muscle lactate dehydrogenase, equilibrium constants were measured to examine the effects of phosphate on interactions pertinent to the enzymic process. Frontal gel-chromatographic studies of the binding of NADH to the enzyme established that the intrinsic association constant is doubled in the presence of 50 mM-phosphate in the buffer (pH 7.4, I0.15). From kinetic studies of the competition between NAD+ and NADH for the coenzyme-binding sites of the enzyme it is concluded that the binding of oxidized nicotinamide nucleotide is also doubled in the presence of 50 mM-phosphate. Competitive-inhibition studies and fluorescence-quenching measurements indicated the lack of a phosphate effect on ternary-complex formation between enzyme-NADH complex and oxamate, a substrate analogue of pyruvate. The equilibrium constant for the interaction between enzyme-NAD+ complex and oxalate, an analogue of lactate, was also shown, by difference spectroscopy, to be insensitive to phosphate concentration. Provided that the effects observed with the substrate analogues mimic those operative in the kinetic situation, the equilibrium constant governing the isomerization of ternary complex is also independent of phosphate concentration. It is concluded that enhanced coenzyme binding is the source of phosphate activation of the rabbit muscle lactate dehydrogenase system.     

Characterisation of a highly hydrophobically modified lactate dehydrogenase

1. Lysine residues of porcine H4 lactate dehydrogenase (L-lactate:NAD+ oxidoreductase EC 1.1.1.27) were modified with methyl-epsilon-(N-2,4-dinitrophenyl)aminocaproimidate - HCl. With increasing incorporation of the reagent a linear decrease of enzymatic activity was noticed. No essential lysyl group with an extraordinary reactivity was modified. 2. The active forms of the modified enzyme with different incorporation values were separated from denatured material by fractional precipitation and gel chromatography. An epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase was obtained with an average incorporation of 38 groups per tetramer and a residual activity of 42%. This material proved to be homogenous in cellulose electrophoresis. 3. The epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase is soluble only in glycine buffer at pH 8 and can be stabilized as ternary complex with NAD+ and sodium sulfite. Gel chromatography and ORD measurements show no strong conformational change. 4. epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase has similar Km values for pyruvate, NADH, lactate and NAD+ as the native enzyme, and shows a lower thermostability due to a diminished stabilization by the hydrate layer on the surface.     

Modulation of lactate dehydrogenase activity by enzyme-protein interaction

Some lactate dehydrogenase modulator proteins have been isolated from the lactate dehydrogenase-free crude mitochondrial fraction of rabbit muscle, beef liver and chicken liver. It was shown that beef and chicken liver mitochondrial extracts exhibited activatory capacity in contrast to the inhibitory capacity of rabbit muscle mitochondrial extracts. All modulators can be precipitated by 80% ammonium sulphate saturation and show high anodic electrophoretic mobility and heat stability. Modulators have higher affinity for alkaline pI lactate dehydrogenase isoenzymes, independent of whether the M and H subunits are predominant. The inhibitor and the activator molecules compete for lactate dehydrogenase since their modulatory capacity was nullified when similar relative amounts were used. This study shows the existence of analogous proteins with an acidic pI in the different mitochondrial fractions which modify lactate dehydrogenase activity.     

The use of auramine O to study ligand binding and subunit cooperativity of lactate dehydrogenase

The tetrameric molecule of pig skeletal muscle lactate dehydrogenase binds a cationic fluorescent probe, auramine O, at four equal non-interacting sites with a dissociation constant of (1.25 +/- 0.2) X 10(-4) M. Fluorescence of the dye/enzyme mixture is strongly pH-dependent, with a maximum at pH 6.3-6.8. Auramine O-binding sites are located outside the active center of the enzyme. The microenvironment of the bound dye changes upon interaction of lactate dehydrogenase with NAD+, NADH, ADP and pyruvate. The binding of specific ligands induces an increase in fluorescence of auramine O-enzyme complex. This effect was used to determine the dissociation constants of the complexes of lactate dehydrogenase with specific ligands. Pyruvate was demonstrated to bind to the apoenzyme-auramine O complex with a dissociation constant of 5.2 X 10(-4) M. With the use of auramine O, it became possible to reveal subunit interactions within the tetrameric molecule of lactate dehydrogenase. They are manifested in the changes of the microenvironment of a dye-binding site located on one of the subunits induced by the binding of ligands in the active center of a neighboring subunit.     

Phosphorylation of lactate dehydrogenase by ATP

Evidence is presented indicating that phosphorylation of porcine muscle lactate dehydrogenase by [gamma-32P] ATP occurs at carboxyl residues of the protein. The phosphoenzyme complex was moderately stable at pH 6.8 and 25 degrees C, with a half-life of 3.5 h. In the presence of NADH rapid dephosphorylation occurred. Formation of an abortive complex with NAD-pyruvate also caused hydrolysis of the phosphoenzyme. The phosphorylated lactate dehydrogenase was shown to serve as a phosphate donor for phosphorylation of ADP.     

Interaction of bovine heart lactate dehydrogenase with erythrocyte lipids

The interaction between bovine heart lactate dehydrogenase and erythrocyte lipid suspension as a function of pH, NAD, NADH, lipid and salt concentration was studied by ultracentrifugation. In the presence of erythrocyte lipid liposomes the enzyme forms two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. The two complexes reveal different dependence of their stability on pH values. Lactate dehydrogenase decreases its specific activity when it binds to the phospholipid molecules. Efficient adsorption of lactate dehydrogenase to liposomes occurs in their pH range 6.0-8.0 and at low ionic strength. The adsorption is diminished in the presence of NAD+ but it is not influenced by NADH. Possible mechanisms of the interaction and implications for the function in vivo are discussed.     

Modification of lactate dehydrogenase by pyridoxal phosphate and adenosine polyphosphopyridoxal

Pyridoxal phosphate reacts with not only the lysyl residue(s) essential for enzymatic activity but also other reactive lysyl residues in rabbit muscle lactate dehydrogenase (EC 1.1.1.27). To raise the specificity of pyridoxal phosphate, adenosine diphospho-, triphospho-, and tetraphosphopyridoxals have been newly synthesized and used for modification of the enzyme. Incubation of the enzyme for 30 min with the diphospho, triphospho, and tetraphospho compounds all at 1 mM followed by reduction by sodium borohydride resulted in the loss of enzymatic activity by 64, 51, and 34%, respectively. NADH almost completely protected the enzyme from inactivation, whereas pyruvate showed no protection. Binding of the reagents to the enzyme subunit in an equimolar amount corresponds to the complete inactivation. The adenosine diphosphopyridoxal modified enzymes with different residual activities were chromatographed on a Blue Toyopearl affinity column. The results showed the presence of at least four enzyme species besides the intact enzyme that are significantly different from one another in the amount of the reagent bound, the affinity for NADH, and the specific activity. The decrease in the affinity of the enzyme for NADH and the loss of enzymatic activity paralleled in the modification by adenosine diphosphopyridoxal, whereas, in the modification by pyridoxal phosphate, the decrease in the affinity for NADH preceded the inactivation. It is concluded that modification by adenosine polyphosphopyridoxal compounds are specific for the active site lysyl residue(s) in lactate dehydrogenase.     

Affinity liquid-liquid extraction of lactate dehydrogenase on a large scale

Rapid liquid-liquid extraction of lactate dehydrogenase from muscle by using a low-cost aqueous bipolymer two-phase system was achieved by using a centrifugal separator. Extraction of the target enzyme into the upper phase was enhanced by including the dye Procion yellow HE-3G (bound to polyethylene glycol). The dye acted as an affinity ligand for the enzyme. The isolation of the enzyme was carried out either by using a cell extract or by homogenizing the muscle directly in the system. The latter approach reduced the preparation time with a factor of 0.25. The two methods gave, respectively, 310 and 360 kU lactate dehydrogenase/kg muscle (measured at 22 degrees C). By using a small centrifugal separator, Alfa Laval LAPX 202, 3-5 kg muscle could be processed/h in a 30-L, two-phase system.     

Interaction between glyceraldehyde-3-phosphate-dehydrogenase and lactate dehydrogenase

Physical interaction between rabbit muscle glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase was detected by means of matrix immobilization technique. Glyceraldehyde-3-phosphate dehydrogenase covalently bound to CNBr-activated Sepharose 4B was capable of forming a complex with soluble lactate dehydrogenase with a stoichiometry of 0.8 mole of lactate dehydrogenase per mole of glyceraldehyde-3-phosphate dehydrogenase and KD of 0.385 microM at pH 6.5. The bienzyme association weakened when pH changed to 7.0 (the KD increased to 1.25 microM).     

Interaction of bovine skeletal muscle lactate dehydrogenase with liposomes. Comparison with the data for the heart enzyme

The effects of pH, salt concentration and the presence of oxidized and reduced forms of coenzyme on the interaction of skeletal muscle lactate dehydrogenase with the liposomes derived from the total fraction of bovine erythrocyte lipids were investigated by ultracentrifugation and were compared with those results obtained using the heart-rate isoenzyme which we have previously studied. Liposomes are good adsorptive systems for both types of isoenzyme. In the presence of erythrocyte lipid liposomes, bovine muscle and heart lactate dehydrogenases form two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. Soluble protein-phospholipid complexes reveal different dependences of their stabilities on pH values and it seems that the nature of the binding site in either isozyme is different. In addition, absorption of the isoenzymes on the liposomes also reveals in difference in the effects of NAD and NADH. While the presence of NAD dissociates LDH-H₄ from the liposomes and NADH does not influence its adsorption, NAD promotes the binding of LDH-M₄, and NADH favors the dissociation.     

Interaction of bovine skeletal muscle lactate dehydrogenase with liposomes. Comparison with the data for the heart enzyme

The effects of pH, salt concentration and the presence of oxidized and reduced forms of coenzyme on the interaction of skeletal muscle lactate dehydrogenase with the liposomes derived from the total fraction of bovine erythrocyte lipids were investigated by ultracentrifugation and were compared with those results obtained using the heart-rate isoenzyme which we have previously studied. Liposomes are good adsorptive systems for both types of isoenzyme. In the presence of erythrocyte lipid liposomes, bovine muscle and heart lactate dehydrogenases form two kinds of complex: lactate dehydrogenase adsorbed to liposomes and soluble lactate dehydrogenase-phospholipid complexes. Soluble protein-phospholipid complexes reveal different dependences of their stabilities on pH values and it seems that the nature of the binding site in either isozyme is different. In addition, absorption of the isoenzymes on the liposomes also reveals in difference in the effects of NAD and NADH. While the presence of NAD dissociates LDH-H4 from the liposomes and NADH does not influence its adsorption, NAD promotes the binding of LDH-M4, and NADH favors the dissociation.     

Immobilization of lactate dehydrogenase on polyacrylamide beads

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.     

Autophosphorylation of lactate dehydrogenase

Incubation of rabbit muscle lactate dehydrogenase in the presence of Mg[alpha-32p]ATP results in the incorporation of the label into the protein. The autophosphorylation reaction is strongly pH-dependent. The maximal phosphorylation is observed at pH 6.8 with 3-4 moles of phosphate bound per mole of tetrameric enzyme. The enzyme-phosphate complex is readily hydrolyzed by hydroxylamine.     

Changes in the lactate dehydrogenase isoenzyme pattern during differentiation of rabbit bone-marrow erythroid cells.

1. Differentiation and maturation of rabbit bone-marrow erythroid cells was accompanied by a 15-fold decrease in lactate dehydrogenase activity from approx. 0.1pmol of NADH utilized/min per cell in basophilic cells to 0.007 pmol of NADH/min per cell in reticulocytes. 2. In early cells, cell division takes place with a corresponding decrease in cell volume, but the concentration of lactate dehydrogenase remains almost constant. 3. When cell division ceases, qualitative as well as quantitative changes in the lactate dehydrogenase isoenzyme pattern become apparent and reticulocytes were found to contain almost exclusively the H4 isoenzyme, whereas early erythroblasts contained also the M4 and hybrid isoenzymes. 4. Extracts from a lysosome-enriched subcellular fraction of bone-marrow erythroid cells specifically degraded the M4 isoenzyme in vitro, but the H4 form was stable. It is suggested that lysosomal enzymes are involved in bringing about the observed changes in lactate dehydrogenase isoenzyme patterns in vivo.     

Regulation of enzyme activity in adsorptive enzyme systems. III. Interaction of pig muscle lactate dehydrogenase with F-actin

The binding of pig skeletal muscle lactate dehydrogenase by F-actin has been studied using the sedimentation method in 10 mM Tris-acetate buffer, pH 6.0 at 20 degrees C. Adsorption capacity of F-actin is equal to (1 +/- 0.1) . 10(-5) moles of lactate dehydrogenase per 1 g of actin. NADH decreases the affinity of F-actin with respect to lactate dehydrogenase. The binding of lactate dehydrogenase by F-actin in diminishing the rate of enzymatic reduction of alpha-ketoglutarate. The microscopic dissociation constant for the complex of the enzyme with F-actin which is estimated from the dependence of the enzymatic reaction rate of F-actin concentration at saturating NADH concentrations is equal (3.0 +2- 0.5) . 10(-7) M. It has been shown that the bound enzyme is characterized by the greater value of Km and the lower value of Vmax in comparison to the free enzyme.     

The disproportionation of glyoxylate by lactate dehydrogenase

Lactate dehydrogenase (EC 1.1.1.27) catalyzes the NAD-dependent oxidation to (oxalate) and reduction (to glycollate) of glyoxylate. The kinetics of this disproportionation are in accord with the usual reaction pathway of lactate dehydrogenase:substrate inhibition with appropriate pH dependence occurs; a steady state in the ratio of NADH to NAD⁺ is set up during the reaction, has the expected dependence on pH, and is independent of the initial glyoxylate, coenzyme, and enzyme concentration. At pH 7 the lactate dehydrogenase-NADH complex is about fivefold more likely to react with and reduce glyoxylate (at a concentration of 100 mm) than to dissociate to produce free NADH, and the ratio of the fraction of the enzyme-NADH complex which dissociates to the fraction which reacts with and reduces glyoxylate varies with glyoxylate concentration and with pH in a manner in agreement with the normal reaction pathway of the enzyme. With all concentrations of glyoxylate and over the pH range 7–9.6 both free (not enzyme bound) NAD⁺ and free NADH are formed in the steady state of the disproportionation. From these results it is apparent that lactate dehydrogenase, like alcohol dehydrogenase (EC 1.1.1.1), catalyzes a disproportionation within the bounds of its normal kinetic reaction pathway.     

Substrate-induced conformational changes in lactate dehydrogenase. Proteolysis of the immobilized enzyme in the presence of specific substrates.

We report here a new approach to the study of the conformation of enzymes in the presence of specific substrates. Rabbit muscle lactate dehydrogenase was attached to CL-Sepharose via a cleavable spacer arm (-NH-(CH2)6NHCO(CH2)2SS(CH2)2CO-). The bound lactate dehydrogenase was digested with subtilisin BPN' in the presence of substrates of lactate dehydrogenase. The use of a flow system permits the maintenance of saturating levels of substrates. Proteolysis was followed by loss of activity of the enzyme column. The time course of proteolysis in the presence of either NADH, NAD+, or pyruvate alone did not differ from the control. However, when NADH and pyruvate were present simultaneously, the enzyme became more susceptible to proteolysis. The initial rate of proteolysis was increased by 40%. The abortive ternary complex (lactate dehydrogenase - NAD+ - pyruvate) also showed an increase in susceptibility to proteolysis. These findings clearly show that the productive ternary complex (lactate dehydrogenase - NADH - pyruvate) is conformationally different from the apoenzyme and binary complexes under optimal catalytic conditions.     

Characterization of a lactate dehydrogenase inhibitor protein from rabbit skeletal muscle mitochondrial fraction

A lactate dehydrogenase inhibitor protein is isolated from rabbit skeletal muscle crude mitochondrial fraction. The molecular weight of the inhibitor is approximately 20,000 as determined by size exclusion HPLC. The inhibitor isoelectricpH is 5.3 as determined by agarose or polyacrylamide gel isoelectric focusing. The amino acid composition of the inhibitor is given. The presence of the inhibitor gives an acidic characteristic to the alkaline M₄ lactate dehydrogenase isozyme and the lactate dehydrogenase-inhibitor complex is more stable than the enzyme alone.