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 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.     

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.     

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.     

Relationship between hydroxypyruvate and the production of oxalate in vitro.

Chicken liver lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC1.1.1.27) catalyses the reversible reduction reaction of hydroxypyruvate to L-glycerate. It also catalyses the oxidation reaction of the hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form of glyoxylate to oxalate and the reduction of the non-hydrated form to glycolate. At pH 8, these latter two reactions are coupled. The coupled system equilibrium is attained when the NAD+/NADH ratio is greater than unity. Hydroxypyruvate binds to the enzyme at the same site as the pyruvate. When there are substances with greater affinity to this site in the reaction medium and their concentration is very high, hydroxypyruvate binds to the enzyme at the L-lactate site. In vitro and with purified preparation of lactate dehydrogenase, hydroxypyruvate stimulates the production of oxalate from glyoxylate-hydrated form and from NAD; the effect is due to the fact that hydroxypyruvate prevents the binding of non-hydrated form of glyoxylate to the lactate dehydrogenase in the pyruvate binding site. At pH 8, THE L-glycerate stimulates the production of glycolate from glyoxylate-non-hydrated form and NADH since hydroxypyruvate prevents the binding of glyoxylate-hydrated form to the enzyme     

Structure of lactate dehydrogenase inhibitor generated from coenzyme.

Two inhibitors of lactate dehydrogenase generated during NADH storage have been isolated by chromatography. One is a dimer of the dinucleotide where the AMP moiety is unmodified. The other is also generated from NAD+ in the presence of a high concentration of phosphate ions at alkaline pH. This inhibitor was proved to be the addition compound of one phosphate group to position C-4 of the nicotinamide ring of NAD+ by NMR spectroscopy, enzymatic cleavage, and dissociation to NAD+ at neutral pH. This compound is a competitive inhibitor with respect to NAD+ in the presence of the lactate dehydrogenase with a Ki of 2 X 10(-7) M. The interaction of this inhibitor with lactate dehydrogenase is discussed relative to the structure of this enzyme.     

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.     

The interaction of pyridine-adenine-dinucleotides containing a hydrazide group with swine muscle lactate dehydrogenase]

Hydrazide group of 4-substituted NAD analogues is shown to interact with functional groups of substrate-binding site in double complexes with pig muscle lactate dehydrogenase (isoenzyme M4). The lactic acid residue, which is structurally incorporated into NAD analogue, improves slightly the binding of dinucleotide, while 2,2,6,6-tetramethylpiperidine-1-oxyl residue considerably decreases the firmless of binding. The comparison of the inhibitory ability of oxamate, incotinic acid hydraxide and their spin-labelled derivatives indicates the restricted and stiff sizes of a substrate-binding site.     

Interactions of glycolytic enzymes with erythrocyte membranes

Partition equilibrium experiments have been used to characterize the interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and lactate dehydrogenase, in 5 mM sodium phosphate buffer (pH 7.4). For each of these tetrameric enzymes a single intrinsic association constant sufficed to describe its interaction with erythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these findings are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.     

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.     

Lactate dehydrogenase isoenzymes of human semen

1. A lactate dehydrogenase isoenzyme present in human spermatozoa and semen was isolated and characterized biochemically in term of its pH for optimum activity and by means of K(m) values for lactate, NAD(+) and NAD analogues. The results were compared with those obtained with the human heart-type and the liver-type lactate dehydrogenase isoenzymes. 2. The enzyme was characterized by its resistance to digestion with different proteolytic enzymes. The time for 50% digestion in terms of residual dehydrogenase activity was compared with times obtained for the H(4)- and M(4)-types.     

The elucidation of the effect of ammonium chloride on pyruvate distribution and pyruvate dehydrogenase interconversion in isolated rat hepatocytes

The distribution of pyruvate between cell compartments measured in isolated hepatocytes in the presence of lactate was in agreement with delta pH across plasma and mitochondrial membranes. In isolated liver mitochondria NH4Cl decreased the transmembrane potential (delta psi) by about 14 mV, whereas no change of delta pH was observed. In the presence of lactate or alanine NH4Cl increased the mitochondrial pyruvate concentration presumably due to the inhibition of the flux through pyruvate carboxylase. In the presence of lactate or alanine changes in the amount of the active form of pyruvate dehydrogenase (PDHa) were correlated with the mitochondrial pyruvate concentration, NH4Cl increased the amount of PDHa by lowering the mitochondrial ATP/ADP and NADH/NAD+ ratios.     

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.     

The binding of oxidized and reduced nicotinamide–adenine dinucleotides to bovine liver uridine diphosphate glucose dehydrogenase

The binding of NAD(+) and NADH to bovine liver UDP-glucose dehydrogenase was studied by using gel-filtration and fluorescence-titration methods. The enzyme bound 0.5mol of NAD(+) and 2 mol of NADH/mol of subunit at saturating concentrations of both substrate and product. The dissociation constant for NADH was 4.3mum. The binding of NAD(+) to the enzyme resulted in a small quench of protein fluorescence whereas the binding of NADH resulted in a much larger (60-70%) quench of protein fluorescence. The binding of NADH to the enzyme was pH-dependent. At pH8.1 a biphasic profile was obtained on titrating the enzyme with NADH, whereas at pH8.8 the titration profile was hyperbolic. UDP-xylose, and to a lesser extent UDP-glucuronic acid, lowered the apparent affinity of the enzyme for NADH.     

Lactate dehydrogenase from the northern krill Meganyctiphanes norvegica: comparison with LDH from the Antarctic krill Euphausia superba

Electrophoretic polymorphism of lactate dehydrogenase (LDH, EC 1.1.1.27) from abdominal muscle is reported in the northern krill Meganyctiphanes norvegica. In the population, from the Gullmarsfjord (west coast of Sweden), LDH was encoded for by two different Ldh-A* and -B* loci. The isoenzymes were named according to their electrophoretic mobilities. Ldh-A* locus was polymorphic. The allelic frequencies were a=0.99, a'=0.002, a"=0.004, a"'=0.004. The level of LDH polymorphism is low. Most individuals possess the same amount of two LDH homopolymers (LDH-A*(4) and LDH-B*(4)). The Meganyctiphanes norvegica LDH-A*(4) and LDH-B*(4) isoenzymes and the predominant LDH-A*(4) isoenzyme from Euphausia superba were purified to specific activities of 294, 306 and 464 micromol NADH min(-1) mg(-1), respectively. In both species the LDH isoenzymes were separated by chromatofocusing. All three isoenzymes are L-specific tetramers with molecular weight of approximately 160 kDa. Northern krill LDH-A*(4) has higher affinity for pyruvate and lactate and is more thermostable than LDH-B*(4). Both isoenzymes are inhibited significantly by high concentration of pyruvate but not lactate. Antarctic krill isoenzyme exhibits high substrate affinities, high NAD inhibition, high inhibition at 10 mM pyruvate, lack of lactate inhibition, and high heat stability and resembles northern krill LDH-A*(4) isoenzyme.     

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.     

Transient-kinetic studies of pig muscle lactate dehydrogenase

1. The very fast pre-steady-state formation of NADH catalysed by pig M(4) lactate dehydrogenase was equivalent to the enzyme-site concentration at pH values greater than 8.0 and to one-half the site concentration at pH6.8. 2. The rate of dissociation of NADH from the enzyme at pH8.0 (450s(-1)) in the absence of other substrates is faster than the steady-state oxidation of lactate (80s(-1)). The latter process is therefore controlled by a step before NADH dissociation but subsequent to the hydride transfer. 3. The oxidation of enzyme-NADH by excess of pyruvate was studied as a first-order process at pH9.0. There was no effect of NADD on this reaction and it was concluded that the ternary complex undergoes a rate-limiting change before the hydride-transfer step. 4. Some conclusions about the reactions catalysed by the M(4) isoenzyme were drawn from a comparison of these results with those obtained with the H(4) isoenzyme and liver alcohol dehydrogenase.     

Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group

1. The formation of the non-enzymic adduct of NAD(+) and sulphite was investigated. In agreement with others we conclude that the dianion of sulphite adds to NAD(+). 2. The formation of ternary complexes of either lactate dehydrogenase or malate dehydrogenase with NAD(+) and sulphite was investigated. The u.v. spectrum of the NAD-sulphite adduct was the same whether free or enzyme-bound at either pH6 or pH8. This suggests that the free and enzyme-bound adducts have a similar electronic structure. 3. The effect of pH on the concentration of NAD-sulphite bound to both enzymes was measured in a new titration apparatus. Unlike the non-enzymic adduct (where the stability change with pH simply reflects HSO(3) (-)=SO(3) (2-)+H(+)), the enzyme-bound adduct showed a bell-shaped pH-stability curve, which indicated that an enzyme side chain of pK=6.2 must be protonated for the complex to form. Since the adduct does not bind to the enzyme when histidine-195 of lactate dehydrogenase is ethoxycarbonylated we conclude that the protein group involved is histidine-195. 4. The pH-dependence of the formation of a ternary complex of lactate dehydrogenase, NAD(+) and oxalate suggested that an enzyme group is protonated when this complex forms. 5. The rate at which NAD(+) binds to lactate dehydrogenase and malate dehydrogenase was measured by trapping the enzyme-bound NAD(+) by rapid reaction with sulphite. The rate of NAD(+) dissociation from the enzymes was calculated from the bimolecular association kinetic constant and from the equilibrium binding constant and was in both cases much faster than the forward V(max.). No kinetic evidence was found that suggested that there were interactions between protein subunits on binding NAD(+).     

31 P nuclear magnetic resonance studies of the interaction of pyridine nucleotide coenzymes with dehydrogenases.

31P nuclear magnetic resonance spectra of the pyrophosphate group in NAD+ and NADH were recorded in the presence of beef heart lactate dehydrogenase and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. At high lactate dehydrogenase concentrations (60 mg/ml), two NADH resonances are observed: a slowly exchanging peak which is shifted to 1.9 ppm downfield (relative to free NADH) and a rapidly exchanging peak with a downfield shift of 0.5-0.6 ppm. At lover concentrations (15 mg/ml) only the rapidly exchanging peak is observed thus indicating that the peak observed at-1.9 ppm is due to coenzyme bound to an aggregated enzyme species. With NAD+, rapid exchange and downfield shifts are observed at both enzyme and concentrations, with shifts of about 1.5 ppm and 0.6 ppm at 60 and 15 mg/ml, respectively. In the presence of glyceraldehydephosphate dehydrogenase, the results are independent of enzyme concentration, and slow exchange and upfield shifts of 0.4-0.6 ppm occur with each coenzyme. These data indicate that the environment of the pyrophosphate group of oxidized and reduced coenzyme is the same for a given dehydrogenase, but is different in one enzyme from the other. The resonances observed with glyceraldehydephosphate dehydrogenase are broader than those observed with lactate dehydrogenase. This is indicative of either shorter relaxation times with the former enzyme, or the presence of multiple, unresolved resonances.     

Interdependence of coenzyme-induced conformational work and binding potential in yeast alcohol and porcine heart lactate dehydrogenases: a hydrogen-deuterium exchange study

Binding of NAD coenzymes to yeast alcohol dehydrogenase (YADH) and porcine heart lactate dehydrogenase (PHLDH) was studied by hydrogen-deuterium exchange with the infrared technique. Conformational changes in the enzymes specific to the coenzymes and their fragments were observed, and the pH dependence of the exchange reaction shows that it conforms to the EX-2 scheme. In both YADH and PHLDH the magnitude of the conformational change of measured by exchange retardation is considerably larger for NAD+ than for NADH. Studies with coenzyme fragments like ADP-ribose, ADP, and AMP also highlight the lack of rigorous correlation between structural features such as charge and size and their influence on exchange behavior. Ternary complexes such as YADH-NAD+-pyrazole, PHLDH-NAD+-oxalate, and PHLDH-NADH-oxamate, which mimic the transition state, have a significantly more pronounced effect on exchange rates than the corresponding binary complexes. The outstanding feature of this study is the demonstration that in the binary enzyme-coenzyme complexes the more loosely bound NAD+ is more effective in retarding exchange than the more firmly bound NADH. These differences are attributed to the unequal structural constraints exerted by the two coenzymes upon the enzymes, which translate to unequal expenditure of transconformational work in the formation of the two complexes. The opposing variation in the free energy of binding and the transconformational work expended can be viewed as an unequal partitioning of the net free energy gain resulting from the protein-ligand interaction into a binding term and that required for conformational change.