Free and membrane-bound lactate dehydrogenase from white driving muscles of skate.

Purified cytoplasmic and membrane-bound lactate dehydrogenases (LDH) from white muscle of skate were characterized, Km for pyruvate and NADH for purified LDH were 150 +/- 16 and 29 +/- 7 microM, and for membrane-bound LDH were 185 +/- 22 and 7.5 +/- 1.5 microM, respectively. The membrane-bound enzyme was not inhibited by high pyruvate concentration (up to 20 mM) in contrast to purified LDH. Part of membrane-bound LDH was released by incubation in solutions with a high level of KCl (up to 1 M) or at alkaline pH. The inactivation rate during trypsin digestion for solubilized LDH was 2-3-fold higher than that for the membrane-bound enzyme.     

Lactate dehydrogenase from gastrocnemius muscle of turtle

Five bands of lactate dehydrogenase (LDH) isoenzymes were seen by polyacrylamide gel electrophoresis in gastrocnemius muscle of the turtle (Kachuga smithi). The major band was of M2H2 type and was partially purified by gel filtration and affinity chromatography. The specific activity of the enzyme was 2.6 units/mg protein. The half-life of the enzyme at 4 degrees C, was about 7 days. The optimum temperature for enzyme activity was 30 degrees C and the enzyme was irreversibly inactivated at 40 degrees C. The optimum pH for the forward reaction (pyruvate to lactate) was 5.5, while for reverse reaction it was between 8.0 to 9.5. The apparent Km values for pyruvate, NADH, lactate and NAD+ were 0.20, 0.013, 25 and 0.333 mM, respectively. Oxalate was found to be the inhibitor of LDH with Ki of about 4.2 mM.     

Regulating effect of mitochondrial lactate dehydrogenase on oxidation of cytoplasmic NADH via an "external" pathway in skeletal muscle mitochondria.

1. The specific activity of lactate dehydrogenase of skeletal muscle mitochondria was found to be 2.5 times lower than specific activity of total NADH-cytochrome c reductase. 2. The specific activity of mitochondrial LDH in skeletal muscle mitochondria was almost equal to the activity of rotenone-insensitive NADH-cytochrome c reductase. 3. Mitochondrial LDH acting as an oxidase of lactate to pyruvate may feed an "external" pathway, but the activity of the mitochondrial enzyme is a limiting factor in oxidation of lactate-derived NADH. 4. Mitochondrial LDH acting as a reductase of pyruvate to lactate successfully competes with an "external" pathway for cytoplasmic NADH. 5. Exogenous NADH oxidation via an "external" pathway was inhibited by pyruvic acid. This inhibition was overcome by addition of oxamic acid or hydrazine.     

Functional Properties of Lactate Dehydrogenase from Dunaliella salina and Its Role in Glycerol Synthesis

The dependence of the catalytic properties of lactate dehydrogenase (LDH, EC 1.1.1.27) from a halophilic alga Dunaliella salina, a glycophilic alga Chlamydomonas reinhardtii, and from porcine muscle on glycerol concentration, medium pH, and temperature was investigated. Several chemical properties of the enzyme from D. salina differentiated it from the LDH preparation obtained from C. reinhardtii and any homologous enzymes of plant, animal, and bacterial origin. (1) V _max of pyruvate reduction manifested low sensitivity to the major intracellular osmolyte, glycerol. (2) The affinity of LDH for its coenzyme NADH dropped in the physiological pH region of 6–8. Above pH 8, NADH virtually did not bind to LDH, while the enzyme affinity for pyruvate did not change considerably. (3) The enzyme thermostability was extremely low: LDH was completely inactivated at room temperature within 30 min. The optimum temperature for pyruvate reduction (32°C) was considerably lower than with the enzyme preparations from C. reinhardtii (52°C) and porcine muscle (61°C). (4) NADH greatly stabilized LDH: the ratio of LDH inactivation constants in the absence of the coenzyme and after NADH addition at the optimum temperature in the preparation from D. salina exceeded the corresponding indices of LDH preparations from C. reinhardtii twelve times and from porcine muscle eight times. The authors believe that these LDH properties match the specific metabolism of D. salina which is set at rapid glycerol synthesis under hyperosmotic stress conditions. The increase of cytoplasmic pH value produced in D. salina by the hyperosmotic shock can switch off the terminal reaction of the glycolytic pathway and thus provide for the most efficient utilization of NADH in the cycle of glycerol synthesis. As LDH is destabilized in the absence of NADH, this reaction is also switched off. In the course of alga adaptation to the hyperosmotic shock, glycerol accumulation and the neutralization of intracellular pH stabilize LDH, thus creating the conditions for restoring the complete glycolytic cycle.     

Molecular Dynamics Simulations of L-Lactate Dehydrogenase: Conformation of a Mobile Loop and Influence of the Tetrameric Protein Environment

L-lactate dehydrogenase (LDH) catalyses the interconversion of pyruvate and L-lactate in the presence of the coenzyme NADH. Molecular dynamics (MD) simulations have been performed for LDH complexed with NADH and the pyruvate-analogue inhibitor oxamate with the aim of characterising important influences on maintaining the geometry and hydrogen bond network of the active site. Two features in particular were found to dominate. First, the tetrameric protein environment is found to play a significant role in maintaining the active-site geometry. Simulations of the monomer alone reproduce the crystallographic structure poorly, and at least part of the neighboring subunit is necessary to prevent water penetration into the active site and to provide rigidity to the α1G-α2G helix immediately adjacent to the active site. These results offer one explanation for the observation that the monomer is not biologically active. Second, the conformation of Arg109 (part of the mobile loop which closes over the active site) is shown to play a key role in maintaining the active-site geometry. In some simulations, a torsional rotation in the side chain of Arg109 results in the breaking of crystallographic hydrogen bonds which are important for polarising the carbonyl bond of the substrate. This conformational change appears to be a trigger for the opening of the mobile loop. Long-range nonbonded interactions are found to be influential in maintaining the proper crystallographic conformation of Arg109. Thus, we conclude that to adequately model LDH, at least part of the neighboring subunit must be included in the MD simulations and nonbonded interactions must be properly represented to ensure that Arg109 remains in the crystallographic conformation. Out of a set of simulation protocols tested here, one meets both these criteria and will be used for the generation of starting structures for future reaction-mechanism calculations.     

Energy enzymes of the flight muscle of the house sparrow, Passer domesticus--III. Comparative physical properties of heart and flight muscle lactate dehydrogenase

Purification of heart (LDH-4) and flight muscle (LDH-2 and LDH-3) lactate dehydrogenase isoenzymes from the house sparrow, Passer domesticus, has been accomplished. Although these isoenzymes electrophoretically migrate reversed to most other vertebrate LDH isoenzymes, comparison of the amino acid compositions of LDH-4 and LDH-2-LDH-3 fails to reveal the basis for their reversed electrophoretic migration. Amino acid compositions did reveal mol. wts between 141,000-142,000 as well as vp of 0.744 ml/g (LDH-4) and 0.745 ml/g (LDH-2-LDH-3). SDS-gel electrophoresis yielded single bands for each preparation with mol. wts of 35,000 suggesting that LDH in this species exists as a tetramer. LDH-4 has a lower Km for both pyruvate (0.005 mM) and NADH (0.002 mM) than does LDH-2-LDH-3 (0.062 mM for pyruvate, 0.013 mM for NADH).     

Thiol groups of chicken liver LDH (author's transl)]

Crystallized chicken liver H4 lactatedehydrogenase with PCBM and DTNB, proved to have sic thiol groups per enzyme molecule. Sulphydryl groups seemed necessary for activity since the enzyme became inactive when the groups were blocked by PCMB, DTNB or by Zn (II), Cu (II) or Hg (II). LDH inhibited by Hg (II) recovered its activity after treatment with beta-mercaptoentanol. LDH reversible inactivation, caused by PCMB, was partially impeded by NAD, NADH hand L-lactate but inactivation caused by DTNB was impeded in any way by coenzymes or substrates. PCMB is a competitive inhibitor with the coenzymes but is non-competitive with the substrates whereas DTNB is a competitive inhibitor with NADH or L-lactate. Kinetic studies of the DTNB inactivation suggest the possible formation of a DTNB-LDH-NADH complex. The formation of LDH-NADH and LDH-NAD pyruvate inactive complexes have been detected by U.V. absorbancy measurements. Such inactive complexes have equally been observed experimenting with the PCMB of Hg (II) previously treated enzyme. The results showed that these essential sulphydryl groups are not involved in th attaching of coenzymes or substrates to the chicken liver LDH molecule, but they seem to suggest the participation of --SH groups during the reversible hydrogen transfer between NADH and pyruvate.     

Ethanol production by anaerobic thermophilic bacteria: regulation of lactate dehydrogenase activity in Clostridium thermohydrosulfuricum

Summary The enzyme lactate dehydrogenase (LDH) in Clostridium thermohydrosulfuricum is controlled by the type and the concentration of the substrate. In batch fermentations an increase of the initial concentration of glucose leads to an increase in the activity of LDH. This increase in activity is related to the accumulation of fructose 1,6-diphosphate (F 1,6-DP), an intermediate of the Embden-Meyerhof-Parnas (EMP) pathway, which stimulates the enzyme by increasing its affinity for pyruvate and NADH. The K _mvalues of LDH for pyruvate and NADH, which are 2.5×10^-3 M and 9.1×10^-5 M respectively in absence of F 1,6-DP, fall considerably in the presence of this substrate. In presence of 0.2 mM of F 1,6-DP we observed a K _mof 3.3×10^-4 M for pyruvate and 4.1×10^-5 M for NADH.     

Catalytic properties of lactate dehydrogenase in Homarus americanus.

Lobster tail and leg lactate dehydrogenases (LDH) have been characterized kinetically. The four binding sites for reduced coenzyme have been shown to be equivalent for the enzyme purified from lobster tail muscle. For the reduced form of 3-acetyl pyridineadenine dinucleotide, the K_a = 1.4 × 10⁷ M^?1 S^?1. The activity of the enzyme purified from the tail muscle is severely inhibited (90%) by high levels of pyruvate (10 mm) when assayed for pyruvate reductase activity at 11 °C; the reductase activity measured using the enzyme from the walking leg muscle was not inhibited by these high levels of pyruvate. Evidence is presented which indicates that the LDH from the tail muscle of the East Coast lobster forms an abortive ternary complex (enzyme-NAD⁺-pyruvate) which accounts for these inhibitory kinetics. The data suggest that the LDH from the tail muscles of the invertebrate lobster represents a “kinetic” heart-type l-specific LDH and that from the walking legs, a “kinetic” muscle-type l-specific LDH.     

An unusual fructose 1,6-bisphosphate-activated lactate dehydrogenase from the ascidian Pyura stolonifera

Lactate dehydrogenase (LDH) was purified from the siphon muscle of the intertidal ascidian Pyura stolonifera. The enzyme is unique among chordate LDHs but resembles some bacterial and platyhelminth LDHs in being activated by fructose 1,6-bisphosphate (FBP). Concentrations of FBP in the range 5μM to 0.5 mM increase V_max of the pyruvate reductase reaction by 130% to 210%, and decrease K_m pyruvate 5 to 11 fold and K_m NADH 2.5 to 5 fold. The enzyme is also activated by inorganic phosphate, but requires a 50 fold higher concentration to attain the maximum activation achieved by 0.5 mM FBP. Of a range of metabolites tested, including other glycolytic sugar phosphates, only FBP and inorganic phosphate activated the enzyme. FBP activation was not observed with 16 representative vertebrate LDH homotetramers, but did occur to a limited extent with LDH from an echinoderm. LDH was the only pyruvate reductase enzyme detected in P. stolonifera siphon muscle, and its activity was much greater than that of phosphorylase or phosphofructokinase. The LDH reaction is utilized by P. Stolonifera during prolonged siphon closure on exposure to air when lactate, but not succinate, accumulates in the siphon muscle. While the ascidian enzyme provides the first example of a FBP activated LDH from a chordate, it remains to be determined if this unusual property has any role in metabolic regulation.     

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.     

A study of binding-solubilization of some glycolytic enzymes in striated muscle in situ

The solubilization of lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and alpha-glycerophosphate dehydrogenase (GAPDH) was studied in pressed muscle as a function of ionic strength and NADH concentration. The results indicate that these factors affect the binding-solubilization of LDH and GAPDH in a similar way to their effect in dilute homogenized tissue. Alpha-glycerophosphate dehydrogenase was included as a typical soluble enzyme, since we have been unable to demonstrate its binding to subcellular fractions under any conditions. As with homogenized tissue, LDH was less susceptible to solubilization by ionic strength than GAPDH. It was demonstrated that LDH isozymes richer in heart-type subunits were more easily removed from muscle by centrifugation-imbibition than isozymes richer in the muscle-type subunits. This was interpreted as indicating that binding of the enzyme to subcellular structures was a major factor in the restricted removal of these enzymes from muscle, since only the muscle-type subunit is capable of binding to subcellular particles. It was further demonstrated that LDH could be taken up into muscle tissue, depleted in the enzyme, against an apparent concentration gradient. This was also interpreted as binding of the enzyme to the particulate structure of the muscle. Furthermore, this uptake of LDH occurred under conditions of ionic strength (0.25) and pH (7.5) that would prevent binding of the enzyme to the particulate fraction of a dilute suspension of homogenized muscle tissue. Thus, physiological conditions of pH and ionic strength do not necessarily induce solubilization of chicken breast muscle LDH in situ. Data obtained with dilute tissue homogenates, therefore, may not necessarily be easily and safely extrapolated to conditions in situ.     

Development of a simple and efficient method for assaying cytidine monophosphate sialic acid synthetase activity using an enzymatic reduced nicotinamide adenine dinucleotide/oxidized nicotinamide adenine dinucleotide converting system

A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay, relative activities toward Sia derivatives have been obtained. The preference of mouse CSS toward Neu5Ac and the ability of the rainbow trout enzyme to activate both KDN and Neu5Ac were confirmed. Thus, this simple and time-saving method is suitable for a systematic comparison of enzyme activity of structurally mutated enzymes based on the relative specific activity.     

Studies of lactate dehydrogenase of Mycoplasma mycoides var. mycoides.

Polyacrylamide gel electrophoresis and isoelectric focusing techniques have been used to compare NAD-dependent L(plus) lactate dehydrogenases (LDH) from ten different strains of Mycoplasma mycoides var. mycoides. The enzymes were not distinguished from one another, or from normal bovine LDH 1 by these methods. The kinetic behaviour of LDH form M. mycoides (T1 vaccine strain) suggested that the enzyme could readily reduce pyruvate or oxidize lactate in a manner which, in vertebrates, requires two different isoenzymes.     

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.     

Analysis of the kinetic and redox properties of the NADH peroxidase R303M mutant: correlation with the crystal structure

The crystal structure of the flavoprotein NADH peroxidase shows that the Arg303 side chain forms a hydrogen bond with the active-site His10 imidazole and is therefore likely to influence the catalytic mechanism. Dithionite titration of an R303M mutant [E(FAD, Cys42-sulfenic acid)] yields a two-electron reduced intermediate (EH(2)) with enhanced flavin fluorescence and almost no charge-transfer absorbance at pH 7.0; the pK(a) for the nascent Cys42-SH is increased by over 3.5 units in comparison with the wild-type EH(2) pK(a) of Cys42-SOH. The crystal structure of the R303M peroxidase has been refined at 2.45 A resolution. In addition to eliminating the Arg303 interactions with His10 and Glu14, the mutant exhibits a significant change in the conformation of the Cys42-SOH side chain relative to FAD and His10 in particular. These and other results provide a detailed understanding of Arg303 and its role in the structure and mechanism of this unique flavoprotein peroxidase.     

Purification and properties of a fructose-1,6-diphosphate activated L-lactate dehydrogenase from Staphylococcus epidermidis.

L-(+)-lactate dehydrogenase (LDH) from Staphylococcus epidermidis ATCC 14990 was purified by affinity chromatography. The purified enzyme was specifically activated by fructose-1,6-diphosphate (FDP). The concentration of FDP required for 50% maximal activity was about 0.15 mM. The enzyme activity was inhibited by adenosine diphosphate (ADP) and oxamate. The inhibition by ADP appeared to be competitive with respect to reduced nicotinamide adenine dinucleotide (NADH). The catalytic activity of the LDH for pyruvate reduction exhibited an optimum at pH 5.6. The enzyme is composed of four, probably identical, subunits. Sephadex gel filtration and sedimentation velocity at pH 5.6 Yielded molecular weights of about 130 000 and 126 000, respectively. The molecular weight at pH 6.5 and 7.0 was found to be only about 68 000. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and sedimentation velocity at pH 2.0 or 8.5 revealed monomeric subunits with an approximate molecular weight of 36000. The thermostability of the heat labile enzyme was increased in the presence of FDP, NADH and pyruvate. The purified LDH exhibited an anomalous type of kinetic behavior. Plots of initial velocity vs. different concentrations of pyruvate, NADH or FDP led to saturation curves with intermediary plateau regions. As a consequence of these plateau regions the Hill coefficient alternated between lower and higher n-values. Some distinguishing properties of the S. epidermidis LDH and other LDHs activated by FDP are discussed.     

The reversibility of skeletal muscle pyruvate kinase and an assessment of its capacity to support glyconeogenesis.

The kinetics of pyruvate phosphorylation by rabbit skeletal muscle pyruvate kinase (EC 2.7.1.40) has been studied with a coupled assay using P-enolpyruvate carboxylase (EC 4.1.1.31) and malate dehydrogenase (EC 1.1.1.37). The reaction sequence is (See journal for formula). Although the equilibrium of the pyruvate kinase reaction by itself strongly favors pyruvate production, the over-all equilibrium of this coupled system favors the depletion of pyruvate, thus greatly reducing the problem of back reaction during the assay. In addition, the oxidation of NADH by malate dehydrogenase makes it possible to monitor the system with a spectrophotometer. The Michaelis constant of pyruvate kinase was found to be 0.9 mM for ATP and 7 mM for pyruvate, values that agree reasonably well with earlier studies using direct assays. However, the maximum velocity is about 6 mumol of pyruvate phosphorylated/min/mg of enzyme, which is very much faster than that indicated by earlier studies. These results suggest that the metabolic significance of the reverse reaction of muscle pyruvate kinase may have been underestimated. In particular, the data given here suggest that its rate in vivo is probably comparable to the observed rate of glycogen synthesis from lactate, making possible glyconeogenesis in muscle by pyruvate kinase reversal without the need for an enzymatic bypass of the kind employed by liver and kidney.     

Trimethylamine-N-oxide counteracts urea effects on rabbit muscle lactate dehydrogenase function: a test of the counteraction hypothesis.

Trimethylamine-N-oxide (TMAO) in the cells of sharks and rays is believed to counteract the deleterious effects of the high intracellular concentrations of urea in these animals. It has been hypothesized that TMAO has the generic ability to counteract the effects of urea on protein structure and function, regardless of whether that protein actually evolved in the presence of these two solutes. Rabbit muscle lactate dehydrogenase (LDH) did not evolve in the presence of either solute, and it is used here to test the validity of the counteraction hypothesis. With pyruvate as substrate, results show that its Km and the combined Km of pyruvate and NADH are increased by urea, decreased by TMAO, and in 1:1 and 2:1 mixtures of urea:TMAO the Km values are essentially equivalent to the Km values obtained in the absence of the two solutes. In contrast, values of k(cat) and the Km for NADH as a substrate are unperturbed by urea, TMAO, or urea:TMAO mixtures. All of these effects are consistent with TMAO counteraction of the effects of urea on LDH kinetic parameters, supporting the premise that counteraction is a property of the solvent system and is independent of the evolutionary history of the protein.     

Trichomonas gallinae: charaterization and regulatory properties of lactic dehydrogenase.

The lactic dehydrogenase (l-lactate: NAD oxidoreductase, EC 1.1.1.27, LDH)of Trichomonas gallinae was characterized and some of its regulatory properties studied. Electrophoretic analysis, with specific enzymatic staining of crude and dialyzed cell-free extracts and dialyzed ammonium sulfate fractions, all revealed a single band of enzymatic activity suggesting only one molecular form of the enzyme. The pH optima were found to be the following: 7.0 in the pyruvate to lactate direction and 9.0 in the reverse direction. Thermal inactivation studies showed a narrow temperature optimum peaking at 35 C. The K_m values for all four reaction components were determined and found to be: NADH, 70 μm; pyruvate, 88 μm; NAD, 65 μm; and l-lactate, 4.6 mM. T. gallinae LDH was absolutely specific for NAD, NADH, l-lactate, and pyruvate. The enzyme exhibited negative cooperativity, with both NADH and l-lactate, as evidenced by curvilinear Lineweaver-Burk kinetics and Hill coefficients of less than one. Several glycolytic intermediates lowered the K_m of NADH with variable effects on the K_m of pyruvate. The regulation of LDH by glycolytic intermediates is discussed.