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.     

Steady-state kinetics of smooth muscle myosin.

The kinetic properties of purified smooth muscle myosin, free of actin, have been examined. Analysis of the steady-state kinetic data revealed an intermediary plateau region on the substrate saturation curves. In addition, these data, when analyzed by Hill and Lineweaver and Burk plots, indicate both positive and negative cooperativity, suggesting at least four substrate binding sites. The plateau region was abolished when the kinetic measurements were made at pH 5.5 and 9.0. Both positive and negative cooperative effects were absent at pH 9.0 and hyperbolic kinetics was observed. In contrast, at pH 5.5, although the plateau region was abolished, the enzyme exhibited positive cooperativity of substrate binding. When either heated or urea treated enzyme was used for kinetic measurements: (i) the plateau region shifted toward higher substrate concentration range; (ii) the cooperativity of binding sites was lost at low substrate concentrations but was instead seen at higher concentrations; and (iii) the Vmax was doubled. These data have been interpreted as due to ligand-induced conformational changes in the enzyme according to J. Teipel and D. E. Koshland, Jr. (1969).     

Acetylcholinesterase. I. Kinetic aspects of interaction with reversible effectors]

Interaction of an effector M with acetylcholinesterase (EC 3.1.1.7) according to the model of Krupka and Laidler was analysed. Some usual functions of [M] : 1/VM, [(VO/VM)-1]/[M] (where VO and VM are the steady state rates in the absence and in the presence of modifiers, respectively), vertical intercept 1/VM, slope KM/VM and absolute value of reciprocal horizontal intercept KM of Lineweaver-Burk plots are investigated and corresponding plots described. It is particularly shown that if Dixon plots are curves concave downwards, plots of [VO/VM)-1]/[M] and 1/VM against [M] are hyperbolas concave upwards and downwards respectively. If Dixon plots are curves concave upwards, plots of [(VO/VM)-1]/[M] and 1/VM versus [M] are hyperbolas concave downwards and upwards respectively. Moreover plots of KM/VM against [M] are linear. However, this model does not explain some observations, under conditions of high ionic strength (gamma/2 greater than or equal to 0,1), where Dixon plots are curves concave upwards, plots of [VO/VM)-1]/[M] versus [M] strainght lines, the plot of 1/VM against [M] is a straight line or a curve concave upwards of positives slopes and the plot of KM/VM versus [M] a curve of positive slope concave upwards. These experimental data might be interpreted by an extension of the preceding model to a mechanism with two enzymatic binding sites under kinetic conditions that are determined.     

Kinetics of suicide substrates. Practical procedures for determining parameters.

Many clinically important or mechanistically interesting inhibitors react with enzymes by a branched pathway in which inactivation of the enzyme and formation of product are competing reactions. The steady-state kinetics for this pathway [Waley (1980) Biochem. J. 185, 771-773] gave equations for progress curves that were cumbersome. A convenient linear plot is now described. The time (t1/2) for 50% inactivation of the enzyme (this is also the time for 50% formation of product), or for 50% loss of substrate, is measured in a series of experiments in which the concentration of inhibitor, [I]0, is varied; in these experiments the ratio of the concentration of enzyme to the concentration of inhibitor is kept fixed. Then a plot of [I]0 X t1/2 against [I]0 is linear, and the kinetic parameters can be found from the slope and intercept. Furthermore, simplifications of the equations for progress curves are described that are valid when the concentration of inhibitors is high, or is low, or when the extent of reaction is low. The use of simulated data has shown that the recommended methods are not unduly sensitive to experimental error.     

Investigations on the kinetic mechanism of octopine dehydrogenase. 2. Location of the rate-limiting step for enzyme turnover.

The kinetic mechanism of octopine dehydrogenase has been investigated by stopped-flow and isotope replacement techniques. When the enzyme is saturated by substrate and coenzyme, both for NADH oxidation and NAD+ reduction, the stationary phase is preceded by a rapid burst. Under these saturation conditions, furthermore, the stationary phase shows a secondary isotope effect when 4S-[4(2)H]NADH is substituted for NADH and when (on the other reaction end) D-[2H] octopine is substituted for D-octopine. The data are taken to indicate that the rate-limiting step for enzyme turnover is a step following a very fast chemical transformation of the reagents. However, when the substrate concentration is lowered below the corresponding Km value keeping the coenzyme concentration at saturating levels, the time course of the reaction shows no burst and the stationary phase has a larger isotope effect. This indicated that under those non-saturating conditions, the enzyme turnover has a larger contribution than the hydrogen-transfer step. Changing the coenzyme concentration alone has very little or no effect on the amplitude of the burst or on the isotope effect. These features are discussed in terms of the other known kinetic properties of the enzyme, and in terms of analogous studies reported in the literature for other dehydrogenases.     

Action of sulphite on the substrate kinetics of chloroplastic NADP-dependent glyceraldehyde-3-phosphate dehydrogenase

The kinetics of NADP-GPD from spinach chloroplasts are biphasic vs NADPH and PGA. Thus, two maximum velocities exist with an intermediary plateau and two K_m values. Activation by NADPH + DTT increases V_max of both sections, but does not change the substrate affinities. Sulphite reduces the maximum activities of both sections vs NADPH, however, it causes normal substrate kinetics vs PGA; even V_max is reduced. Sulphite, present only during the activation process, suppresses the enzyme form with the higher V_max. The kinetics vs NADH are also biphasic; the activity is strongly reduced by preincubation of the chloroplasts with NADH + DTT or at NADH concentrations > 0.4mM. Using NADH as cofactor, inverted peaks in the kinetics vs PGA occur; sulphite is active in a similar way as when NADPH is used as cofactor. The biphasic kinetics are discussed with respect to additional potential for regulation of enzyme activity according to illumination and NADPH concentrations respectively.     

The effect of pH on the cooperative behavior of aspartate transcarbamylase from Escherichia coli.

Saturation curves of activity versus concentration were determined for aspartate transcarbamylase from Escherichia coli (EC 2.1.3.2) for the substrate L-aspartate at saturating carbamyl phosphate (4.8 mM) in buffered solution at pH values from 6.0 to 12.0. Hill coefficients were obtained from the sigmoidal curves. At pH values from 7.8 to 9.1, where substrate inhibition causes difficulties in the Hill approximation, our kinetic scheme includes substrate inhibition and residual activity in the abortive enzyme-substrate complex. The plot of Hill coefficient versus pH has pKalpha values of 7.4 and 9.8 at the half-maximum positions of the curve which has a plateau from pH 8.1 to 9.1. These pKalpha values may be associated with functional groups involved in the allosteric transition which activates the enzyme. A plot of [S]0.5 versus pH shows a pKalpha of 8.5, which may belong to a residue either at or near the aspartate binding site. At 50 mM aspartate concentration the pH-rate profile shows maxima at pH values of 8.8 and 10.0 (cf. Weitzman, P.D.J., and Wilson, I.B.(1966)J. Biol. Chem. 2418 5481-5488, who used 100 mM aspartate). However, when the pH-dependent substrate inhibition is included, the calculated Vmax--H curve is bell-shaped like that of the isolated catalytic subunit.     

Kinetic mechanism of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with conversion of NAD+ to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. IMPDH is a target for antitumor, antiviral, and immunosuppressive chemotherapy. We have determined the complete kinetic mechanism for IMPDH from Tritrichomonas foetus using ligand binding, isotope effect, pre-steady-state kinetic, and rapid quench kinetic experiments. Both substrates bind to the free enzyme, which suggests a random mechanism. IMP binds to the enzyme in two steps. Two steps are also involved when IMP binds to a mutant IMPDH in which the active site Cys is substituted with a Ser. This observation suggests that this second step may be a conformational change of the enzyme. No Vm isotope effect is observed when [2-2H]IMP is the substrate which indicates that hydride transfer is not rate-limiting. This result is confirmed by the observation of a pre-steady-state burst of NADH production when monitored by absorbance. However, when NADH production was monitored by fluorescence, the rate constant for the exponential phase is 5-10-fold lower than when measured by absorbance. This observation suggests that the fluorescence of enzyme-bound NADH is quenched and that this transient represents NADH release from the enzyme. The time-dependent formation and decay of [14C]E-XMP intermediates was monitored using rapid quench kinetics. These experiments indicate that both NADH release and E-XMP hydrolysis are rate-limiting and suggest that NADH release precedes hydrolysis of E-XMP.     

NADH dehydrogenase from bovine neutrophil membranes. Purification and properties

A membrane-associated NADH dehydrogenase from beef neutrophils was purified to homogeneity, using detergent (cholate plus Triton X-100) extraction and chromatography on DEAE-Sepharose CL-6B, agarose-hexane-NAD, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 17,500, but the enzyme was highly aggregated (Mr greater than 450,000) in nondenaturing gels containing 0.1% Triton X-100. The protein band in nondenaturing gels was also stained for activity using NADH and nitro blue tetrazolium. The enzyme showed greatest electron acceptor activity with ferricyanide (100%), followed by cytochrome c (3.5%), dichloroindophenol (2.7%), and cytochrome b5 (0.34%). No activity was seen with oxygen. The Km values for NADH and ferricyanide were 18 and 9.5 microM, respectively, and NAD+ was a weak competitive inhibitor (Ki = 118 microM). No activity was seen with NADPH. No effects were seen with mitochondrial respiratory inhibitors such as azide, cyanide, or rotenone, but p-chloromercuribenzoate was strongly inhibitory and N-ethylmaleimide was weakly inhibitory. No free flavin was detectable in enzyme preparations. Based upon kinetic, physical, and inhibition properties, this NADH dehydrogenase differs from those previously described in microsomes and erythrocyte plasma membrane.     

Mitochondrial NADH dehydrogenase in cystic fibrosis: enzyme kinetics in cultured fibroblasts.

Differences among cystic fibrosis (CF) genotypes (CF, obligate carriers for CF [HZ], and controls) in mitochondrial calcium pool size, oxygen (O2) consumption, and rotenone inhibition of O2 consumption led to examination of mitochondrial NADH dehydrogenase (NADH: [acceptor] oxidoreductase, E.C. 1.6.99.3). pH optima of mitochondrial NADH dehydrogenase were different in enzyme derived from whole cell homogenates of cultured skin fibroblasts of subjects with CF, HZ, and controls. We describe here apparent binding of substrate to the enzyme (Km [NADH]) in cell fractions. Km (NADH) for CF ranged from 10.9 to 16.1 micro M (no. = 7); for HZ from 20.9 to 26.3 microM (no. = 5). With three exceptions, Km for controls (no. = 12) ranged from 31.8 to 42.8 microM. Km of the three exceptional controls were 21.5, 23.7, and 22.4 microM (the latter two are identical twins). pH optima of enzyme from these three strains were no different from that of known HZ. The correlation between two kinetic parameters of an enzyme and the three CF genotypes suggests an association between the CF gene and mitochondrial NADH dehydrogenase.     

Fluorescence and CD spectroscopic analysis of the alpha-chymotrypsin stabilization by the ionic liquid, 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]amide

The stability of alpha-chymotrypsin in the ionic liquid, 1-ethyl-3-methyl-imidizolium bis[(trifluoromethyl)sulfonyl]amide ([emim][NTf2]), was studied at 30 and 50 degrees C and compared with the stability in other liquid media, such as water, 3 M sorbitol, and 1-propanol. The kinetic analysis of the enzyme stability pointed to the clear denaturative effect of 1-propanol, while both 3M sorbitol and [emim][NTf2] displayed a strong stabilizing power. For the first time, it is shown that enzyme stabilization by ionic liquids seems to be related to the associated structural changes of the protein that can be observed by differential scanning calorimetry (DSC) and fluorescence and circular dichroism (CD). The [emim][NTf2] enhanced both the melting temperature and heat capacity of the enzyme compared to the other media assayed. The fluorescence spectra clearly showed the ability of [emim][NTf2] to compact the native structural conformation of alpha-chymotrypsin, preventing the usual thermal unfolding which occurs in other media. Changes in the secondary structure of this beta/beta protein, as quantified by the CD spectra, pointed to the great enhancement (up 40% with respect to that in water) of beta-strands in the presence of the ionic liquid, which reflects its stabilization power.     

Thiol groups of Escherichia coli citrate synthase and their influence on activity and regulation.

The modification of Escherichia coli citrate synthase (citrate oxaloacetatelyase(pro-3S-CH2.COO- leads to acetyl-CoA, EC 4.1.3.7) with 5,5'-dithiobis-(2-nitrobenzoic acid) has been investigated. (1) In low ionic strength (20 mM Tris.HCl, pH 8.0): (A) Eight thiol groups per tetramer of the native enzyme reacted with Nbs2. (b) Two of the eight accessible thiols were modified rapidly with the loss of 26% enzyme activity but with no change in the NADH inhibition. The remaining six were modified more slowly, resulting in a further 60% loss of activity and complete densensitization to NADH. (c) The 2nd-order rate constant for the modification of the rapidly reacting thiols is 2.5.10(4) M-1.min-1. At the reagent concentrations used (0.1 to 0.2 mM) the modification of the six thiols in the slow kinetic set appeared to be 1st-order; at 0.1 mM dithionitrobenzoic acid their rate of modification was approximately 30 times slower than the thiols in the fast kinetic set. (2) In high ionic strength (20 mM Tris.HCl, pH 8.0, 0.1 M KCl): (a) Four thiol groups were modified in a single kinetic set and it appeared that these thiols are four of the six slowly modified in the absence of KCl. (b) The modification resulted in 70% loss of enzyme activity and complete loss of NADH inhibition. (3) From the kinetic analysis it is proposed that the four thiol groups accessible to dithionitrobenzoic acid in the absence and presence of 0.1 M KCl are those involved in the response of NADH. Modification of any one of these four groups produced no reduction in the inhibition; instead, loss of NADH sensitivity was coincident with the appearance of tetrameric protein possessing three substituted thiols, whereas enzyme with one or two modified groups was still fully inhibited by NADH.     

Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

Salmonella typhimurium histidinol dehydrogenase produces histidine from the amino alcohol histidinol by two sequential NAD-linked oxidations which form and oxidize a stable enzyme-bound histidinaldehyde intermediate. The enzyme was found to catalyze the exchange of 3H between histidinol and [4(R)-3H]NADH and between NAD and [4(S)-3H]NADH. The latter reaction proceeded at rates greater than kcat for the net reaction and was about 3-fold faster than the former. Histidine did not support an NAD/NADH exchange, demonstrating kinetic irreversibility in the second half-reaction. Specific activity measurements on [3H]histidinol produced during the histidinol/NADH exchange reaction showed that only a single hydrogen was exchanged between the two reactants, demonstrating that under the conditions employed this exchange reaction arises only from the reversal of the alcohol dehydrogenase step and not the aldehyde dehydrogenase reaction. The kinetics of the NAD/NADH exchange reaction demonstrated a hyperbolic dependence on the concentration of NAD and NADH when the two were present in a 1:2 molar ratio. The histidinol/NADH exchange showed severe inhibition by high NAD and NADH under the same conditions, indicating that histidinol cannot dissociate directly from the ternary enzyme-NAD-histidinol complex; in other words, the binding of substrate is ordered with histidinol leading. Binding studies indicated that [3H]histidinol bound to 1.7 sites on the dimeric enzyme (0.85 site/monomer) with a KD of 10 microM. No binding of [3H]NAD or [3H]NADH was detected. The nucleotides could, however, displace histidinol dehydrogenase from Cibacron Blue-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of lactic acid oxidation catalyzed by lactate dehydrogenase from the tail muscle of Homarus americanus.

The mechanism of lactic acid oxidation in the tail muscles of Homarus americanus was studied. In solutions of intermediate ionic strength (0.55) time-course progress curves for lactic acid oxidation as catalyzed by lactate dehydrogenase exhibited a lag period. Evidence is presented which indicates that the lactate dehydrogenase found in the tail muscles of the lobster exists in two distinct physical and kinetic forms. The equilibrium of these forms is dependent upon the ionic strength of the reaction mixture. In low ionic strength solutions, the enzyme exists as a tetrameric species with an apparent K_m for lactic acid of 1.1 m; in high ionic strength solutions, the enzyme exists as a dimer and the corresponding K_m is 0.028 m. At intermediate ionic strengths, an equilibrium between the two physical and kinetic species exists which is modulated by the NADH mole-fraction ($\text{[}\text{NADH}\text{]}\text{[}\text{NADH}\text{+}\text{NAD}{}^{\mathrm{+}}\text{]}$) and, in turn, this modulation results in sigmoidal time-course progress curves. The role of this enzyme is discussed as affected by in vivo ionic strength, temperature and levels of oxidized and reduced nicotine adenine dinucleotides.     

Malate dehydrogenase of spinach: Substrate kinetics of different forms

Sephadex G-200 gel filtration of an ammonium sulfate fraction, containing the bulk of NAD-dependent malate dehydrogenase, yields forms of differing MW. Both Mg²⁺ and NADH stabilize the 127000 daltons MW form. K⁺, or incubation with dithioerythritol, cause splitting and partial reaggregation, resulting in MWs ranging between 35000 and 180000 daltons. Chromatography in the presence of dithioerythritol and NADH results in an enzyme with a non-linear reaction rate at low substrate concentrations. Plots of initial velocity vs substrate and cofactor concentration respectively are characterized by two slopes of positive cooperativity separated by an intermediary plateau of negative cooperativity. Gel chromatography in the presence of Mg²⁺ or K⁺ or drastic dilution of the enzyme results in an enzyme with linear reaction rates also at low substrate concentration. Its kinetics are consistent with the view that the enzyme undergoes conformational changes when the substrate concentration is varied.     

Mechanism of transfer of reduced nicotinamide adenine dinucleotide among dehydrogenases. Transfer rates and equilibria with enzyme-enzyme complexes

The direct transfer of NADH between A-B pairs of dehydrogenases and also the dissociation of NADH from individual E-NADH complexes have been investigated by transient stopped-flow kinetic techniques. Such A-B transfers of NADH occur without the intermediate dissociation of coenzyme into the aqueous solvent environment [Srivastava, D.K., & Bernhard, S.A. (1985) Biochemistry 24, 623-628]. The equilibrium distributions of limiting NADH among aqueous solvent and A and B dehydrogenase sites have also been determined. At sufficiently high but realizable concentrations of dehydrogenases, both the transfer rate and the equilibrium distribution of bound NADH are virtually independent of the excessive enzyme concentrations; at excessive E2 concentration, substantial NADH is bound to the E1 site. These results further substantiate earlier kinetic arguments for the preferential formation of an EA-NADH-EB complex, within which coenzyme is directly transferred between sites. The unimolecular specific rates of coenzyme transfer from site to site are nearly invariant among different A-B dehydrogenase pairs. The equilibrium constants for the distribution of coenzyme within the EA X EB complexes are near unity. At high [E2] and for [E2] greater than [E1] greater than [NADH], E1-NADH X E2 and E1 X NADH-E2 are virtually the only coenzyme-contained species. In contrast to the nearly invariant unimolecular NADH transfer rates within EA X EB complexes, unimolecular specific rates of dissociation of NADH from E-NADH into aqueous solution are highly variable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic properties of sorbitol dehydrogenase from calf liver cell cytoplasm

The kinetic properties of sorbitol dehydrogenase from calf liver cell cytoplasm during sorbitol oxidation were studied at pH 7.0, 7.5, 8.0, 9.0 and 10.0. It was found that the shape of kinetic curves for NADH accumulation depends on pH and substrate concentration. At pH 7.0, 7.5 and 8.0 the enzymatic reaction obeys the Michaelis-Menten kinetics with Km of 3.3 x 10(-3) M. 2.3 x 10(-3) M and 2.08 x 10(-3) M, respectively. At pH 9.0 and 10.0 the vovs [So] curves have an "intermediate plateau". The Hill plots for this reaction reveal two slopes that are dependent on substrate concentration. The nH values for sorbitol (up to 2 mM) are 1.0 and 1.16 at pH 9.0 and 10.0, respectively. With a further rise in the substrate concentration, the nH value increases up to 2.4 and 2.18 at pH 9.0 and 10.0, respectively. This is suggestive of the existence of a slowly dissociating enzymatic system of the Np in equilibrium P type (where P is the oligomeric and p the monomeric forms of the enzyme); N approximately greater than 2. The vovs NAD plots are S-shaped at all pH values studied. The data obtained are discussed in terms of regulatory effects of sorbitol and acidity on association-dissociation of sorbitol dehydrogenase from liver cell cytoplasm.     

Kinetic manifestations of allosteric interactions in models of regulatory enzymes with "indirect" co-operativity

The shape of the plots of initial reaction rate (ν) versus initial substrate concentration ([S]₀) and versus initial concentration of allosteric effector ([F]₀) for the model of allosteric enzyme of Monod, Wyman &; Changeux (1965) and for the model of dissociating regulatory enzyme has been analysed by means of the inconstant exponent (q) for substrate or effector concentration, respectively. It has been shown that allosteric interactions in above-mentioned models with “indirect” co-operativity may be manifested not only by the sigmoidal shape of the plot of ν versus [S]₀ or ν versus [F]₀ (with one point of inflexion) but also by the increase in the magnitude of exponent q in progress of saturation process of the enzyme by the substrate or by the effector in the absence of the sigmoidal shape of these plots. It has been shown also that the plot of ν versus [S]₀ has two inflexion points when the parameters have certain definite values. One of these inflexion points (or even both at definite values of the parameters) is hardly discernible. At certain definite values of the parameters two inflexion points may be kinetically manifested by such phenomenon as “negative” co-operativity (q < 1). This is possible if one of the interconvertable enzyme forms exceeds another not only in the affinity to the substrate but also in the value of the rate constant for catalytic breakdown of the enzyme-substrate complex.     

The influence of ionic strength on the binding of a water soluble porphyrin to nucleic acids.

The ionic strength dependences of the binding of tetrakis (4-N-methylpyridyl)porphine (H2TMpyP) to poly(dG-dC) and calf thymus DNA have been determined. For the former system the results are typical of other intercalators, i.e., a plot of log K vs log [Na+] is linear albeit with a slope which suggests that the "effective charge" of the porphyrin is closer to two than the formal charge of +4. For calf thymus DNA, the binding profile is not completely compatible with the predictions of condensation theory. Whereas the avidity of binding does decrease with increasing [Na+] as predicted, of greater interest is the relocation of the porphyrin from GC-rich regions to AT-rich regions as the ionic strength increases.     

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 130000 and 126000 respectively. The molecular weight at pH 6.5 and 7.0 was found to be only about 68000. 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.