Electron Transfer in the Dissimilatory Iron-reducing Bacterium Geobacter metallireducens

To investigate electron transport in the dissimilatory iron-reducing isolate Geobacter metallireducens strain GS-15, assays for redox enzymes and characterizations of cytochromes were performed. G. metallireducens produced 1.56 g dry cell weight per mol e⁻transferred when grown on benzoate and contained the following citric acid cycle enzymes (activities in nkat per mg cell protein); isocitrate dehydrogenase (0.84), coenzyme A-dependent 2-oxoglutarate: methyl viologen oxidoreductase (2.80), succinate dehydrogenase (0.80), and malate dehydrogenase (8.35). An oxygen-sensitive, soluble coenzyme A-dependent 2-oxoglutarate: ferredoxin oxidoreductase (0.14) with no NAD(P)-activity was observed. In cell suspensions NADPH, but not NADH, could reduce methyl viologen (2.45). Isocitrate and malate dehydrogenase activities were soluble enzymes that coupled with NADP and NAD, respectively. NADPH (0.94) and NADH (1.85) oxidation activities were observed in detergent solubilized, whole-cell suspensions using the artificial electron acceptor menadione. Menaquinone was observed at 1.2 μmol per g cell protein. The triheme c₇cytochrome was purified and 37 amino acids were determined. The mass observed by mass spectroscopy was 9684±10 Da. The average mid-point potential for the three hemes was measured at −91 mV. The growth yield, redox reactions, and electron transfer components are discussed with regards to possible sites of energy conservation during growth on iron(III).     

A novel biotin protein required for reductive carboxylation of 2-oxoglutarate by isocitrate dehydrogenase in Hydrogenobacter thermophilus TK-6

Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2-oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP-Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N-terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase. Isocitrate dehydrogenase, the novel biotin protein, ATP-Mg and NADH were essential for the reductive carboxylation of 2-oxoglutarate. These observations indicate that the novel biotin protein is an ATP-dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase.     

Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

Cloning, Expression, and Enzymatic Characterization of Isocitrate Dehydrogenase from Helicobacter pylori

Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate with NAD(P) as a cofactor in the tricarboxylic acid cycle. As a housekeeping protein in Helicobacter pylori, IDH was considered as a possible candidate for serological diagnostics and detection. Here, we identified a new icd gene encoding IDH from H. pylori strain SS1. The recombinant H. pylori isocitrate dehydrogenase (HpIDH) was cloned, expressed, and purified in E. coli system. The enzymatic characterization of HpIDH demonstrates its activity with k _cat of 87 s^?1, K _m of 124 μM and k _cat/K _m of 7 × 10⁵ M^?1s^?1 toward isocitrate, k _cat of 80 s^?1, K _m of 176 μM and k _cat/K _m of 4.5 × 10⁵ M^?1s^?1 toward NADP. The optimum pH of the enzyme activity is around 9.0, and the optimum temperature is around 50 °C. This current work is expected to help better understand the features of HpIDH and provide useful information for H. pylori serological diagnostics and detection.     

Isolation of a sn-glycerol 3-phosphate: NAD oxidoreductase from spinach leaves.

An NAD-dependent glycerol 3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD oxidoreductase; EC 1.1.1.8) has been purified from spinach leaves by a three-step procedure involving ion-exchange, gel filtration, and affinity chromatography. The enzyme has been purified over 10,000-fold to a specific activity of 38. It has a molecular weight of approximately 63,500. The pH optimum for the reduction of dihydroxyacetone phosphate is 6.8 and for glycerol 3-phosphate oxidation it is 9.5. During dihydroxyacetone phosphate reduction hyperbolic kinetics were observed when either NADH or dihydroxyacetone phosphate was the variable substrate, but concentrations of NADH greater than 150 μm were inhibitory. Michaelis constants were 0.30–0.35 mm for dihydroxyacetone phosphate and 0.01 mm for NADH. Glycerol 3-phosphate oxidation obeyed Michaelis-Menten kinetics with a K_m of 0.19 mm for NAD and 1.6 mm for glycerol 3-phosphate. The enzyme was specific for those substrates, and dihydroxyacetone, glyceraldehyde, glyceraldehyde 3-phosphate, NADPH, NADP, and glycerol were not utilized. The spinach leaf enzyme appears to be in the cytoplasm and probably functions for the production of glycerol 3-phosphate from dihydroxyacetone phosphate.     

The effects of calcium and lanthanide ions on the activity of bovine heart nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase

(i) The activity of purified NAD-specific isocitrate dehydrogenase from bovine heart was stimulated by free Ca²⁺ in the presence of ADP and subsaturating levels of magnesium isocitrate, but not in absence of ADP. However, Ca²⁺ was not absolutely required for ADP activation. This was particularly apparent when free Mg²⁺ was kept low (0.0024–0.020 mm) and the substrate magnesium dl-isocitrate ranged from 0.07–0.25 mm. When kinetic constants were determined at pH 7.4 under these conditions and in the absence of ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetate, Ca²⁺ had little or no effect on K_m (app) for ADP; the stimulation of rate by Ca²⁺ was mainly due to increased V (app). With subsaturating ADP, there was an interdependence in the interaction of the enzyme with substrate and Ca²⁺. Thus, with ADP constant (0.30 mm) the values of K_m (app) for magnesium dl-isocitrate declined from 0.35 mm at zero Ca²⁺ to 0.19 mm with saturating Ca²⁺ without affecting V; K_m (app) for free Ca²⁺ declined with increasing magnesium isocitrate to a limiting K_m of 0.3 μm. (ii) Ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetate, frequently used as a calcium buffer, inhibited enzyme activity with and without ADP. (iii) The enzyme was not inhibited by the calmodulin inhibitors trifluoperazine and chlorpromazine. Inhibition by lanthanide ions of the isocitrate dehydrogenase was competitive with magnesium isocitrate and not with respect to Ca²⁺. The values of K_is (1.8 to 3.1 μm) for La³⁺, Yb³⁺, Gd³⁺, Eu³⁺, Tb³⁺, and Er³⁺ were about two orders of magnitude smaller than K_m for magnesium dl-isocitrate.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

Horse liver alcohol dehydrogenase SS: purification and characterization of the homogenous isoenzyme.

Alcohol dehydrogenase SS was prepared from horse liver by salt fractionation, ion-exchange chromatography, and affinity chromatography. The purified isoenzyme is free from extraneous protein and other alcohol dehydrogenase isoenzyme contaminants and contains four Zinc atoms per molecule. The substrate specificity with saturated aliphatic alcohols and aldehydes of two to six carbon chain lengths has been investigated. The K_m values and turnover numbers at maximal velocity (k_cat) are presented. Values of k_cat are constant within a substrate category and independent of the substrate chain length, while the K_m values decrease with the increase of the substrate chain length. The K_m values for two- and three-carbon substrates are large, that for ethanol (40 mm) is two orders of magnitude larger than that reported for classical preparations of horse liver alcohol dehydrogenase. At pH 7, the k_cat values for alcohol oxidation are almost 30 times smaller than for aldehyde reduction. The enzyme has been characterized with regard to specific activity with several nonsteroidal substrates and with two steroids: 3-oxo-5β-androstan-17β-ol and 5β-pregnan-21-ol-3,20-dione hemisuccinate. NAD(H) is the preferred coenzyme. Values of K_m for NADH with constant steroidal substrates are an order of magnitude smaller than the corresponding K_m values with nonsteroidal substrates. A possible explanation for this observation is presented.     

Isocitrate lyase in germinating spores of the fern Anemia phyllitidis

Isocitrate lyase was partially purified from germinating spores of the fern Anemia phyllitidis. The enzyme requires Mg²⁺ and thiol compounds for maximal activity and has a pH optimum between 6.5 and 7.5. The K_m of the enzyme for threo-Δ_s-isocitrate is 0.5 mM. Succinate inhibits the enzyme non-competitively (K_i. 1.8 mM). The increase of isocitrate lyase activity is closely correlated with the induction of the germination process. The fall of enzyme activity during germination is associated with the decline in triglyceride reserves.     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.     

Isolation and properties of watermelon isocitrate lyase

The glyoxylate cycle enzyme, isocitrate lyase (EC 4.1.3.1) was purified from cotyledons of Citrullus vulgaris (watermelon). The final preparation, which had been 97-fold purified with a specific activity of 16.1 units/mg protein in a yield of 36%, was homogeneous by gel- and immunoelectrophoretic criteria. The tetrameric enzyme had: a molecular weight of 277 000, a sedimentation coefficient of 12.4 s, and a K_m for D_s-isocitrate equal to 0.25 mM. Isocitrate lyase from this source is not a glycoprotein as shown by total carbohydrate content after precipitation by trichloroacetic acid of the purified enzyme. Reduction of the enzyme with thiols increased activity and maximal activity was obtained with at least 5 mM dithiothreitol. EDTA partially substituted for thiol in freshly isolated enzyme. Watermelon isocitrate lyase was also protected against thermal denaturation at 60° for at least 1 hr by 5 mM Mg²⁺ plus 5 mM oxalate. Oxalate was a competitive inhibitor with respect to isocitrate (K_i: 1.5 μM, pH 7.5, 30°).     

NAD(P)+-dependent isocitrate dehydrogenases in mitochondria purified from Picea abies seedlings

Isocitrate dehydrogenase (IDH) activities were measured in mitochondria isolated from aerial parts of 21-day-old spruce (Picea abies L. Karst.) seedlings. Mitochondria were purified by two methods, involving continuous and discontinuous Percoll gradients. Whatever the method of purification, the mitochondrial outer membrane was about 69% intact, and the mitochondria contained very low cytosolic, chloroplastic and peroxisomal contaminations. Nevertheless, as judged by the recovery of fumarase activity, purification on a continuous 28% Percoll gradient gave the best yield in mitochondria, which exhibited a high degree of inner membrane intactness (91%). The purified mitochondria oxidized succinate and malate with good respiratory control and ADP/O ratios. The highest oxidation rate was obtained with succinate as substrate, and malate oxidation was improved (+ 60%) by addition of exogenous NAD⁺. Experiments using standard respiratory chain inhibitors indicated that, in spruce mitochondria, the alternative pathway was present. Both NAD⁺-isocitrate dehydrogenase (EC 1.1.1.41) and NADP⁺-isocitrate dehydrogenase (EC 1.1.1.42) were present in the mitochondrial matrix fraction, and NAD⁺-IDH activity was about 2-fold higher than NADP⁺-IDH activity. The NAD⁺-IDH showed sigmoidal kinetics in response to isocitrate and standard Michaelis-Menten kinetics for NAD⁺ and Mg²⁺. The NADP⁺-IDH, in contrast, displayed lower K_m values. For NAD⁺-IDH the pH optimum was at 7.4, whereas NADP⁺-IDH exhibited a broad pH optimum between 8.3 and 9. In addition, NAD⁺-IDH was more thermolabile. Adenine nucleotides and 2-oxoglutarate were found to inhibit NAD(P)⁺-IDH activities only at high concentrations.     

Purification and characterization of two glutamate dehydrogenase isoenzymes from Brassica napus

Glutamate dehydrogenase (L-glutamate: NAD⁺ oxidoreductase, EC 1.4.1.2) was purified from Brassica napus leaves. Isoenzyme 1 (GDH1), with the lowest, and isoenzyme 7 (GDH7) with the highest electrophoretic mobility were characterized. The native GDH was estimated to have a molecular mass of about 239 kDa and consisted of six identical 41.4-kDa subunits for GDH1 and 42.4-kDa subunits for GDH7. The pH optima of both isoenzymes in amination and deamination reactions were 9.0 and 9.5, respectively. At optimum pH, the K_m values for ammonium, 2-oxoglutarate, NADH, NAD and glutamate did not differ between the two isoenzymes. Addition of 10 mM EGTA inhibited the amination activity of GDH1, but that of GDH7 remained at about 30 %. Cellular fractionation experiments showed that both GDH1 and GDH7 localized in mitochondria with a loose association with the mitochondrial membrane.     

Purification and properties of malic dehydrogenase from Trichomonas gallinae

The malic dehydrogenase (MDH², l-malate: NAD oxidoreductase, E.C. 1,1.1.37) of Trichomonas gallinae was purified 215-fold and characterized. The molecular weight was found to be 72,000 and the enzyme protein contained essential cations and sulfhydryl groups. Polyacrylamide gel electrophoresis before and after extensive purification yielded a single band of malic dehydrogenase activity strongly suggesting only one molecular form of the enzyme. Analysis of kinetic data yielded the following K_m values: oxalocetate, 16 μM; malate, 200 μM; NADH 11 μM; and NAD, 70 μM. The enzyme was absolutely specific for l-malic acid, NAD, and NADH. The enzyme exhibited a broad band of heat stability with an optimum of 51 C. The pH optimum in the direction of oxalacetate reduction was 9.0. The pH optima in the reverse direction were 9.0 and 10.5 A role for this enzyme in T. gallinae metabolism is discussed.     

Nondecarboxylating and Decarboxylating Isocitrate Dehydrogenases: Oxalosuccinate Reductase as an Ancestral Form of Isocitrate Dehydrogenase

Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate and NAD⁺) toward the enzyme were extremely low compared to other known ICDHs. The binding activities of isocitrate and NAD⁺ were not independent; rather, the binding of one substrate considerably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decarboxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring higher substrate affinities.     

Hymenolepis microstoma: lactate and malate dehydrogenases of the adult worm.

Lactate and malate dehydrogenases (EC 1.1.1.27 and EC 1.1.1.37, respectively) were precipitated with ammonium sulfate, redissolved in 100 mM phosphate buffer, and the kinetic parameters of each enzyme determined. Lactate dehydrogenase: The enzyme preparation had a specific activity of 0.35 μmole NADH oxidized/min/mg protein for pyruvate reduction, and 0.10 μmole NAD reduced/min/mg protein for lactate oxidation. K_m values for the substrates and cofactors were as follows: pyruvate = 0.51, mM; lactate = 3.8 mM; NADH = 0.011 mM; and NAD = 0.17 mM. NADPH, NADP, or d(?)-lactate would not replace NADH, NAD, or l(+)-lactate, respectively. The enzyme was relatively stable at 50 C for 45 min, but much less stable at 60 C; repeated freezing and thawing of the enzyme preparation had little effect on LDH activity. Both p-chloromercuribenzoate (p-CMB) and N-ethylmaleimide (NEM) significantly inhibited LDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least two LDH isoenzymes in the unpurified enzyme preparation. The molecular weight was estimated at 160,000 by gel chromatography. Malate dehydrogenase: The enzyme preparation had a specific activity of 6.70 μmole NADH oxidized/min/mg protein for oxaloacetate reduction, and 0.52 μmole NAD reduced/ min/mg protein for malate oxidation. K_m values for substrates and cofactors were as follows: l-malate = 1.09 mM; oxaloacetate = 0.0059 mM; NADH = 0.017 mM; and NAD = 0.180 mM. NADP and NADPH would not replace NAD and NADH, respectively, d-malate was oxidized slowly when present in high concentrations (>100 mM). Significant substrate inhibition occurred with concentrations of l-malate and oxaloacetate above 40 mM and 0.5 mM, respectively. The enzyme was unstable at temperatures above 40 C, but repeated freezing and thawing of the enzyme preparation had little effect on MDH activity. Only p-CMB inhibited MDH activity. Polyacrylamide gel electrophoresis demonstrated the presence of at least three MDH isoenzymes in the unpurified enzyme preparation, and the molecular weight was estimated at 49,000 by gel chromatography.     

Oxalacetate decar☐ylase and pyruvate car☐ylase activities,and effect of sulfhydryl reagents in malic enzyme from Sulfolubus solfataricus

Malic enzyme (S)-malate: NADP⁺ oxidoreductase (oxaloacetate-decar☐ylating, EC 1.1.1.40) purified from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4, catalyzed the metal-dependent decar☐ylation of oxaloacetate at optimum pH 7.6 at a rate comparable to the decar☐ylation of l-malate. The oxaloacetate decar☐ylase activity was stimulated about 50% by NADP but only in the presence of MgCl₂, and was strongly inhibited by l-malate and NADPH which abolished the NADP activation. In the presence of MnCl₂ and in the absence of NADP, the Michaelis constant and V_m for oxaloacetate were 1.7 mM and 2.3 μmol·min⁻¹·mg⁻¹, respectively. When MgCl₂ replaced MnCl₂, the kinetic parameters for oxaloacetate remained substantially unvaried, whereas the K_m and V_m values for l-malate have been found to vary depending on the metal ion. The enzyme carried out the reverse reaction (malate synthesis) at about 70% of the forward reaction, at pH 7.2 and in the presence of relatively high concentrations of bicarbonate and pyruvate. Sulfhydryl residues (three cysteine residues per subunit) have been shown to be essential for the enzymatic activity of the Sulfolobus solfataricus malic enzyme. 5,5′-Dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate and N-ethylmaleimide caused the inactivation of the oxidative decar☐ylase activity, but at different rates. The inactivation of the overall activity by p-hydroxymercuribenzoate was partially prevented by NADP singly or in combination with both l-malate and MnCl₂, and strongly enhanced by the car☐ylic acid substrates; NADP + malate + MnCl₂ afforded total protection. The inactivation of the oxaloacetate decar☐ylase activity by p-hydroxymercuribenzoate treatment was found to occur at a slower rate than that of the oxidative decar☐ylase activity.     

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.     

The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex.

1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.     

Microbial oxidation of methanol: Purification and properties of formaldehyde dehydrogenase from a Pichia sp. NRRL-Y-11328

An NAD⁺-linked, reduced glutathione-dependent formaldehyde dehydrogenase was purified to homogeneity from soluble extracts of methanol-grown yeast, Pichia sp. Formaldehyde and methylglyoxal are oxidized in the presence of NAD⁺ as an electron acceptor. NADP⁺ could not replace NAD⁺. Other straight chain aldehydes (C₂–C₆ tested), branched-chain aldehydes (e.g., isobutyaldehyde), aromatic aldehydes (e.g., salicylal-dehyde, benzaldehyde), glutyraldehyde, glyceraldehyde, glycoaldehyde, and glyoxal-dehyde tested were not oxidized by the purified formaldehyde dehydrogenase. The product of formaldehyde oxidation by purified enzyme was demonstrated to be S-for-mylglutathione by measuring the absorption at 240 nm due to the formation of thioester of formaldehyde and reduced glutathione. The K_m values for NAD⁺, formaldehyde, and reduced glutathione were 0.12, 0.31, and 0.16 mm, respectively, for the forward reaction at pH 8.0. The purified formaldehyde dehydrogenase also catalyzed the reduction of S-formylglutathione in the presence of NADH. Formate was not reduced by the purified enzyme. The K_m values for S-formylglutathione and NADH were 0.60 and 0.25 mm, respectively, for the reverse reaction at pH 6.0. Formaldehyde dehydrogenase has a molecular weight of 84,000 as determined by gel filtration and subunit molecular weight of 41,000 as determined by sodium dodecyl sulfate-gel electrophoresis. S-Formylglutathione, a product of formaldehyde oxidation, was oxidized by the partially purified formate dehydrogenase from Pichia sp. Formate dehydrogenase has a higher affinity toward S-formylglutathione (K_m value 1.8 mm) than toward formate (K_m value 25 mm). Antiserum prepared against the purified formaldehyde dehydrogenase from Pichia sp. NRRL-Y-11328 forms strong precipitin bands with isofunctional enzymes from methanol-grown Pichia pastoris NRRL-Y-7556 and Torulopsis candida Y-11419 and weak precipitin bands with Hansenula polymorpha NRRL-Y-2214. No cross-reaction was observed with isofunctional enzyme derived from methanol-grown Kloeckera sp.