Evidence that the gene YLR070c of Saccharomyces cerevisiae encodes a xylitol dehydrogenase.

The open reading frame YLR070c of Saccharomyces cerevisiae has high sequence similarity to S. cerevisiae sorbitol dehydrogenase and to xylitol dehydrogenase of Pichia stipitis. Overexpression of this open reading frame in S. cerevisiae resulted in xylitol dehydrogenase activity. The enzyme is specific for NADH. The following Michaelis constants were estimated: D-xylulose, 1.1 mM; NADH, 240 microM (at pH 7.0); xylitol, 25 mM; NAD, 100 microM (at pH 9.0). Xylitol dehydrogenase activity with the same kinetic properties can also be induced by xylose in wild type S. cerevisiae cells.     

Biochemical characteristics of erythrocyte sorbitol dehydrogenase from selected mammals

The erythrocyte sorbitol dehydrogenase (EC 1.1.1.14) activity, regarding its action on sorbitol oxidation to fructose, was studied in 19 species of mammals, showing a striking variability, with high activity in rodents. Enzyme activity was studied against other polyols, namely xylitol, inositol, manitol and dulcitol. Most animals showed activity against all the polyols studied, but hamster and red deer only presented activity on sorbitol and xylitol. Michaelis-Menten constant determinations for sorbitol were performed, and it was observed that animals which presented high activity had a high Km. pH curves were obtained from 8 animals, with an optimum pH ranging from pH 8.0 to pH 10.0; four of the animals presented an optimum pH at 8.5.     

Polyol-pathway enzymes of human brain. Partial purification and properties of sorbitol dehydrogenase.

Sorbitol dehydrogenase was isolated from human brain and purified 690-fold, giving a final specific activity of 11.1 units/mg of protein. The enzyme preparation was nearly homogeneous, but was unstable at most temperatures. It exhibited a broad pH optimum of 7.5-9.0 in the forward reaction (i.e. sorbitol leads to fructose), and of 7.0 in the reverse reaction (i.e. fructose leads to sorbitol). Substrate-specificity studies demonstrated that the enzyme had the capability to oxidize a wide range of polyols and that the enzyme had a higher affinity for substrates in the forward reaction than in the reverse reaction, e.g. Km for sorbitol was 0.45 mM, and that for fructose was 480 mM. However, the Vmax. was 10 times greater in the reverse reaction. At high concentrations of fructose (500 mM) the enzyme exhibited substrate inhibition in the reverse reaction. The enzyme mechanism was sequential, as determined by the kinetic patterns arising from varying the substrate concentrations. In addition, both fructose and NADH protected the enzyme against thermal inactivation. These findings, together with product-inhibition data, suggested that the mechanism is random rapid equilibrium with two dead-end complexes.     

Kinetic properties of aldehyde dehydrogenase from sheep liver mitochondria.

The kinetics of the NAD+-dependent oxidation of aldehydes, catalysed by aldehyde dehydrogenase purified from sheep liver mitochondria, were studied in detail. Lag phases were observed in the assays, the length of which were dependent on the enzyme concentration. The measured rates after the lag phase was over were directly proportional to the enzyme concentration. If enzyme was preincubated with NAD+, the lag phase was eliminated. Double-reciprocal plots with aldehyde as the variable substrate were non-linear, showing marked substrate activation. With NAD+ as the variable substrate, double-reciprocal plots were linear, and apparently parallel. Double-reciprocal plots with enzyme modified with disulfiram (tetraethylthiuram disulphide) or iodoacetamide, such that at pH 8.0 the activity was decreased to 50% of the control value, showed no substrate activation, and the plots were linear. At pH 7.0, the kinetic parameters Vmax. and Km NAD+- for the oxidation of acetaldehyde and butyraldehyde by the native enzyme are almost identical. Formaldehyde and propionaldehyde show the same apparent maximum rate. Aldehyde dehydrogenase is able to catalyse the hydrolysis of p-nitrophenyl esters. This esterase activity was stimulated by both NAD+ and NADH, the maximum rate for the NAD+ stimulated esterase reaction being roughly equal to the maximum rate for the oxidation of aldehydes. The mechanistic implications of the above behaviour are discussed.     

Biosynthesis of branched-chain amino acids in Schizosaccharomyces pombe: regulatory properties of threonine deaminase

Biosynthetic threonine deaminase (TD) from Schizosaccharomyces pombe has been partially purified from crude extracts by treatment with protamine sulfate, ammonium sulfate precipitation, and gel filtration through Sephadex G-25. In both crude extracts and purified preparations, TD showed marked stimulation by pyridoxal phosphate. A pH optimum for activity was found at pH 9.0, whereas the inhibition caused by the natural feedback inhibitor, l-isoleucine, was maximal at pH 7.4. l-Threonine exhibits homotropic cooperative effects at low pH (7.0-8.0), which are eliminated at pH 9.0, and the affinity for substrate (in terms of K(m)) increased with increasing pH. Enzyme activity could be completely inhibited by isoleucine over a pH range of 7.4 to 9.0; the amount of isoleucine required for 50% inhibition increased with increasing pH. Isoleucine inhibition was pseudocompetitive with respect to substrate and increased the cooperative effects of threonine. l-Valine was found to reverse isoleucine inhibition; it also activated the enzyme in a pH range of 7.0 to 8.0 by eliminating the cooperative effects of threonine, thus normalizing the substrate saturation curves at these pH values. l-Leucine was shown to be a competitive inhibitor with respect to threonine, and to be able partially to reverse isoleucine inhibition. Treatment of TD with mercurials did not result in desensitization to isoleucine inhibition. However, at pH 10, virtually no sensitivity of the enzyme to isoleucine was observed while activity remained strong, which suggests the existence of separate sites on the TD molecule for binding threonine and isoleucine. A tentative model is presented which unifies the kinetic results reported here in terms of the interactions of TD with its effector molecules.     

Comparative properties of multiple forms of rabbit muscle phosphofructokinase

Some properties of three interconvertible forms of rabbit muscle phosphofructokinase specifically eluted from DEAE-cellulose with 19 mM citrate in 0.1 M tris-phosphate buffer, pH 8,0 (I), with 0,3 M buffer (II) and 1.5 M NaCl (III) are compared. Forms I-III differ in specific activities, alpha-helices content and sedimentation properties. The kinetic behaviour of forms I and III in 25 mM glycylglycine-beta-glycerophosphate, pH 8.3, at inhibitory ATP concentrations is characterized by biphasic velocity versus fructose-6-phosphate concentration curves with nH = 1.0 and 2.3, but with different V and [S]0.5 for the respective forms. At pH 6.8 from I is characterized by the kinetic curves with a lag period, while form III--by that with a burst. Form I reveals negative cooperativity in initial and stationary velocities at low substrate concentrations. The stationary velocity of form III is characterized by negative cooperativity within the whole concentration range studied. At pH 7.0 both forms are inhibited by citrate according to the initial and stationary velocities; however, the Ki values are different. The complex kinetic behaviour of phosphofructokinase corresponds to its complex chromatographic and sedimentation behaviour. The multiplicity of the enzyme forms seems to be due to a complex set of its oligomers and conformers and a hysteretic type of transitions between them as well as to its phosphorylation and possible binding of ligands.     

Some kinetic properties of gamma-glutamyltransferase from rabbit liver

gamma-Glutamyltransferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2) of rabbit liver (detergent form) was purified 1100-fold in order to study its kinetic properties. Kinetic studies were conducted from pH 6.0 to 12.0 in the absence and presence of the acceptor substrate glycylglycine using gamma-glutamyl-3-carboxy-4-nitroanilide as the donor. The existence of more than one binding site for both donor and acceptor is postulated on kinetic evidence such as donor substrate activation, donor substrate inhibition and acceptor substrate activation. Homotropic interaction is also observed, in the form of negative cooperativity, in donor substrate binding, in the absence of acceptor at pH less than 9.0 and positive cooperativity (n = 2), in the absence or presence of acceptor at pH greater than 9.0. Hydrolase reaction reaches a maximum of activity at pH 10 (pK 8.6). Transferase activity under conditions of maximal velocity is maximal at pH 9.0 (pK 7.1). The ratio of transferase activity/hydrolase activity is maximal at pH 7.0-7.5. At low donor substrate concentrations, maximal activity is attained at pH 7.5.     

Inhibition of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide

Renal hyperosmotic conditions may produce reactive oxygen species, which could have a deleterious effect on the enzymes involved in osmoregulation. Hydrogen peroxide was used to provoke oxidative stress in the environment of betaine aldehyde dehydrogenase in vitro. Enzyme activity was reduced as hydrogen peroxide concentration was increased. Over 50% of the enzyme activity was lost at 100 μM hydrogen peroxide at two temperatures tested. At pH 8.0, under physiological ionic strength conditions, peroxide inhibited the enzyme. Initial velocity assays of betaine aldehyde dehydrogenase in the presence of hydrogen peroxide (0-200 μM) showed noncompetitive inhibition with respect to NAD(+) or to betaine aldehyde at saturating concentrations of the other substrate at pH 7.0 or 8.0. Inhibition data showed that apparent V(max) decreased 40% and 26% under betaine aldehyde and NAD(+) saturating concentrations at pH 8.0, while at pH 7.0 V(max) decreased 40% and 29% at betaine aldehyde and NAD(+) saturating concentrations. There was little change in apparent Km(NAD) at either pH, while Km(BA) increased at pH 7.0. K(i) values at pH 8 and 7 were calculated. Our results suggest that porcine kidney betaine aldehyde dehydrogenase could be inhibited by hydrogen peroxide in vivo, thus compromising the synthesis of glycine betaine.     

The effect of pancreatic procolipase and colipase on pancreatic lipase activation

Intestinal fat digestion is carried out by the concerted action of pancreatic lipase and its protein cofactor colipase. Colipase is secreted from pancreas as a procolipase and is transformed into colipase by the trypsin cleavage of the Arg5-Gly6 bond during liberation of an N-terminal pentapeptide. The kinetic parameters for the lipase-colipase system compared to the lipase-procolipase system has been compared using trioctanoin and Intralipid as substrates. It was found that at pH 7.0 the Kmapp using Intralipid as substrate was the same for procolipase and colipase, 0.06 mM and 0.05 mM, respectively. At pH 8.0, however, the Kmapp were different-0.23 mM for procolipase and 0.08 mM for colipase. In a similar way the binding between colipase and lipase had a dissociation constant of 2.4 x 10(-6) M at pH 7.0, while for procolipase--lipase binding the dissociation constant was 4.1 x 10(-6) M with no significant difference. At pH 8.0 the binding between colipase and lipase was stronger, Kd being 2.0 x 10(-7) M, while weaker for procolipase and lipase, Kd being 1.0 x 10(-5) M. It is concluded that at the physiological pH value as is found in the intestine, the activation of procolipase to colipase has no influence on the hydrolysis of trioctanoin or Intralipid in the presence of bile salt.     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol.

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

Purification and characterization of polyethylene glycol dehydrogenase involved in the bacterial metabolism of polyethylene glycol

Polyethylene glycol (PEG) dehydrogenase in crude extracts of a PEG 20,000-utilizing mixed culture was purified 24 times by precipitation with ammonium sulfate, solubilization with laurylbetaine, and chromatography with diethylamino-ethyl-cellulose, hydroxylapatite, and Sephadex G-200. The purified enzyme was confirmed to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme, which appeared to consist of four identical subunits, was 2.4 X 10(5). The enzyme was stable below 35 degrees C and in the pH range of 7.5 to 9.0. The optimum pH and temperature of the activity were around 8.0 and 60 degrees C, respectively. The enzyme did not require any metal ions for activity and oxidized various kinds of PEGs, among which PEG 6,000 was the most active substrate. The apparent Km values for tetraethylene glycol and PEG 6,000 were about 10.0 and 3.0 mM, respectively.     

In situ behaviour of D(-)-lactate dehydrogenase from Escherichia coli

Some kinetic properties of the D(-)-lactate dehydrogenase (EC 1.1.1.28) of Escherichia coli have been investigated. There were marked differences between the kinetic properties of the enzyme studied in situ compared with the in vitro D(-)-lactate dehydrogenase. D(-)-Lactate dehydrogenase in situ showed high substrate inhibition with pyruvate over the pH range 6.0–7.0, whereas the enzyme in vitro did not. The pH optimum for pyruvate reduction by the in situ D(-)-lactate dehydrogenase ranged between pH 7.5 and 7.8, whereas the in vitro enzyme showed its pH optimum between pH 6.8 and 7.0. The pK values of the prototropic groups that controlled the enzymatic activity shift to the acidic region for the in vitro enzyme with respect to the in situ enzyme. In vitro D(-)-lactate dehydrogenase exhibits homotropic interactions with its substrate, pyruvate and its coenzyme, NADH, at pH values ranging between pH 6.0 and 8.5, but the in situ enzyme showed homotropic interactions neither with pyruvate nor with NADH at all pH values studied.     

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).     

Increase in stability of xylanase from an alkalophilic thermophilic Bacillus (NCIM 59)

Summary The cellulase-free xylanase from an alkalophilic thermophilic Bacillus was stable at pH 7.0 to 10.0 at 50 ° for 3 days.At 60 ° the enzyme showed a decrease in stability with a half- life of 3 h. Addition of various additives had no effect on the enzyme stability at 60 °. Glycine (0.5M) increased the enzyme half-life 6-fold at pH 7.0 to 9.0 and at 60 and 70 °. Xylan could offer protection against thermoinactivation of the xylanase at pH 7.0 and 8.0 at 60 ° and only a marginal increase at pH 9.0 at 70 ° was observed.     

Coenzyme-induced slow transitions of NADP-sorbitol dehydrogenase from Gluconobacter oxydans]

The kinetic properties of NADP-dependent sorbitol dehydrogenase from G. oxydans cell extract were studied at pH 8.8 and 9.3 in the direction of D-sorbitol oxydation. It was shown that the shape of the kinetic curves of NADPH accumulation in time is characterised by initial burst whose magnitude depends on the concentration of the enzyme extract used. Preincubation of the enzyme with NADP or D-sorbitol eliminated the initial burst on these curves and transformed them into straight lines coming from the start of co-ordinates. The dependence of the stationary reaction rate on the enzyme extract concentration is not a linear one. The kinetic dependences of stationary rate of the reaction catalysed by the enzyme on the concentration of D-sorbitol and NADP at pH 8.8 and 9.3 were examined under all conditions studied; the shape of these kinetic curves altered to considerable extent with the alteration of the enzyme extract concentration in the reaction mixture and pH. At pH 9.3 several intermiediate plateaux were found on the curves of the D-sorbitol concentration dependent stationary rate of the reaction. The preincubation of the enzyme extract with NADP during 1.5 h removed the intermediate plateau on these curves and made them hyperbolic. Disk-electrophoresis of the enzyme extract in PAAG concentration gradient showed that at pH 8.8 the enzyme exists in one active form, while at pH 9.3 it exists in three major and three minor active forms of the enzyme differing in their molecular weights are found. It is assumed that the enzyme from G. oxydans cell extract can exist in a great number of molecular equilibrium forms, the rate of quilibrium being comparable or significantly less than that of the enzymatic reaction. NADP significantly influences on the equilibrium of the molecular forms of the enzyme.     

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves. Kinetic properties of different oligomeric structures

NADP-dependent malate dehydrogenase (decarboxylating) from sugar cane leaves was inhibited by increasing the ionic strength in the assay medium. The inhibitory effect was higher at pH 7.0 than 8.0, with median inhibitory concentrations (IC50) of 89 mM and 160 mM respectively, for inhibition by NaCl. Gel-filtration experiments indicated that the enzyme dissociated into dimers and monomers when exposed to high ionic strength (0.3 M NaCl). By using the enzyme-dilution approach in the absence and presence of 0.3 M NaCl, the kinetic properties of each oligomeric species of the protein was determined at pH 7.0 and 8.0. Tetrameric, dimeric and monomeric structures were shown to be active but with different V and Km values. The catalytic efficiency of the oligomers was tetramer greater than dimer greater than monomer, and each quaternary structure exhibited higher activity at pH 8.0 than 7.0. Dissociation constants for the equilibria between the different oligomeric forms of the enzyme were determined. It was established that Kd values were affected by pH and Mg2+ levels in the medium. Results suggest that the distinct catalytic properties of the different oligomeric forms of NADP-dependent malate dehydrogenase and changes in their equilibrium could be the molecular basis for an efficient physiological regulation of the decarboxylation step of C4 metabolism.     

Kinetics of L-gamma-Glu-pNA hydrolysis by destabilase, the enzyme from the medicinal leech Hirudo medicinalis

The molecular mass of destabilase isolated from the medicinae leech Hirudo medicinalis was found to be equal to 12.3 kDa. A kinetic analysis of the sole presently known synthetic substrate, L-gamma-Glu-pNA, showed that the enzyme is relatively stable to heating (5 min, 70 degrees C); the pH optimum lies at 7.0-8.5. The enzyme has a specific activity of 0.15 x 10(-9) mol.s-1.mg-1; Km = 2.2 x 10(-4) M, kcat is 3.53 x 10(-3) s-1 (pH 8.0, 37 degrees C).     

Conversion of a glutamate dehydrogenase into methionine/norleucine dehydrogenase by site-directed mutagenesis.

In earlier attempts to shift the substrate specificity of glutamate dehydrogenase (GDH) in favour of monocarboxylic amino-acid substrates, the active-site residues K89 and S380 were replaced by leucine and valine, respectively, which occupy corresponding positions in leucine dehydrogenase. In the GDH framework, however, the mutation S380V caused a steric clash. To avoid this, S380 has been replaced with alanine instead. The single mutant S380A and the combined double mutant K89L/S380A were satisfactorily overexpressed in soluble form and folded correctly as hexameric enzymes. Both were purified successfully by Remazol Red dye chromatography as routinely used for wild-type GDH. The S380A mutant shows much lower activity than wild-type GDH with glutamate. Activities towards monocarboxylic substrates were only marginally altered, and the pH profile of substrate specificity was not markedly altered. In the double mutant K89L/S380A, activity towards glutamate was undetectable. Activity towards L-methionine, L-norleucine and L-norvaline, however, was measurable at pH 7.0, 8.0 and 9.0, as for wild-type GDH. Ala163 is one of the residues that lines the binding pocket for the side chain of the amino-acid substrate. To explore its importance, the three mutants A163G, K89L/A163G and K89L/S380A/A163G were constructed. All three were abundantly overexpressed and showed chromatographic behaviour identical with that of wild-type GDH. With A163G, glutamate activity was lower at pH 7.0 and 8.0, but by contrast higher at pH 9.0 than with wild-type GDH. Activities towards five aliphatic amino acids were remarkably higher than those for the wild-type enzyme at pH 8.0 and 9.0. In addition, the mutant A163G used L-aspartate and L-leucine as substrates, neither of which gave any detectable activity with wild-type GDH. Compared with wild-type GDH, the A163 mutant showed lower catalytic efficiencies and higher K(m ) values for glutamate/2-oxoglutarate at pH 7.0, but a similar k(cat)/K(m) value and lower K(m) at pH 8.0, and a nearly 22-fold lower S(0.5) (substrate concentration giving half-saturation under conditions where Michaelis-Menten kinetics does not apply) at pH 9.0. Coupling the A163G mutation with the K89L mutation markedly enhanced activity (100-1000-fold) over that of the single mutant K89L towards monocarboxylic amino acids, especially L-norleucine and L-methionine. The triple mutant K89L/S380A/A163G retained a level of activity towards monocarboxylic amino acids similar to that of the double mutant K89L/A163G, but could no longer use glutamate as substrate. In terms of natural amino-acid substrates, the triple mutant represents effective conversion of a glutamate dehydrogenase into a methionine dehydrogenase. Kinetic parameters for the reductive amination reaction are also reported. At pH 7 the triple mutant and K89L/A163G show 5 to 10-fold increased catalytic efficiency, compared with K89L, towards the novel substrates. In the oxidative deamination reaction, it is not possible to estimate k(cat) and K(m) separately, but for reductive amination the additional mutations have no significant effect on k(cat) at pH 7, and the increase in catalytic efficiency is entirely attributable to the measured decrease in K(m). At pH 8 the enhancement of catalytic efficiency with the novel substrates was much more striking (e.g. for norleucine approximately 2000-fold compared with wild-type or the K89L mutant), but it was not established whether this is also exclusively due to more favourable Michaelis constants.     

The kinetic mechanism of sheep liver sorbitol dehydrogenase.

The relations between the kinetic parameters for both sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase show that a Theorell-Chance compulsory order mechanism operates from pH 7.4 to 9.9. This is supported by many parallels with the kinetics of horse liver alcohol dehydrogenase, which operates by this classical mechanism. An isotope-exchange study using D-(2H8)sorbitol confirmed the existence of ternary complexes and that, under maximum velocity conditions, their interconversion is not rate-determining. Substrate inhibition at high concentrations of D-sorbitol or D-fructose confirmed rate-determining enzyme--coenzyme product dissociation, slowed by the existence of more stable abortive ternary enzyme-coenzyme product complexes with substrate. The effect of the inhibitor/activator 2,2,2-tribromoethanol showed the existence of enzyme-NAD-CBr3CH2OH complexes inhibiting the first phase of reaction and enzyme-NADH-CBr3CH2OH complexes dissociating more rapidly than the usual rate-determining enzyme-NADH coenzyme product dissociation in the final phase. Inhibition studies with dithiothreitol also confirmed an ordered binding of coenzymes and second substrates to sorbitol dehydrogenase. Neither D-sorbitol nor D-fructose had any effect on enzyme inactivation by the affinity labelling reagent DL-2-bromo-3-(5-imidazolyl)propionic acid, thus giving no evidence for their existence as binary enzyme-substrate complexes. Several alternative polyol substrates for sorbitol dehydrogenase gave the same maximum velocity as sorbitol. This indicated a common rate-limiting binary enzyme-NADH product dissociation and a similarity of mechanism. An enzyme assay for pH 7.0 and 9.9 is given which enables the concentration of sorbitol dehydrogenase to be determined from initial rate measurements of enzyme activity.     

Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides

A polyol dehydrogenase was detected in cell extracts of the facultative phototrophic bacterium Rhodobacter sphaeroides strain Si 4 grown on D-glucitol (sorbitol) as the sole carbon source. The enzyme was purified 150-fold to apparent homogeneity by steps involving fractionated (NH4)2SO4 precipitation, chromatography on Q-Sepharose and phenyl-Sepharose, and FPLC on Superose 12. The relative molecular mass (Mr) of the native polyol dehydrogenase was 47,200 as calculated from its Stokes' radius (rs = 2.76 nm) and sedimentation coefficient (s20, w = 4.15 S). SDS/PAGE resulted in one single band representing a polypeptide with a Mr of 52,200, indicating that the native protein is a monomer. The isoelectric point of the polyol dehydrogenase was determined to be pH 4.3. The enzyme was specific for NAD+ and oxidized both D-glucitol and D-mannitol to D-fructose, as well as D-arabinitol to D-ribulose. The pH optimum of substrate oxidation was pH 9.0 in 0.1 M Tris/HCl and that of substrate reduction was pH 6.5 in 0.1 M potassium phosphate. The reactions exhibited normal Michaelis-Menten kinetics allowing the estimation of KM values for NAD+ (0.18 mM) in the presence of D-glucitol, and for D-glucitol (31.8 mM), D-mannitol (0.29 mM) and D-arabinitol (1.8 mM), respectively. The KM value for D-fructose was 16.3 mM and that for NADH 0.02 mM. The equilibrium constants determined for the conversion of D-mannitol, D-glucitol and D-arabinitol were 4.5 nM, 0.58 nM and 80 pM, respectively. Based on the catalytic preference of the polyol dehydrogenase for D-mannitol, an enzymatic assay for D-mannitol was elaborated.