Purification and Properties of Dextransucrase from Streptococcus mutans

The dextransucrase (EC 2.4.1.5) activity from cell-free culture supernatants of Streptococcus mutans strain 6715 has been purified approximately 1,500-fold by ammonium sulfate precipitation, hydroxylapatite chromatography, and isoelectric focusing. The enzyme was eluted as a single peak of activity from hydroxylapatite, and isoelectric focusing of the resulting preparation gave a single band of dextransucrase activity which focused at a pH of 4.0. The final enzyme preparation contained two distinct, enzymatically active proteins as judged by assay in situ after polyacrylamide gel electrophoresis. One of the proteins represented 90% of the total dextransucrase activity and 53% of the total protein. The molecular weight of the enzyme was estimated by gel filtration to be 94,000. The temperature optimum of the enzyme was broad (34 to 42 C) and its pH range was rather narrow, with optimal activity at pH 5.5. The K(m) for sucrose was 3 mM, and fructose competitively inhibited the enzyme reaction with a K(i) of 27 mM.     

Activation and inhibition of dextransucrase by calcium

Initial rate kinetics of dextran synthesis by dextransucrase (sucrose:1,6-alpha-D-glucan-6-alpha-D-glucosyltransferase, EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F showed that below 1 mM, Ca2+ activated the enzyme by increasing Vmax and decreasing the Km for sucrose. Above 1 mM, Ca2+ was a weak competitive inhibitor (Ki = 59 mM). Although it was an activator at low concentration, Ca2+ was not required for dextran synthesis, either of main chain or branch linkages. Neither was it required for sucrose hydrolysis, acceptor reactions, or enzyme renaturation after SDS-polyacrylamide gel electrophoresis. A model for dextran synthesis is proposed in which dextransucrase has two Ca2+ sites, one activating and one inhibitory. Ca2+ at the inhibitory site prevents the binding of sucrose.     

Purification and some properties of free and cell-associated dextransucrase from Streptococcus sanguis.

Dextransucrase of Streptococcus sanguis occurred in cell-free and cell-associated forms. Cell-free dextransucrase was purified by four successive chromatographies on Bio-Gel P 60, DEAE-cellulose, and Bio-Gel P 200 from the culture supernatant. The purification of cell-associated dextransucrase was made from the pellet of Streptococcus sanguis culture. Bacterial pellet was extracted with 1 M phosphate buffer (pH 6.0) and chromatographied by using an immunosorbent column. The two enzymes gave single bands in polyacrylamide gel electrophoresis. The molecular weight determined by sodium dodecyl sulfate polyacrylamide gel was about 100 000 daltons for the two forms of dextransucrases. The optimum pH of the cell-free and cell-associated enzymes was around 6 and the temperature optimum was broad for the two enzymes. The KM values for sucrose were respectively 2 mM and 3 mM for cell-free and cell-associated enzymes. When primer dextran was added, the reaction velocity increased but the KM for sucrose remained the same, and the KA for dextran was 200 muM for the two dextransucrases. Trehalose and maltose acted also as glucosyl residue acceptors. Purified enzymes had dextran synthesising activity and invertase-like activity. The same properties of the two forms of enzymes and the positive cross reaction against anti free and anti cell-associated globulins stongly suggest the identity of the two enzymes.     

Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli.

Phosphorylated NADP+-isocitrate dehydrogenase (EC 1.1.1.42) has been purified to electrophoretic homogeneity from in vivo 32P-labeled Escherichia coli. The cells used as the source of phosphorylated enzyme were harvested 1 h after the addition of 5 mCi of [32P]orthophosphoric acid and 25 mM sodium acetate to cultures grown to early stationary phase on a low phosphate medium with limiting glucose. Double immunodiffusion and autoradiography demonstrated immunological identity between the 32P-labeled NADP+-isocitrate dehydrogenase and the enzyme isolated from glucose-grown E. coli. The phosphoenzyme had an apparent subunit molecular weight of 51,000 as determined by denaturing acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the radioactivity co-electrophoresed with NADP+-isocitrate dehydrogenase activity when purified enzyme was subjected to nondenaturing gel electrophoresis. [32P]Phosphoserine was identified following partial acid hydrolysis of the purified phosphoenzyme.     

Purification and characterization of three soluble invertases from barley (Hordeum vulgare L.) leaves.

Three soluble isoforms of invertase (beta-fructofuranosidase; EC 3.2.1.26) were purified from 7-d-old primary leaves of barley (Hordeum vulgare L.). Invertase I, a monomeric protein of 64 kD, was purified to apparent homogeneity as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Invertases IIA and IIB, multimeric proteins with molecular masses of the 116 and 155 kD, were purified 780- and 1370-fold, respectively, but were not yet homogeneous. Extracts of epidermal strips of leaves contained only invertase IIB. The specific activity of invertase was more than 100-fold higher in the epidermis than in the mesophyll. All three isoforms were acidic invertases, with pH optima of around 5.0 and little activity in the alkaline range. Invertase I had a Km for sucrose of 8.1 mM, and invertases IIA and IIB had much lower values of 1.0 and 1.7 mM, respectively. Invertase I was more than 2-fold more resistant than the other two invertases to the inhibitors HgCl2 and pyridoxal. All three constitutive invertases were found to act also as sucrose-sucrose fructosyltransferases when supplied with high concentrations of sucrose, forming 1-kestose as principal product. However, the fructosyltransferase activity of all three enzymes was inhibited by pyridoxal in the same way as their invertase activity. This characteristic clearly differentiates them from the inducible sucrose-sucrose fructosyltransferase of barley leaves, the activity responsible for the initial steps of fructan biosynthesis, which has previously been shown to be insensitive to pyridoxal.     

Purification and characterization of an invertase produced by Aspergillus ochraceus TS.

Purification and characterization of an extracellular invertase produced by Aspergillus ochraceus TS are reported. The enzyme was purified (42-fold) from culture filtrate by salt precipitation, ion-exchange and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single band of molecular mass 66 kDa. The molecular mass of the native enzyme was found to be 130 kDa by gel filtration. The purity of the protein was also checked against its antiserum raised in rabbits by two-dimensional immunodiffusion in agarose gel and Western blot that showed a single band. It is a glycoprotein with mannose as its carbohydrate residue. The enzyme showed high affinity for sucrose with a Km of 3.5 mM. The amino acid analysis revealed a high proportion of acidic residues but it had a low content of cysteine, histidine and arginine comparable to other fungal invertases.     

Purification and some properties of phospholipase C (alpha-toxin) of Clostridium perfringens.

1. Phospholipase C[EC 3.1.4.3] was purified from the culture filtrate of Clostridium perfringens by successive chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-100. During the purification it was noted that, beside the monomer form of the enzyme which was purified, a part of the enzyme existed in active polymerized forms. 2. The purified preparation gave a single band on polyacrylamide gel electrophoresis and gave a single precipitin line in immunodiffusion with the National Standard gas gangrene (C. perfringens) antitoxin, indicating the homogeneity of the preparation. 3. The specific lecithin-hydrolyzing activity of the purified preparation was comparable to that of a preparation obtained by affinity chromatography, which had the highest specific activity previously reported. 4. The molecular weight of the purified enzyme was estimated to be 43,000 by SDS-polyacryl-amide gel electrophoresis, although the same preparation gave a molecular weight of 31,000 as determined by gel filtration on Sephadex G-150. From this and the above finding that a part of the enzyme exists in active polymerized forms, the discrepancy among reported values for the molecular weight of C. perfringens phospholipase C can be accounted for. 5. For maximum hydrolytic activity toward lecithin, the enzyme required sodium deoxycholate (SDC) and Ca2+ ions. In the presence of 6 mM Ca2+, the optimal molar ratio of SDC to lecithin for maximal hydrolytic activity was about 0.5 for dipalmitoyl lecithin and about 1.0 for egg lecithin. The effects of various divalent cations on the enzymatic hydrolysis were also investigated. 6. The effects of sodium deoxycholate and Ca2+ ions on the enzymatic hydrolysis are discussed, based on their possible roles in mixed micelle formation.     

Cytosolic and cell-wall-bound acid invertases from leaves of Urtica dioica L.: a comparison

Two different forms of acid invertase (EC 3.2.1.26) were extracted from expanding leaves of the stinging nettle (Urtica dioica L.). One form was soluble and could be localized within the cytosol, whereas the other was ionically bound to the cell-wall and could not be detected in protoplasts. Both forms were purified, the latter to homogeneity. Western blotting with antibodies against the pure enzyme from cell walls was positive with the cell-wall enzyme but negative with the soluble form of acid invertase. Both forms are glycoproteins with identical molecular weights of 58 kDa. The K_m values for sucrose (raffinose) are 5 mM (4.8 mM) for the soluble and 1.2 mM (3.6 mM) for the cell-wall-bound enzyme. The pH optimum of the latter is slightly more acidic (4.5) than that of the soluble invertase (5.5). Both forms could easily be distinguished by their isoelectric points which were determined at pH 4.6 for the soluble and pH 9.3 for the wall-bound enzyme. When extraction and purification were carried out in the absence of protease inhibitors, both acid invertases showed microheterogeneity (multiple forms). However, with benzamidine and phenylmethylsulfonylfluoride as protease inhibitors each invertase produced only one protein band upon isoelectric focusing and gel electrophoresis, respectively.Abbreviations B benzamidine - Con A concanavalin A - FPLC fast protein liquid chromatography - IEF isoelectric focusing - kDa kilodalton - pI isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the Sonderforschungsbereich 137.     

Invertases in young and mature leaves of Citrus sinensis

The sucrose catabolic enzymes acid invertase (EC 3.2.1.26) and alkaline invertase (EC 3.2.1.27) were studied in young and mature Citrus sinensis leaf tissue. In young, expanding leaves (60 % final length) soluble acid invertase activity predominated, while soluble alkaline invertase activity predominated in mature leaves. The acid and alkaline invertase activities were separated on Sephadex G-200. The acid invertase had an M_r of approximately 60 000, pH maximum of 4.5 and apparent K_m of 3.3 mM sucrose. The alkaline invertase had an M_r of approximately 200 000, pH maxima of 6.8 and an apparent K_m of 20 mM sucrose. Alkaline invertase was strongly inhibited by 10 mM Tris while acid invertase was not. Possible physiological roles for the two invertases are discussed.     

Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls.

Alkaline invertase from sprouting soybean (Glycine max) hypocotyls was purified to apparent electrophoretic homogeneity by consecutive use of DEAE-cellulose, green 19 dye, and Cibacron blue 3GA dye affinity chromatography. This protocol produced about a 100-fold purification with about a 11% yield. The purified protein had a specific activity of 48 mumol of glucose produced mg-1 protein min-1 (pH 7.0) and showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (58 kDa) and in native PAGE, as indicated by both protein and activity staining. The native enzyme molecular mass was about 240 kDa, suggesting a homotetrameric structure. The purified enzyme exhibited hyperbolic saturation kinetics with a Km (sucrose) near 10 mM and the enzyme did not utilize raffinose, maltose, lactose, or cellibose as a substrate. Impure alkaline invertase preparations, which contained acid invertase activity, on contrast, showed biphasic curves versus sucrose concentration. Combining equal activities of purified alkaline invertase with acid invertase resulted in a biphasic response, but there was a transition to hyperbolic saturation kinetics when the activity ratio, alkaline: acid invertase, was increased above unity. Alkaline invertase activity was inhibited by HgCl2, pridoxal phosphate, and Tris with respective Ki values near 2 microM, 5 microM, and 4 mM. Glycoprotein staining (periodic acid-Schiff method) was negative and alkaline invertase did not bind to two immobilized lectins, concanavalin A and wheat germ agglutinin; hence, the enzyme apparently is not a glycoprotein. The purified alkaline invertase, and a purified soybean acid invertase, was used to raise rabbit polyclonal antibodies. The alkaline invertase antibody preparation was specific for alkaline invertase and cross-reacted with alkaline invertases from other plants. Neither purified soybean alkaline invertases nor the crude enzyme from several plants cross-reacted with the soybean acid invertase antibody.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Production,purification, and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

The production of dextransucrase fromLeuconostoc mesenteroides NRRL B-512F was stimulated 2-fold by the addition of 0.005% of calcium chloride to the medium; levansucrase levels were unaffected. Dextransucrase was purified by concentration and dialysis of the culture supernatant with a Bio-Fiber 80 miniplant, and by treatment with dextranase followed by chromatography on Bio-Gel A-5m. A 240-fold purification, with a specific activity of 53 U/mg, was obtained. Contaminating enzyme activities of levansucrase, invertase, dextranase, glucosidase, and sucrose phosphorylase were decreased to non-detectable levels. Poly(acrylamide)-gel electrophoresis of the purified enzyme showed only two protein bands, both of which had dextransucrase activity. These bands also gave a carbohydrate stain, indicating that the dextransucrase could be a glycoprotein. Acid hydrolysis, followed by paper chromatography, of the purified enzyme showed that the major carbohydrate was mannose. ConcanavaIin A completely removed dextransucrase activity from solution, confirming the mannoglycoprotein character of the enzyme. Dextransucrase activity was not altered by the addition of 0.008?4 mg/ml of dextran, but its storage stability was increased by the addition of 4 mg/ml of dextran. As previously shown by others, the activity of dextransucrase was decreased by EDTA, and was restored by the addition of calcium ions. Zinc, cadmium, lead, mercury, and copper ions were inhibitory to various degrees.     

Initial characterization of sucrose-6-phosphate hydrolase from Streptococcus mutans and its apparent identity with intracellular invertase.

An intracellular enzyme catalyzing the hydrolysis of sucrose-6-phosphate to glucose-6-phosphate and fructose has been identified in extracts of $\text{Streptococcus}\text{mutans}$ 6715-10. The preparation was purified chromatographically and found to have an apparent molecular weight of 42,000. The enzyme has as a K_m for sucrose-6-phosphate of 0.21 mM, a pH optimum of 7.1, is quite stable and requires no added cofactors or metal ions. Sucrose is a competitive inhibitor of sucrose-6-phosphate hydrolysis (K_i = 8. 12 mM). A previously described intracellular invertase copurifies with the enzyme and could not be separated from it by disc gel electrophoresis. It is concluded that intracellular invertase is a sucrose-6-phosphate hydrolase with a low catalytic activity for hydrolysis of sucrose.     

Molecular, functional and ultrastructural characterisation of plastids from six species of the parasitic flowering plant genus Cuscuta

 Hydroponically cultivated barley plants were exposed to nitrogen (N)-deficiency followed by N-resupply. Metabolic and genetic regulation of fructan accumulation in the leaves were investigated. Fructan accumulated in barley leaves under N-deficiency was mobilized during N-resupply. The enhanced total activity of fructan-synthesizing enzymes, sucrose:sucrose 1-fructosyltransferase (EC 2.4.1.99) and sucrose:fructan 6-fructosyltransferase (6-SFT; EC 2.4.1.10) caused by N-deficiency decreased with the mobilization of fructan during N-resupply. The activity of the barley fructan-degrading enzyme, fructan exohydrolyase (EC 3.2.1.80) was less affected by the N status. The low level of foliar soluble acid invertase activity under N-deficiency conditions was maintained during the commencement of N-resupply but increased subsequently. Further analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot and northern blot demonstrated that the fructan accumulation and the total activity of fructan-synthesizing enzymes correlated with the 6-SFT mRNA level. We suggest that the changes in fructan levels under N stress are intimately connected with the regulation of fructan synthetic rate which is mostly controlled by 6-SFT. Received: 25 October 1999 / Accepted: 15 February 2000     

Purification and properties of goat liver catalase: two pH optima

Goat liver catalase (EC 1.11.1.6) has been purified to homogeneity using the techniques of ammonium sulfate fractionation, DEAE-cellulose chromatography and gel-filtration through Ultrogel AcA-34 involving two alternating steps of column chromatography. The homogeneity of the purified enzyme was tested by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunodiffusion and immunoelectrophoresis. The enzyme is a tetramer having a subunit molecular weight of 58,000 +/- 3000, contains six sulfhydryl groups per mole of the enzyme and shows pH optima at pH 6.8 and 7.7. The kinetic data show no cooperativity between the substrate binding sites. Tryptophan, indoleacetic acid, cysteine, formaldehyde and sodium azide inhibit the enzyme non-competitively with Ki values of 4 +/- 1, 2.5 +/- 0.8, 6 +/- 1.5, 0.48 +/- 0.15 and 0.0013 +/- 0.0003 mM, respectively. Sulfhydryl group binding agents as well as thiol reagents inhibit the enzyme activity.     

Electrophoretic analysis of the multiple forms of dextransucrase from Leuconostoc mesenteroides

Multiple forms of the extracellular dextransucrase [EC 2.4.1.5] from Leuconostoc mesenteroides NRRL B-512F strain were characterized by polyacrylamide gel electrophoresis. Based on the Rm (Relative mobility) values, a newly devised simple plot of log (Rm X 10/(1-Rm)) vs. degree of association of the enzyme showed a good correlation with the results obtained by the Hedrick-Smith method. Both results indicated that the B-512F dextransucrase aggregates were a mixture of two types of forms, i.e., oligomers of a 65 kDa protomer and their charge isomers. Boiling and treatment of the enzyme at pH 10.5 suggested that enzyme aggregates contained dextran or its fragments bound to the enzyme and the enzyme-dextran complex showed the charge isomerism. Since the highly aggregated forms showed higher activity for dextran synthesis than the dissociated forms, the endogenous dextran may serve as a source of primer and may stabilize the enzyme molecule. Besides allosteric regulation of the activity, the occurrence of oligomeric forms of the enzyme may play an important role in the control of dextran synthesis in vivo.     

Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

Isolation and characterization of the major form of beta-glucuronidase from human seminal plasma

Human seminal plasma contain two forms of beta-glucuronidase (beta-D-glucuronidase glucuronosohydrolase, EC 3.2.1.31) which are present in the ratio of 4:1. The major form of beta-glucuronidase with a slow moving band in electrophoresis was purified to homogeneity as revealed by polyacrylamide gel electrophoresis, double immunodiffusion and immunoelectrophoresis. The major form of beta-glucuronidase shows dual optimum pH at 4.3 and 4.7 with a dip in the activity at pH 4.5. The Km of this form of beta-glucuronidase is dependent on pH and was found to be 0.95, 3.08 and 0.67 mM at pH 4.4, 4.5 and 4.7, respectively. The major form of beta-glucuronidase from seminal plasma is stable at 55 degrees C for 30 min but it denatures at 65 degrees C. Heat denaturation is faster at acidic pH (4.7) than at alkaline pH (7.8). However, the activity of enzyme increased linearly with increase in temperature up to 70 degrees C during incubation with substrate. Cu, Ag, Hg and Ni salts inhibited enzyme activity significantly at 0.1 and 1.0 mM concentration, but the inhibition of HgCl2 was protected by cysteine. 1,4-D-Saccharic acid lactone and ascorbic acid inhibited seminal beta-glucuronidase competitively, yielding Ki values of 1.7 . 10(-3) mM and 10.3 mM, respectively. Though fructose and mannose also showed significant inhibition of beta-glucuronidase at 10-100 mM, glucose did not show any effect. The molecular weight of the major form of beta-glucuronidase was found to be 279 000, and it appears to be composed of four subunits each having a molecular weight of 74 000.     

Purification and properties of an NADP-specific 6-phosphogluconate dehydrogenase from Streptococcus faecalis.

A procedure is described for the purification of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating) EC 1.1.1.44) from cell extracts of Streptococcus gaecalis. A 180-fold purification was achieved with an over-all yield of about 12% and an average specific activity of 14. The enzyme was homogeneous as determined by polyacrylamide gel electrophoresis, immunoelectrophoresis, and sedimentation equilibrium, studies. Its weight average molecular weight, as measured by sedimentation equilibrium, was 108,000 +/- 3,600. Other methods employed for molecular weight determinations gave values that ranged between 106,000 and 115,000. An analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed it to be a dimer composed of subunits having equal molecular weight. The amino acid composition of the streptococcal enzyme is reported. The apparent Km values for NADP and 6-phosphogluconate were calculated from kinetic data and found to be 0.015 mM and 0.024 mM, respectively. Kinetic studies also indicated that the binding of one substrate did not affect the apparent affinity of the enzyme for the other substrate.     

Purification and properties of aldose reductase form Pachysolen tannophilus

Aldose reductase (EC 1.1.1.21) from Pachysolen tannophilus IFO 1007 was purified 15 fold from the crude enzyme in a yield of 0.9% by pH 5 treatment, protamine sulfate precipitate, ammonium sulfate fractionation, and G-100 gel chromatography. The purified enzyme was entirely homogeneous on disc gel electrophoresis. The optimum pH and temperature were 5–6 and 50°C, and it was stable at pH 6–8 and up to 35°C. Its activity was enhanced slightly by Na₂SO₄, glycylglycine, glutathione, and cysteine, and inhibited remarkably by SH inhibitors such as AgNO₃, HgCl₂, lead acetate and iodo-acetate. Its K_m values were determined ad follows: 0.97 mM for d-glyceraldehyde, 1.7 mM for dl-glyceraldehyde, 3.5 mM for d-erythrose, 12 mM for d-xylose, 18mM for l-arabinose, 25 mM for galactose, 33 mM for valeraldehyde, 33 mM for 2-deoxy-d-glucose, 50 mM for propionaldehyde, 67 mM for d-ribose, 200 mM for d-mannose, and 280 mM for acetaldehyde. The enzyme also reduced glucose, l-sorbose, butylaldehyde, and benzaldehyde. Its molecular weight was estimated to be 40,650 by sedimentation equilibrium, 40,000 by SDS polyacrylamide gel electrophoresis and 43,000 by Sephadex G-200 column chromatography.