Diethylaminoethyl Sephadex Research Articles

Biotransformation and elimination of digoxin with normal and minimal renal function

Six subjects with normal renal function (NRF) and 6 patients with minimal renal function (MRF) on 3 times weekly hemodialysis received 150 muCi3H-digoxin-12 alpha orally. Serial urine collections were made for five days or more. Digoxin and metabolites were separated using diethylaminoethyl Sephadex LH-20 column chromatography. Mean cumulative percentages of the ingested radioactivity excreted over five days in NRF and MRF groups were: digoin, 54.5% and 14.7%; bis-digitoxoside of digoxigenin, 2.0% and 0.59%; mono-digitoxoside, 0.8% and 0.19%; digoxigenin, 0.25% and 0.03%; and dihydrodigoxin, 0.3% and 0.03%. Half-lives based on the mean rates of disappearance from urine comparing NRF and MRF groups were: for digoxin 40 hr and 120 hr; for bis-digitoxoside, 11.5 hr and 46 hr; for mono-digitoxoside, 8.5 hr and 12 hr; for digoxigenin, 2 hr and 7.5 hr; and for dihydrodigoxin, 1.2 hr and 7.0 hr. Considering the relationships of the five-day cumulative excretion and half-lives of digoxin and metabolites in the NRF and MRF groups, it appears unlikely that there is a major alteration in the biotransformation of digoxin in advanced renal failure when there appears to be a shift from renal to slower biliary excretion.

Purification and characterization of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli

A simple procedure is described for the purification in high yields of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli strain MRE 600. IF2 was separated from IF1 and IF3 by ammonium sulfate fractionation and was purified by column chromatography on phosphocellulose and diethylaminoethyl (DEAE) Sephadex. IF1 and IF3 were separated by phosphocellulose column chromatography. IF1 was purified by molecular sieve chromatography, and IF3 by phosphocellulose column chromatography in urea buffer. Each factor was analyzed by sodium dodecyl sulfate or urea polyacrylamide gel electrophoresis and was greater than 98% pure. Only one form of IF1 and IF3 was found, with molecular weights of 8,500 and 22,500, respectively. Two forms of IF2 were isolated: IF2a with a molecular weight of 118,000 and IF2b with a molecular weight of 90,000. The amino acid composition of each factor was determined, and their stimulation in a variety of assays for initiation of protein synthesis is reported.

Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus.

Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 47,000, indicating a tetramer of identical subunits. The isoelectric point of the bifunctional enzyme was 5.8. Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutasedehydratase by DEAE-cellulose chromatgraphy. Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The molecular weight of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis. Its isoelectric point was pH 6.6. In the overall conversion of chorismate to phenylpyruvate, free prephenate was formed which accumulated in the reaction mixture. The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.

Further studies on the peptido-galactomannan from the yeast form of Cladosporium werneckii: Identification of O-acetyl substituents and isolation of the peptide components following β-elimination

The pathogenic yeast Cladosporium werneckii produces a surface peptido-phosphogalactomannan (PPGM) with a peptide backbone rich in serine and threonine to which three types of carbohydrate chains are linked. These chains are: Type a, glactomannan units linked through phosphodiester bonds to produce long chains of molecular weight about 50,000; type b more numerous short mannosyl oligosaccharide units, and type c, more infrequent, long galactomannan chains. The first two are linked to the peptide through alkali-labile bonds to serine and threonine of the peptide whereas type c chains are linked through alkali-stable bonds (Lloyd, K. O. (1972) Biochemistry, 11, 3884–3890). The PPGM sample can be separated into three or four major components by diethylaminoethyl (DEAE-) Sephadex chromatography. By means of sequential degradations with alkali and acid, the structural basis for this heterogeneity has been demonstrated. It is due to the presence of different proportions of the three types of chains in the various fractions. The presence of O-acetyl groups, mainly on the a chains, was demonstrated in PPGM by chemical analysis and by proton and 13C nuclear magnetic resonance spectroscopy. Purified a chains were isolated from PPGM following Pronase digestion. By taking advantage of the alkali lability of the carbohydrate-protein linkages it has been possible to cleave the peptide moiety away from the carbohydrate. By sequential chromatography on Bio-Gel P-100, Dowex 1 and Bio-Gel P-100, five modified peptide fractions were isolated. The molecular weights of these fractions varied from 9,500 to 18,500 as judged by polyacrylamide-gel electrophoresis. Unlike the original peptide, the modified peptides contained low amounts of serine and threonine. The predominant amino acids were alanine, glycine, aspartic acid and glutamic acid which together make up between 51 and 55% of the peptides. The high content of the last two amino acids accounts for the acidic nature of the peptides. It appears that each fraction consists of a slightly heterogeneous population of peptides very similar in amino acid composition. It is not clear whether the compositional and size heterogeneity exists in the original peptide or whether it arose during the isolation procedure.

Isolation and Characterization of the Multiple 7S Globulins of Soybean Proteins

Two major proteins (the 7S and 11S globulins) of soybean (Glycine max) were simultaneously isolated by a simple method based on their different solubilities in dilute tris (hydroxymethyl) aminomethane buffers. The purified 7S globulins, which represented essentially the entire 7S soybean protein fraction capable of dimerization at 0.1 ionic strength, were fractionated into five components by diethylaminoethyl Sephadex A-50 column chromatography. The five 7S components were characterized by disc-electrophoresis.

Partial purification and characterization of thymidylate kinase from embryonic chick liver

Thymidylate kinase from the livers of 18-day-old chick embryos was concentrated 423-fold. The purification procedure included acid precipitation, ammonium sulfate fractionation, gel filtration on Sephadex G-100 and G-75 Super Fine, and ion-exchange chromatography on Diethylaminoethyl Sephadex A-50. This enzyme was found to be very labile but could be stabilized for long periods of time by its substrate (thymidine 5′-monophosphate) in the presence of 2-mercaptoethanol. Enzymes responsible for the formation of thymidine 5′-diphosphate and thymidine 5′-triphosphate, respectively, were separated during fractionation procedures. Thymidylate kinase from chick embryo liver was found to be a single protein having a molecular weight of approximately 46,000, Michaelis constant approximately 8 × 10 −5 m, and a broad pH optimum between 6.6 and 8.6. A 2–3 m m requirement of Mg 2+ above the adenosine 5′-triphosphate concentration was shown to be necessary for maximum enzyme activity. The enzyme appears to be competitively inhibited by thymidine, thymidine 5′-diphosphate, and thymidine 5′-triphosphate and noncompetitively inhibited by adenosine 5′-diphosphate. Thymidylate kinase enzymes isolated from two stages of developing embryonic liver and adult chick liver were shown to be identical.

Separation of Creatine Kinase Isoenzymes in Serum by Ion-Exchange Column Chromatography (Mercer's Method, Modified to Increase Sensitivity)

We describe our experience with Mercer's method [Clin. Chem. 20, 36 (1974)] for separating isoenzymes of creatine kinase (EC 2.7.3.2) in serum by ion-exchange chromatography. By using diethylaminoethyl cellulose rather than diethylaminoethyl Sephadex in the column and by changing the ionic strength of the eluting buffer, we could detect a significant proportion of the MB isoenzyme of serum creatine kinase at normal activities of creatine kinase in serum, even in the absence of cardiac disease.

Comparative Studies on Nitrate Reductase in Agrostemma githago Induced by Nitrate and Benzyladenine

NADH-nitrate reductase activity in excised embryos of Agrostemma githago develops in response to nitrate as well as benzyladenine. Induction of nitrate reductase by benzyladenine was much more susceptible to inhibition by a mixture of amino acid analogues and by cordycepin than induction by nitrate. In contrast, only induction of nitrate-nitrate reductase was decreased by chloramphenicol.NADH-cytochrome c reductase and reduced flavin mono-nucleotide-nitrate reductase activities were found to be associated with NADH-nitrate reductase and were induced by both nitrate and benzyladenine. When a partially purified enzyme sample was centrifuged in a linear 5 to 20% sucrose density gradient, a minor and a major band of NADH-cytochrome c reductase activity were observed. NADH-nitrate reductase cosedimented with the major band.The characteristics of nitrate-nitrate reductase and benzyl-adenine-nitrate reductase were compared by four methods but no differences could be detected: (a) Both enzymes sedimented with the same velocity during sucrose density gradient centrifugation. (b) Their distribution among fractions obtained by differential precipitation with (NH(4))(2)SO(4) was identical. (c) The elution profile of nitrate-nitrate reductase and benzyl-adenine-nitrate reductase after chromatography on diethyl-aminoethyl Sephadex A-25 columns showed no significant difference. (d) On polyacrylamide gel, the electrophoretic migration of the two enzymes was also identical.

The Structure of Plant Cell Walls

The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed "amyloid" xyloglucans.Xyloglucan-or fragments of xyloglucan-and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 m urea, endoglucanase, or 0.5 n NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the sycamore cell wall.The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The structure of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of beta-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues.

Oxygen equilibrium of hemoglobin E.

Oxygen equilibrium was determined on hemoglobin of individuals both heterozygous and homozygous for hemoglobin E. The whole blood oxyhemoglobin dissociation curve of AE blood was identical to that of normal AA blood. E hemoglobin, isolated by diethylaminoethyl Sephadex and carboxymethyl cellulose column chromatography, had oxygen affinity, heme-heme interaction, and Bohr effect identical to those of hemoglobin A prepared from the same column. Furthermore, the two hemoglobins had equal reactivity with 2,3-diphosphoglycerate. Phosphate-free hemolysates of blood from E and A homozygotes also had identical oxygen saturation curves. These results do not confirm earlier reports that hemoglobin E has an abnormally low oxygen affinity.

Thiosulfate oxidase from an Alcaligenes grown on mercaptosuccinate.

The soil isolate Alcaligenes sp. (M1) was able to use mercaptosuccinate (MS) as a sole source of carbon, sulfur, and energy. Resting cells oxidized the sulfur moiety of MS completely to sulfate. Thiosulfate oxidase, rhodanese, sulfite oxidase, and adenosine-5-phosphosulfate reductase were all detected in crude extracts. However, only thiosulfate oxidase appeared to be induced by growth of the organism on mercaptosuccinate. The intracellular thiosulfate oxidase was purified 139 to 170-fold by a procedure which included MnCl2 precipitation, treatment with calcium phosphate gel, and successive column chromatography on diethylaminoethyl (DEAE) Sephadex and Sephadex G-150, respectively. The resultant enzyme had a pH optimum which appeared to be below 5.0 and an optimum temperature of 40 °C using ferricyanide as electron acceptor. However, with cytochrome c as electron acceptor the pH optimum was 7.2 to 7.6. No activity was observed with other electron acceptors such as methylene blue, nicotinamide-adenine dinucleotide, 2,6-dichlorophenol-indophenol, and flavine-adenine dinucleotide. Enzymatic activity was inhibited 100% by 10−3 M mercuric chloride and 90% by 10−3 M p-chloromercuribenzoate (p-CMB). The molecular weight of the enzyme was found to be about 90 000. Resting cells and thiosulfate oxidase converted thiosulfate labeled in the inner sulfur atom to tetrathionate as the only detectable product, thereby suggesting that thiosulfate oxidase does not play a direct role in sulfate formation from mercaptosuccinate.

Subunits of Luteinizing Hormone from Human Pituitary Glands

Abstract Three species of luteinizing hormone (LH) designated as LH-A, -B, and -C, and containing 9, 8, and 4 NIH-LH-S1 units per mg were isolated from human pituitary glands by isoelectric focusing. The LH-A, -B, and -C each sedimented as a single boundary in the ultracentrifuge with s20,w of 2.34, 2.24, and 2.70 S, respectively. Amino acid analyses of LH-A, -B, and -C were very similar, although minor differences, whose significance remains to be determined, were observed. LH-A, -B, and -C contained 13.3, 13.2, and 6.9% of neutral sugars and 0.5, 1.3, and 1.8% of sialic acid, respectively. Minor differences in the physicochemical properties of the three forms of LH suggested microheterogeneity of the LH molecule caused by the modifications in the glycosidic linkages involving labile carbohydrate and sialic acid residues. Incubation of LH in 8 m urea followed by ion exchange chromatography on diethyl amino ethyl Sephadex A-50 resulted in the separation of α and β subunits. The α and β subunits were further purified by gel filtration on Sephadex G-100 and contained 0.8 and 1.1 units of LH activity per mg, respectively. Incubation of the α and β subunits in equal quantities (w/w) showed a 4-fold augmentation of the biological activity thus suggesting the recombination of the subunits. The gel filtration on Sephadex G-100, disc electrophoreses, amino acid analyses, and bioassays indicated that the α and β subunits of LH represent two nonidentical and noncovalently linked components of LH. The α subunits of human LH, follicle-stimulating hormone, and human chorionic gonadotropin could be substituted for each other in combination with the β subunit of each hormone suggesting that the β subunits are hormone specific and the α subunits of LH, follicle-stimulating hormone, and human chorionic gonadotropin are homologous.

Combined Use of Clofibrate and Cholestyramine or DEAE Sephadex in Hypercholesterolaemia

A comparison was made of the effect of DEAE (diethylaminoethyl) Sephadex (an anion exchange resin) and cholestyramine (Questran) with and without the addition of clofibrate in normal and hypercholesterolaemic patients. DEAE Sephadex (12-15 g/day) alone appeared to be as effective as cholestyramine in lowering the plasma cholesterol by 12-15%. Clofibrate acted synergistically with DEAE Sephadex and increased the activity of the latter by over twofold. This combination proved superior to that of clofibrate and cholestyramine and has the greatest potential use in the treatment of type II pattern hyperlipoproteinaemia.

Dissociation Phenomenon and Subunit Nature of Follicle-stimulating Hormone from Human Pituitary Glands

Abstract Sedimentation rate as a function of protein concentration obtained by ultracentrifugation has revealed the dissociation of follicle-stimulating hormone at concentrations below 0.5%; extrapolation to zero protein concentration indicated two s20,w values of 1.98 and 2.89 S, suggesting the existence of a monomer-dimer system. With the s20,w of 2.89 S, diffusion coefficient of 7.504 x 10-7 cm2 sec-1, and a partial specific volume of 0.72 calculated from the amino acid analysis, the molecular weight of the undissociated follicle-stimulating hormone was calculated to be 32,600, which is in good agreement with the value of 31,000 ± 600 obtained for the weight average molecular weight by sedimentation equilibrium studies. Stokes radii of the unlabeled and 131I-labeled follicle-stimulating hormones were determined by molecular sieve chromatography to be 31.3 and 23.7 A, respectively, supporting the dissociation of the follicle-stimulating hormone at lower concentrations. Treatment of the follicle-stimulating hormone with 8 m urea yielded an s20,w of 0.61 ± 0.10 S. Ion exchange chromatography of the urea-treated hormone on diethyl amino ethyl Sephadex A-50 resulted in the separation of two subunits designated as α and β. Incubation of the α and β subunits (1:1 w/w) resulted in a 400% augmentation of the biological activity of the subunits, exhibiting recombination of the subunits. Disc electrophoreses of the α and β subunits showed two basic and three acidic bands, respectively, suggesting microheterogeneity. Acid hydrolysates (24-, 48-, and 72-hour) of the follicle-stimulating hormone, and 24-hour acid hydrolysates of the subunits were analyzed for amino acids. Amino acid analysis of the follicle-stimulating hormone revealed a total of 236 amino acid residues for a molecular weight of 32,600. A comparison of the amino acid analyses of the subunits indicates higher quantities of histidine, methionine, and glycine, and lower quantities of proline, cysteine, and tyrosine in the α subunit than in the β subunit. The disc electrophoreses, amino acid analyses, and bioassays indicate that the α and β subunits are two nonidentical, noncovalently linked components of follicle-stimulating hormone.

Endonuclease I of Proteus mirabilis: I. PURIFICATION AND DEMONSTRATION OF LIMITED ENDONUCLEASE ACTIVITY

Abstract Endonuclease I was purified from a lysate of Proteus mirabilis by dihydrostreptomycin sulfate precipitation, ammonium sulfate precipitation, ion exchange chromatography on diethylaminoethyl Sephadex, and isoelectric density gradient electrophoresis. Purified endonuclease I sediments as a 3 S molecule and degrades double stranded P. mirabilis chromosomal DNA to acid-soluble products. In the presence of Escherichia coli tRNA the enzyme sediments as 7.2 and 6 S molecular forms and degrades double stranded chromosomal DNA to fragments that sediment as 8 S DNA molecules. The 7.2 and 6 S forms of endonuclease I that can be generated by the addition of tRNA to the purified enzyme also are observed in the dihydrostreptomycin sulfate precipitate of the crude extract of the bacteria. The limited digestion of DNA in vitro to the 8 S fragments is not caused by insufficient enzyme or end product inhibition by the 8 S DNA products. The 8 S DNA fragments also can be extracted from intact P. mirabilis cells grown under certain conditions.

Separation of Mycobacterial Soluble Antigens by Isoelectric Focusing

Ion-exchange chromatography and gel filtration have been reported to yield partial separation of mycobacterial antigens. These procedures were used in combination with isoelectric focusing in an attempt to purify antigens of Mycobacterium tuberculosis strain H37Ra. The fractionating action of isoelectric focusing is dependent upon differences in the isoelectric points of the proteins to be separated. Culture filtrate of M. tuberculosis H37Ra was chromatographed on Sephadex G-200. This resulted in two widely separated peaks. The first peak, presumably containing high-molecular-weight substances, was then fractionated on a diethylaminoethyl Sephadex anion-exchange column. Three peaks were collected, and each was subjected to isoelectric focusing. Each peak was further separated into two or more fractions. The serological reactivity of each fraction was determined by immunodiffusion and immunoelectrophoresis. Sensitized guinea pigs were also skin-tested with the fractions. Two of the fractions contained only a single precipitinogen. One fraction contained two precipitinogens. A fourth fraction contained three precipitinogens and was also the only fraction to display sensitin activity. Four of the fractions were inactive either as precipitinogens or sensitins. The results suggest that the methods described are useful for the separation of mycobacterial antigens.

Glycosidases of Aspergillus niger: II. PURIFICATION AND GENERAL PROPERTIES OF 1,2-α-l-FUCOSIDASE

Abstract A highly purified preparation of 1,2-α-l-fucosidase, free of β-galactosidase and β-N-acetylglucosaminidase activities, has been obtained from a commercial preparation of Aspergillus niger by a simple isolation procedure involving ammonium sulfate precipitation, pressure dialysis, repeated gel filtration on Sephadex G-150, and chromatography on diethylaminoethyl Sephadex A-50. The enzyme has a pH optimum of 3.8 ± 0.2 and Km and Vmax values of 8.3 x 10-5 m and 16.0 µmoles per mg per hour, respectively, at 37° for methyl 2-O-α-l-fucopyranosyl-β-d-galactoside as substrate. The detailed specificity studies indicate that it is highly specific for nonreducing terminal l-fucose residues linked to d-galactose residues by 1 → 2-α linkage. It hydrolyzes fucose residues quantitatively from 2-O-α-l-fucopyranosyl-d-galactose, 2-O-α-l-fucosyllactose, and lacto-N-fucopentaose I. It does not split p-nitrophenyl-α-l-fucoside, 2-O-, 3-O-, and 4-O-α-l-fucopyranosylfucoses, and 3-O- and 4-O-α-l-fucopyranosyl-d-galactoses. It also does not release any focuse from oligosaccharides of human milk such as lacto-N-fucopentaose II, lacto-N-fucopentaose III, and 3'-O-α-l-fucosyllactose. The enzyme liberates 80 to 90% of fucose residues from porcine and canine submaxillary mucins. It has no action on orosomucoid, human chorionic gonadotropin, and fucan sulfate. The enzyme is extremely active against human blood group substance H, destroying virtually all of the detectable activity.

The quantitative separation of 3′,5′-cyclic adensine monophosphate, adenosien-5′-monophosphate, adenosien-5′-diphosphate, and] adenosine-5′-triphosphate by ion-exchange chromatography on diethylaminoethyl Sephadex

The quantitative separation of 3′,5′-cyclic adensine monophosphate, adenosien-5′-monophosphate, adenosien-5′-diphosphate, and] adenosine-5′-triphosphate by ion-exchange chromatography on diethylaminoethyl Sephadex

Cholera toxins: immunogenicity of the rabbit ileal loop toxin and related antigens.

A method of assay of immunogenic potency of the cholera gut toxin is described; it is based on the relation of dose of antigen to neutralizing antibody titer produced in the rabbit under defined conditions and allows quantification of immunogenicity as immunogenic units per milligram of protein. Evidence, based on immunogenicity and rabbit ileal loop toxicity, is presented which indicates that the positively charged fraction of liquid-culture supernatant fluid eluted in deionized water from diethylaminoethyl Sephadex, or in electrolyte from carboxymethyl Sephadex, is a complex made up of a nonantigenic toxic moiety, a nontoxic protein component which elicits the formation of toxin-neutralizing antibody, and an inactive fraction. The complex may also be dissociated in high-salt concentrations with apparent recombination of the toxic moiety with a nondialyzable constituent of peptone to give a negatively charged complex. The immunogenic component is found in nontoxic supernatant fluids of cultures grown at pH 6.5 or in media deficient in peptone. It is also present in the nontoxic fraction eluted from diethylaminoethyl Sephadex in electrolyte or in deionized water from carboxymethyl Sephadex. When separated from the positively charged toxic moiety, the net charge of the antigen is reduced as shown by immunoelectrophoresis. On primary fractionation, the antigen may be associated with a minor antigenic component of the negatively charged complex containing a major antigen eliciting vibriocidal antibody formation, but antisera to the toxin antigen preparations, either in this form or freed of antigenic contamination by recycling, do not contain vibriocidal antibody. It is suggested that this antigen be designated the T (toxin) antigen, and the antigen producing vibriocidal antibody the V antigen. These two antigens would appear to represent the major antigenic specificities associated with the antitoxic and antibacterial elements of the immune response to infection.

Hexuronic acid dehydrogenase of Agrobacterium tumefaciens.

Growth of Agrobacterium tumefaciens on d-glucuronic acid (GlcUA) or d-galacturonic acid (GalUA) induces formation of hexuronic acid dehydrogenase [d-aldohexuronic acid: nicotinamide adenine dinucleotide (NAD) oxidoreductase]. The dehydrogenase, which irreversibly converts GlcUA or GalUA to the corresponding hexaric acid with the concomitant reduction of NAD, but not of nicotinamide adenine dinucleotide phosphate was purified 60-fold by MnCl(2) treatment, (NH(4))(2)SO(4) fractionation, chromatography on diethylaminoethyl Sephadex and negative adsorption with Ca(3)(PO(4))(2) gel. The pH optimum is 8.0. Other uronic acids, aldohexoses, aldopentoses, and polyols, are not substrates. Reduced nicotinamide adenine dinucleotide is an inhibitor strictly competitive with NAD. Kinetic data indicate that the dehydrogenase induced by growth on GlcUA may not be identical with that induced by growth on GalUA.