Enzymatic metabolism of 3-deoxyglucosone,a Maillard intermediate

Summary In order to verify the formation of endogenous 3-deoxyglucosone (3-DG), an intermediate compound in the Maillard reaction, we tried to detect 3-deoxyfructose (3-DF) which is main metabolite of 3-DG. Endogenous 3-DF was detected in the urine of normal and diabetic rats by the oral administration of 3-DG-free feed. Metabolizing activities of crude extracts prepared from porcine organs were examined using methylglyoxal (MG) and 3-DG as substrates. NAD- or NADP-dependent 2-oxoaldehyde dehydrogenase activity was detected in liver, kidney, small intestine and lung. On the other hand, NADH- or NADPH-dependent 2-oxoaldehyde reductase activity was detected in all porcine organs in which liver and kidney contained higher activity of NADPH-dependent enzyme than the other organs. The reductase which catalyzes the reduction of 3-DG to 3-DF and MG to acetol, was purified and characterized from porcine kidney. The enzyme was the same to NADPH-dependent-2-oxoaldehyde reductase from porcine liver, which is speculated to prevent the advanced stage of the Maillard reaction as a self-defense enzyme.     

Kinetic properties of glucose-6-phosphate dehydrogenase from lamb kidney cortex

Glucose-6-phosphate dehydrogenase is the key regulatory enzyme of the pentose phosphate pathway and one of the products of this enzyme; NADPH has a critical role in the defence system against the free radicals. In this study, glucose-6-phosphate dehydrogenase from lamb kidney cortex kinetic properties is examined. The purification procedure is composed of two steps after ultracentrifugation for rapid and easy purification: 2', 5'-ADP Sepharose 4B affinity and DEAE Sepharose Fast Flow anion exchange chromatography. Previously, we used this procedure for the purification of glucose-6-phosphate dehydrogenase from bovine lens. The double reciprocal plots and product inhibition studies showed that the enzyme obeys 'Ordered Bi Bi' mechanism: K(m NADP+)K(m G-6-P) and K(i G-6-P) (dissociation constant of the enzyme--G-6-P complex) were found to be 0.018 +/- 0.002, 0.039 +/- 0.006 and 0.029 +/- 0.005 mM, respectively, by using nonlinear regression analysis. The enzyme was stable at 4 degrees C for a week.     

Affinity chromatography of putrescine oxidase from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens.

Putrescine oxidase [EC 1.4.3.4], putrescine : oxygen oxidoreductase (deaminating) (flavin-containing), from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens were adsorbed on amine-Sepharose 4B in which one of the terminal amino groups of diamine or triamine was covalently bound to Sepharose 4B leaving the other terminal amino group(s) free. The affinities of these enzymes for the amine-Sepharose 4B increased on increasing the chain length of the methylene groups in the immobilized amines and fell upon addition of the substrate. The affinity of putrescine oxidase modified with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC) was reduced in comparison with that of the native enzyme so far as 1,12-diaminododecane-Sepharose 4B was concerned. From these results, it can be concluded that the interactions between the enzyme and the amine-Sepharose result from specific affinities mediated through the active sites of the enzymes. It is suggested that spermidine dehydrogenase as well as putrescine oxidase has as anionic point and a hydrophobic region in the active site. On the basis of these results, the applicability of the enzyme affinities to purification procedures was examined. When partially purified enzymes were subjected to affinity chromatography, the following results were obtained. Putrescine oxidase gave a purification factor of 40-fold with about 100% recovery on a 1,12-diaminododecane-Sepharose column. In the case of spermidine dehydrogenase, the purification factor and recovery on a 1,8-diaminooctane-Sepharose column were about 1,200-fold and 86%, respectively. By introducing affinity chromatography as a purification step, each enzyme could be purified more simply and with higher recovery.     

Purification of L-3-glycerophosphate dehydrogenase from rat liver mitochondria

A rapid three-step procedure is presented for the purification of flavin-linked L-3-glycerophosphate dehydrogenase (E.C. 1.1.99.5.) from rat liver mitochondria. Solubilization of the enzyme is achieved selectively by digitonin, at a detergent-to-protein ratio of 0.7 mg/mg (mitochondrial protein concentration 10 mg/ml). The procedure involves chromatography on hydroxymethyl-hexamethylenediamine-succinyl-hexamethylenediamin e Sepharose 4B, followed by anion exchange chromatography using a FPLC technique. Subunit molecular weight of the enzyme was found to be 77,000 when prepared in the presence of the protease inhibitor phenylmethylsulphonyl fluoride. The Kmapp value for glycerophosphate was not influenced by the purification, and the ability of the enzyme to be activated by Ca2+ was preserved as well.     

Purification and characterization of histidinol dehydrogenase from cabbage

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms.     

Purification and molecular properties of malate dehydrogenase from the marine diatom Nitzschia alba.

Malate dehydrogenase (EC 1.1.1.37) was purified to homogeneity from the marine diatom Nitzschia alba. The purification steps consisted of (NH4)2SO4 precipitation, ion-exchange chromatography, Blue Sepharose affinity chromatography and gel filtration. A typical procedure provided 685-fold purification with 58% yield. The Mr of the holoenzyme was estimated to be 322,000 by gel filtration and 316,000 by ultracentrifugation. The enzyme migrated as a single polypeptide spot on two-dimensional polyacrylamide-gel electrophoresis with an Mr of 38,500, suggesting that the holoenzyme consists of eight identical subunits. This is the first case where malate dehydrogenase has been shown to be a homo-octamer; malate dehydrogenases from other sources are predominantly homodimers, with two homotetramers reported so far. The amino acid composition of the enzyme was determined and the N-terminal sequence of the subunit polypeptide was found to be Arg-Lys-Val-Ala-Val-Met-Gly-Ala-Ala-Gly-Gly-Ile-Gly-Gln-Pro-Leu-Ser-Leu- Leu-Leu - Lys-Leu-Ser-Pro-Gln-Val-Thr-Glu-Leu-Ser-Lys-Tyr-. For the first 21 amino acid residues, near-identical sequences were reported for the enzymes isolated from pig heart, Escherichia coli, yeast and watermelon. Other physicochemical and catalytic properties, such as sedimentation coefficient, partial specific volume, Stokes radius, excitation and emission maxima, Michaelis constants, pH optima, pH stability range and activation energy, of this enzyme are also presented.     

Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.     

Purification of xanthine dehydrogenase from rat liver: a rapid procedure with high enzyme yields

Xanthine dehydrogenase (EC 1.2.1.37) was purified approximately 1000-fold from liver homogenates of adult male Sprague-Dawley rats. Enzyme recovery was good (greater than 20% of the starting activity was obtained), and the homogeneously pure enzyme had a molecular mass of approximately 300,000 Da. The purified protein exhibited a specific activity of 2470 units/mg protein and spectral properties identical to those of the best preparations of this enzyme reported by other investigators. Routine preparations of this enzyme also possess higher dehydrogenase:oxidase ratios (typically between 5 and 6) than do other xanthine dehydrogenase preparations so far reported in the literature. Maximum dehydrogenase:oxidase ratios, greater than 10, could be obtained from this procedure if only peak dehydrogenase fractions from the chromatography columns were saved. The present small-scale purification method, which can be completed in 48-60 h, utilizes ammonium sulfate fractionation, Sephadex G-200 column chromatography, Blue Dextran-Sepharose column chromatography, and preparative gel electrophoresis.     

Morganella morganii J-8羰基不对称还原酶的分离纯化及性质研究

由本实验室筛选得到的摩尔摩根氏菌J-8菌株可将底物1-苯基-2-甲氨基丙酮专一性地转化为d-伪麻黄碱。以M.morganiiJ-8为出发菌株,菌体超声破碎后,经硫酸铵沉淀、Phenyl Superose疏水柱层析、DEAD阴离子柱层析和非变性凝胶电泳四步纯化获得电泳纯羰基不对称还原酶。亚基分子质量为42.5kD,高效液相色谱分析酶的分子质量约为84.1kD,初步认为该酶为二聚体蛋白。对所得到的部分纯化酶的酶学性质做了初步研究,纯酶进行基质辅助激光解析电离-飞行质谱分析,比对结果显示为与亮氨酸脱氢酶蛋白有很高相似性。     

Coenzyme binding capacity of yeast alcohol dehydrogenase.

Commercial lyophilized preparations of yeast alcohol dehydrogenase from Boehringer G.m.b.H. (Mannheim, Germany) bind 2 mols of reduced coenzyme/144000 g of enzyme (1). After the purification by a DEAE-Sephadex column chromatography, the coenzyme binding capacity is raised to 4 mols of NADH/mol of enzyme. Commercial preparations and ionexchange-purified preparations are homogeneous on the ionexchange column chromatography and the disc gel electrophoresis, after reduction with thioglycolic acid. Ionexchange chromatography does not increase the -SH titer, zinc content and the specific activity of enzyme. It is suggested that ionexchange chromatography raises the NADH-binding capacity by removing some impurities present in commercial enzyme preparations.     

Rapid purification of glucose-6-phosphate dehydrogenase from mammal's erythrocytes

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.     

A multifunctional fermentative alcohol dehydrogenase from the strict aerobe Alcaligenes eutrophus: purification and properties

A NAD (P)-linked alcohol dehydrogenase was isolated from the soluble extract of the strictly respiratory bacterium Alcaligenes eutrophus N9A. Derepression of the formation of this enzyme occurs only in cells incubated under conditions of restricted oxygen supply for prolonged times. The purification procedure included precipitation by cetyltrimethylammonium bromide and ammonium sulfate and subsequent chromatography on DEAE-Sephacel, Cibacron blue F3G-A Sepharose and thiol-Sepharose. The procedure resulted in a 120-fold purification of a multifunctional alcohol dehydrogenase exhibiting dehydrogenase activities for 2,3-butanediol, ethanol and acetaldehyde and reductase activities for diacetyl, acetoin and acetaldehyde. During purification the ratio between 2,3-butanediol dehydrogenase and ethanol dehydrogenase activity remained nearly constant. Recovering about 20% of the initial 2,3-butanediol dehydrogenase activity, the specific activity of the final preparation was 70.0 U X mg protein-1 (2,3-butanediol oxidation) and 2.8 U X mg protein-1 (ethanol oxidation). The alcohol dehydrogenase is a tetramer of a relative molecular mass of 156000 consisting of four equal subunits. The determination of the Km values for different substrates and coenzymes as well as the determination of the pH optima for the reactions catalyzed resulted in values which were in good agreement with the fermentative function of this enzyme. The alcohol dehydrogenase catalyzed the NAD (P)-dependent dismutation of acetaldehyde to acetate and ethanol. This reaction was studied in detail, and its possible involvement in acetate formation is discussed. Among various compounds tested for affecting enzyme activity only NAD, NADP, AMP, ADP, acetate and 2-mercaptoethanol exhibited significant effects.     

New quinoproteins in oxidative fermentation

Several quinoproteins have been newly indicated in acetic acid bacteria, all of which can be applied to fermentative or enzymatic production of useful materials by means of oxidative fermentation. (1) D-Arabitol dehydrogenase from Gluconobacter suboxydans IFO 3257 was purified from the bacterial membrane and found to be a versatile enzyme for oxidation of various substrates to the corresponding oxidation products. It is worthy of notice that the enzyme catalyzes D-gluconate oxidation to 5-keto-D-gluconate, whereas 2-keto-D-gluconate is produced by a flavoprotein D-gluconate dehydrogenase. (2) Membrane-bound cyclic alcohol dehydrogenase was solubilized and purified for the first time from Gluconobacter frateurii CHM 9. When compared with the cytosolic NAD-dependent cyclic alcohol dehydrogenase crystallized from the same strain, the reaction rate in cyclic alcohol oxidation by the membrane enzyme was 100 times stronger than the cytosolic NAD-dependent enzyme. The NAD-dependent enzyme makes no contribution to cyclic alcohol oxidation but contributes to the reduction of cyclic ketones to cyclic alcohols. (3) Meso-erythritol dehydrogenase has been purified from the membrane fraction of G. frateurii CHM 43. The typical properties of quinoproteins were indicated in many respects with the enzyme. It was found that the enzyme, growing cells and also the resting cells of the organism are very effective in producing L-erythrulose. Dihydroxyacetone can be replaced by L-erythrulose for cosmetics for those who are sensitive to dihydroxyacetone. (4) Two different membrane-bound D-sorbitol dehydrogenases were indicated in acetic acid bacteria. One enzyme contributing to L-sorbose production has been identified to be a quinoprotein, while another FAD-containing D-sorbitol dehydrogenase catalyzes D-sorbitol oxidation to D-fructose. D-Fructose production by the oxidative fermentation would be possible by the latter enzyme and it is superior to the well-established D-glucose isomerase, because the oxidative fermentation catalyzes irreversible one-way oxidation of D-sorbitol to D-fructose without any reaction equilibrium, unlike D-glucose isomerase. (5) Quinate dehydrogenase was found in several Gluconobacter strains and other aerobic bacteria like Pseudomonas and Acinetobacter strains. It has become possible to produce dehydroquinate, dehydroshikimate, and shikimate by oxidative fermentation. Quinate dehydrogenase was readily solubilized from the membrane fraction by alkylglucoside in the presence of 0.1 M KCl. A simple purification by hydrophobic chromatography gave a highly purified quinate dehydrogenase that was monodispersed and showed sufficient purity. When quinate dehydrogenase purification was done with Acinetobacter calcoaceticus AC3, which is unable to synthesize PQQ, purified inactive apo-quinate dehydrogenase appeared to be a dimer and it was converted to the monomeric active holo-quinate dehydrogenase by the addition of PQQ.     

A rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens.

This paper describes a simple and rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, together with analysis of the kinetic behaviour and some properties of the enzyme. The purification consisted of two steps, 2',5'-ADP-Sepharose 4B affinity chromatography and DEAE Sepharose Fast Flow ion exchange chromatography in procedure which took two working days. The enzyme was obtained with a yield of 13.7% and had a specific activity of 2.64 U/mg protein. The overall purification was about 19,700-fold. The molecular weight of the enzyme was found to be 62 +/- 3 kDa by Sephadex G-200 gel filtration chromatography. A protein band corresponding to a molecular weight of 69.2 +/- 3.2 kDa was obtained on SDS polyacrylamide slab gel electrophoresis. On chromatofocusing, lens glucose-6-phosphate dehydrogenase gave a single peak at pI 5.14. The activation energy of the reaction catalyzed by the enzyme was calculated from Arrhenius plot as Ea = 5.88 kcal/mol. The pH versus velocity curve had two peaks at pH 7.7 and 9.6. By the double-reciprocal plots and the product inhibition studies, it was shown that the enzyme follows 'Ordered Bi Bi' sequential kinetics. From the graphical and statistical analyses, KmNADP+, KmG-6-P, KiNADPH, Ki6-PGA were estimated to be 0.008 +/- 0.002, 0.035 +/- 0.013, 0.173 +/- 0.007 and 1.771 +/- 0.160 mM, respectively. The observed kinetic behaviour of glucose-6-phosphate dehydrogenase from bovine lens was in accordance with the enzyme from other sources.     

A rapid and efficient new method of purification of glutamate dehydrogenase by affinity chromatography on GTP-sepharose

The very high affinity for GTP of glutamate dehydrogenase was used to purify this enzyme by affinity chromatography. After periodic acid oxidation, GTP was covalently bound to an activated Sepharose. When crude mitochondrial extracts were applied on a column of this GTP-Sepharose, glutamate dehydrogenase was retained with very few other proteins. Glutamate dehydrogenase from rat liver was eluted with a KCl gradient with only one contaminating protein. From a pig heart mitochondrial extract the enzyme was purified 300-fold in one step. A chromatography on hydroxyapatite was sufficient to achieve the purification. This very simple technique avoids the long and troublesome crystallization steps generally involved in glutamate dehydrogenase purification.     

Some observations on the NADP+-linked oxidation of methylglyoxal catalysed by 2-Oxoaldehyde dehydrogenase.

In the oxidation of methylglyoxal by 2-oxoaldehyde dehydrogenase, the apparent Km value for NADP+ was about 2.5 times lower than the corresponding Km for NAD+; the apparent Km values for methylglyoxal and for the amine activator L-2-aminopropan-1-ol, with NADP+ as cofactor, were also different from those obtained with NAD+. In the presence of NADP+, the enzyme was not activated by P1, in contrast with the activation of the enzyme when NAD+ was used. The significance of the results is discussed.     

Purification and properties of D-3-hydroxybutyrate dehydrogenase from Paracoccus denitrificans

D-3-Hydroxybutyrate dehydrogenase from Paracoccus denitrificans has been purified to near homogeneity. The enzyme was prepared using DEAE-cellulose chromatography, affinity chromatography on immobilized Cibacron blue (Matrex Gel Blue A) and gel permeation chromatography. The pure enzyme was obtained by chromatofocusing as the final isolation step. The purification procedure yielded the enzyme with a specific activity of about 100 units/mg protein. The enzyme is specific for D-3-hydroxybutyrate and NAD and it exhibits anomalous kinetics (hysteresis) at low enzyme and coenzyme concentrations. It is relatively stable in the presence of EDTA at pH 7–8 higer salt concentrations. D-3-Hydroxybutyrate dehydrogenase is a tetramer with a molecular weight of 130 000 ± 10 000, its isoelectric point equals 5.10 ± 0.05. The enzyme is applicable to the determination of acetoacetate and D-3-hydroxybutyrate concentrations.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

Purification and characterization of glucose 6-phosphate dehydrogenase from sheep erythrocytes and inhibitory effects of some antibiotics on enzyme activity

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2', 5'-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and KM and Vmax values for NADP+ and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4 degrees C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I50 values were determined from Activity%-[Drug] graphs and Ki values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

The novel disulfide reductase bis-gamma-glutamylcystine reductase and dihydrolipoamide dehydrogenase from Halobacterium halobium: purification by immobilized-metal-ion affinity chromatography and properties of the enzymes.

An NADPH-specific disulfide reductase that is active with bis-gamma-glutamylcystine has been purified 1,900-fold from Halobacterium halobium to yield a homogeneous preparation of the enzyme. Purification of this novel reductase, designated bis-gamma-glutamylcystine reductase (GCR), and purification of halobacterial dihydrolipoamide dehydrogenase (DLD) were accomplished with the aid of immobilized-metal-ion affinity chromatography in high-salt buffers. Chromatography of GCR on immobilized Cu2+ resin in buffer containing 1.23 M (NH4)2SO4 and on immobilized Ni2+ resin in buffer containing 4.0 M NaCl together effected a 120-fold increase in purity. Native GCR was found to be a dimeric flavoprotein of Mr 122,000 and to be more stable to heat when in buffer of very high ionic strength. DLD was chromatographed on columns of immobilized Cu2+ resin in buffer containing NaCl and in buffer containing (NH4)2SO4, the elution of DLD differing markedly in the two buffers. Purified DLD was found to be a heat-stable, dimeric flavoprotein of Mr 120,000 and to be very specific for NAD. The utility of immobilized-metal-ion affinity chromatography for the purification of halobacterial enzymes and the likely cellular function of GCR are discussed.