L-fucose metabolism in mammals. Kinetic studies on pork liver 2-keto-3-deoxy-L-fuconate:NAD+ oxidoreductase.

Pork liver 2-keto-3-deoxy-L-fuconate:NAD+ oxidoreductase has been shown to convert 2-keto-3-deoxy-L-fuconate to a 6-carbon acid tentatively identified as 2,4(or 5)-diketo-5(or 4)-monohydroxyhexanoate. The enzyme has a pH optimum of 10. 5 or higher. It is stabilized by dithiothereitol and inhibited by p-hydroxymercuribenzoate and heavy metals (Ag+, Hg2+, Co2+, Cd2+, Pb2+, Zn2+, and Cu2+), suggesting the presence of a functionally essential sulfhydryl group; pre-treatment of enzyme with NAD+ prevents inhibition by p-hydrocymercuribenzoate and heavy metals indicating that this sulfhydryl group may be near the NAD+ binding site. The enzyme has an absolute requirement for NAD+; NADP+ is an ineffective coenzyme. Several lines of evidence indicate that the same enzyme acts on both 2-keto-3-deocy-L-fuconate and 2-keto-3-deoxy-D-arabonate; thus, the pure enzyme acts on both substrates, the two substrates have very similar kinetic parameters (Km values are: 2-keto-3-deocy-L-fuconate, 0.20 mM; 2-keto-3-deoxy-D-arabonate, 0.25 mM; NAD+ for either substrate, 0.22 to 0.25 mM), the two substrates show identical pH and temperature profiles and the two substrates compete for common enzyme active sites. A large number of other sugars and sugar acids, including several 2-keto-3-deoxyaldonates, were ineffective as substrates. The dehydrogenase was also found in calf, beef, lamb, mouse, and rat liver. These studies when considered together with previous studies on the metabolism of L-fucose in pork liver indicate the presence of a soluble enzyme pathway capable of converting L-fucose to 2,4(or 5)-diketo-5(or 4)-monohydroxyhexanoate; this pathway can also convert D-arabinose, and probably L-galactose, to the analogous derivatives (diketomonohydroxypentanoate and diketodihydroxyhexanoate, respectively.     

NAD+-kinase from Neurospora crassa: isolation and properties

The NAD+ kinase (EC 2.7.1.23) of the filamentous fungus N. crassa is localized in cytosol. The activity in the dialyzed cell free extract has a pH optimum 8.3; it utilizes only ATP but not inorganic polyphosphates as a phosphoryl donor. A method for 200-fold purification of NAD+ kinase with a 20% yield has been developed. The procedure includes 105000 g centrifugation, fractionation with (NH4)2SO4, isoelectrofocusing in a Ultrodex layer and preparative electrophoresis in polyacrylamide gel. The molecular heterogeneity of NAD+ kinase was demonstrated by polyacrylamide gradient electrophoresis and by gel filtration through Sephadex G-200. The molecular weights of four individual forms of the enzyme are: 330000-338000, 305000-306000, 215000-229000 and 203000 Da. The Km values for the reaction catalyzed by purified NAD+ kinase for NAD+ and ATP are 3.0 X 10(-4) M and 0.9 X 10(-3) M, respectively.     

Candida utilis NAD+ kinase: purification, properties and affinity gel studies

1. NAD+ kinase (ATP:NAD+ 2' phosphotransferase, EC 2.7.1.23) has been purified to apparent enzymic homogeneity on Blue Sepharose CL-6B. 2. The molecular weight of the active species is about 260,000 as determined by PAGE and gel chromatography. Protein staining (PAGE) revealed minor bands with molecular weight values of 40,000, 140,000 and 550,000. Subunit studies (SDS-PAGE) gave evidence of a single band of molecular weight approximately 32,000. 3. On the basis of the release patterns of this enzyme from several affinity gels, an elution diagram is proposed as a device to assess the contribution of any of the several displacing agents that can be used to manipulate the desorption of a (enzyme) ligate from an immobilized ligand.     

NAD+ kinase--a review

NAD+ kinase catalyzes the only (known) biochemical reaction leading to the production of NADP+ from NAD+. Most evidence indicates it is found in the cytoplasm, but reports of its presence in (other) cell bodies can not be discounted. Viewed as a protein, our knowledge of NADK composition and architecture is rudimentary. Though recognized as a large multimeric protein, no agreement is evident for the molecular weight (Mr = approximately 4-65 X 10(4] of the native protein. Is calmodulin an integral subunit of (some, all) NAD+ kinases (analogous to phosphorylase kinase in skeletal muscle)? Or is it an external modulator? Consensus is evident that a subunit of molecular weight 30-35 X 10(3) is a component of the mammalian and yeast kinase. In one case (rabbit liver) two types of subunits are reported to give rise to oligomers differing in molecular weight and catalytic activities. Viewed as an enzyme it is not known why such a complex aggregate is needed for what might otherwise appear to a routine phosphorylation reaction. Rapid equilibrium random (for pigeon liver and C. utilis preparations) and ping-pong (for A. vinelandii kinase) mechanisms have been proposed for the reaction, with multiple reactant binding sites indicated for the random cases. From the perspective of enzyme modulation, the demonstration that green plant and sea urchin egg kinases are targets for calmodulin regulation by intracellular Ca2+ links NADP+ production in these sources to the multi-level discriminatory control functions inherent to this Ca2+-protein complex. Significant questions arise from the results of various investigators considered in this review. These queries offer fertile ground for the selective design of key experiments directed to a better understanding of NAD+ kinase function and pyridine nucleotide biochemistry.     

Isolation and some properties of NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii.

NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder0 452, 411, and 385 nm (the 411 nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119,000. The enzyme catalyzes the reduction of NAD+ and NADP+ by photoreduced spinach ferredoxin or reduced benzyl viologen...     

Purification and properties of an NAD(P)+-linked formaldehyde dehydrogenase from Methylococcus capsulatus (Bath).

Crude soluble extracts of Methylococcus capsulatus strain Bath, grown on methane, were found to contain NAD(P)+-linked formaldehyde dehydrogenase activity. Activity in the extract was lost on dialysis against phosphate buffer, but could be restored by supplementing with inactive, heat-treated extract (70 degrees C for 12 min). The non-dialysable, heat-sensitive component was isolated and purified, and has a molecular weight of about 115000. Sodium dodecyl sulphate gel electrophoresis of the protein suggested there were two equal subunits with molecular weights of 57000. The heat-stable fraction, which was necessary for activity of the heat-sensitive protein, was trypsin-sensitive and presumed to be a low molecular weight protein or peptide. A number of thiol compounds and other common cofactors could not replace the component present in the heat-treated soluble extract. The purified formaldehyde dehydrogenase oxidized three other aldehydes with the following Km values: 0.68 mM (formaldehyde); 0.075 mM (glyoxal); 7.0 mM (glycolaldehyde); and 2.0 mM (DL-glyceraldehyde). NAD+ or NADP+ was required for activity, with Km values of 0.063 and 0.155 mM respectively, and could not be replaced by any of the artificial electron acceptors tested. The enzyme was heat-stable at 45 degrees C for at least 10 min and had temperature and pH optima of 45 degrees C and pH 7.2 respectively. A number of metal-binding agents and substrate analogues were not inhibitory. Thiol reagents gave varying degrees of inhibition, the most potent being p-hydroxymercuribenzoate which at 1 mM gave 100% inhibition. The importance of possessing an NAD(P)+-linked formaldehyde dehydrogenase, with respect to M. capsulatus, is discussed.     

Purification of soluble guanylate cyclase from rat lung.

The soluble form of guanylate cyclase from rat lung has been purified approximately 23,000-fold to homogeneity by isoelectric precipitation, GTP-Sepharose chromatography, and preparative gel electrophoresis. A single protein-staining band is observed after analytical gel electrophoresis on either 4 or 7.5% polyacrylamide gels. The final purified enzyme has a specific activity of about 700 nmol of cyclic GMP formed/min/mg of protein at 37 degrees C in the presence of 4.8 mM MnCl2 and 100 micrometer GTP. Bovine serum albumin appears to slightly increase guanylate cyclase activity, but mainly stabilizes the purified enzyme; in its presence, specific activities in excess of 1 mumol of cyclic GMP formed/min/mg of enzyme protein can be obtained. When Mg2+ or Ca2+ are substituted for Mn2+, specific activities decrease to approximately 21 and 40 nmol of cyclic GMP formed/min/mg of protein, respectively. The apparent Michaelis constant for MnGTP in the presence of 4.8 mM MnCl2 is 10.2 micrometer. Kinetic patterns on double reciprocal plots as a function of free Mn2+ are concave downward. The native enzyme has a molecular weight of approximately 151,000 as determined on Sephacryl S-200; sodium dodecyl sulfate-polyacrylamide gel electrophoresis results in two protein-staining bands with approximate molecular weights of 79,400 and 74,000. Thus, it appears that the soluble form of guanylate cyclase from rat lung exists as a dimer.     

Human alpha-N-acetylglucosaminidase. 1. Purification and properties.

alpha-N-Acetylglucosaminidase was purified from human urine to a state of apparent homogeneity. alpha-N-Acetylglucosaminidase is a glycoprrotein with an extensive charge heterogeneity. The molecular weight determined by gel filtration is 307000. Polycarylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate indicates molecular weight heterogeneity of isocharged forms of the purified enzyme. The enzyme has a pH optimum of 4.5 +/- 0.3 and KM and V values of 0.14-0.74 mM, and 1.04-3.68 mumol mg-1 min-1 for three aryl 2-acetamido-2-deoxy-alpha-D-glucosides and UDP-N-acetylglucosamine. Heparan sulfate, heparin and dermatan sulfate are competitive inhibitors. The enzyme is inhibited by Hg2+ and Cu2+. --SH-protective reagents and thiol reagents have no effect on the enzyme activity. Heating at 65 degrees C and pH values below 5 inactivate the enzyme rapidly.     

Pigeon-liver NAD kinase. The structural and kinetic basis of regulation of NADPH.

NAD kinase was purified from pigeon liver by an improved procedure which included chromatography on phosphocellulose. The resultant preparation was homogeneous as judged by gel electrophoresis, but electrofocusing gave indications of heterogeneity. The enzyme appeared to be of molecular weight 270000, and to consist of subunits of molecular weight 34000; it may therefore be an octomer. Kinetic studies over a wide range of substrate concentrations revealed departures from Michaelis-Menten behaviour with the substrate NAD+; these were interpreted tentatively in terms of negative homotropic interactions between identical binding sites, since thermal and chemical inactivation studies revealed no evidence for more than one type of catalytic site. The significance of the kinetics and of the type of inhibition produced by NADPH is discussed in terms of the regulation of NAD kinase activity in vivo.     

Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver. Purification, characterization and subcellular localization

1. Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity. 2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. 3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000-56,000 as determined by sodium dodecyl sulphate gel electrophoresis. 4. The maximum of enzyme activity was obtained in the presence of 3-5 mM ZnCl2 at pH 6-6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The Km for p-nitrophenylphosphate was 0.28 x 10(-3)M at pH 6 in 50 mM sodium acetate/100 mM NaCl. 5. Phosphate is a competitive inhibitor (Ki = 0.5 x 10(-3)M) whereas ATP seems to be a non-competitive inhibitor (Ki = 0.35 x 10(-3)M). The isoelectric point determined by isoelectric focusing on polyacrylamide gel is 7.5. 6. Cell fractionation studies indicate that the Zn2+-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.     

Purification of NAD malic enzyme from potato and investigation of some physical and kinetic properties

A procedure is described for purification of NAD malic enzyme (EC 1.1.1.39) to near homogeneity from potato tuber mitochondria. The purified enzyme is active with either NAD or NADP, and functions with either Mg²⁺ or Mn²⁺. V_app is greatest when the enzyme is assayed with Mg²⁺ and NAD. When Mn²⁺ replaces Mg²⁺ the V_app of the NAD-linked reaction decreases but the K_m values for all substrates drop substantially. When NADP is used in place of NAD, the V_app of the Mg²⁺-linked reaction decreases and the K_m values for most substrates increase. The pH optimum of the enzyme depends on the metal ion and cofactor used and varies between 6.4 and 6.8. At pH 6.8, with saturating levels of Mg²⁺ and NAD, the turnover number of the enzyme is 37,000 min^?1. The shape of the pH profile indicates the involvement of two to three protons in the activation of the enzyme, whereas only one proton is involved in the inactivation process. The molecular weight of the enzyme in the presence of 5 mm dithiothreitol and 2 mm MgCl₂ is 490,000 as determined by gel filtration. A lower molecular weight form of the enzyme predominates in gel filtration at lower levels of dithiothreitol and in native gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis of the enzyme reveals two main bands with molecular weights of 61,000 and 58,000, suggesting that the subunit stoichiometry of the high-molecular-weight form may be α₄β₄. However, given the possibility that the smaller subunit may be a proteolytic artifact, the enzyme may prove to be an octamer of identical subunits.     

In vivo inactivation of glycerol dehydrogenase in Klebsiella aerogenes: properties of active and inactivated proteins.

Glycerol:oxidized nicotinamide adenine dinucleotide (NAD+) 2-oxidoreductase (EC 1.1.1.6), an inducible enzyme for anaerobic glycerol catabolism in Klebsiella aerogenes, was purified and found to have a molecular weight of 79,000 by gel electrophoresis. The protein seemed to be enzymatically active either as a dimer of a 40,000-dalton peptide at pH 8.6 or as a tetramer of 160,000 molecular weight at pH 7.0. The enzyme activity was present at high levels in cells growing anaerobically on glycerol, but disappeared with a half-life of about 45 min if molecular oxygen was introduced to the culture. In contrast, no such phenomenon occurred with dihydroxyacetone kinase activity, the second enzyme in the pathway. Immunochemical analysis showed that the inactivation of the oxidoreductase did not involve degradation of the protein. Furthermore, subunits of the active and inactive forms of the enzyme were indistinguishable in size on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and had similar isoelectric points (pH 4.7). Inactivation did, however, alter the gel filtration properties of the enzyme protein and, more importantly, reduced its affinity for the dye Cibacron F3GA and the coenzyme NAD+.     

Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes.

Human erythrocyte aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) was purified to apparent homogeneity. The native enzyme has a molecular weight of about 210,000 as determined by gel filtration, and SDS-polyacrylamide gel electrophoresis of this enzyme yields a single protein and with a molecular weight of 51,500, suggesting that the native enzyme may be a tetramer. The enzyme has a relatively low Km for NAD (15 microM) and a high sensitivity to disulfiram. Disulfiram inhibits the enzyme activity rapidly and this inhibition is apparently of a non-competitive nature. In kinetic characteristic and sensitivity to disulfiram, erythrocyte aldehyde dehydrogenase closely resembles the cytosolic aldehyde dehydrogenase found in the liver of various species of mammalians.     

ADP-ribosyl cyclase: an enzyme that cyclizes NAD+ into a calcium-mobilizing metabolite.

Cyclic ADP-ribose (cADPR) is a metabolite of NAD+ that is as active as inositol trisphosphate (IP3) in mobilizing intracellular Ca2+ in sea urchin eggs. The activity of the enzyme responsible for synthesizing cADPR is found not only in sea urchin eggs but also in various mammalian tissue extracts, suggesting that cADPR may be a general messenger for Ca2+ mobilization in cells. An aqueous soluble enzyme, thought to be an NADase, has been purified recently from the ovotestis of Aplysia californica (Hellmich and Strumwasser, 1991). This paper shows that the Aplysia enzyme catalyzes the conversion of NAD+ to cADPR and nicotinamide. The Aplysia enzyme was purified by fractionating the soluble extract of Aplysia ovotestis on a Spectra/gel CM column. The purified enzyme appeared as a single band of approximately 29,000 Da on SDS-PAGE but could be further separated into multiple peaks by high-resolution, cation-exchange chromatography. All of the protein peaks had enzymatic activity, indicating that the enzyme had multiple forms differing by charge. Analysis of the reaction products of the enzyme by anion-exchange high-pressure liquid chromatography (HPLC) indicated no ADP-ribose was produced; instead, each mole of NAD+ was converted to equimolar of cADPR and nicotinamide. The identification of the product as cADPR was further substantiated by proton NMR and also by its Ca(2+)-mobilizing activity. Addition of the product to sea urchin egg homogenates induced Ca2+ release and desensitized the homogenate to authentic cADPR but not to IP3. Microinjection of the product into sea urchin eggs elicited Ca2+ transients as well as the cortical exocytosis reaction. Therefore, by the criteria of HPLC, NMR, and calcium-mobilizing activity, the product was identical to cADPR. To distinguish the Aplysia enzyme from the conventional NADases that produce ADP-ribose, we propose to name it ADP-ribosyl cyclase.     

NAD+ kinase: molecular weight determination by low-angle laser-light scattering

Low-angle laser-light scattering (LALLS) was employed to measure the absolute molecular weight of chicken liver NAD+ kinase (NADK). The weight-average molecular weight (Mw) was found to be 275 000 +/- 15 000. The corresponding value for the second virial coefficient was -1.65 X 10(-3) ml X mol X g2. The value for Mw is in close accord with estimates reported for pigeon liver (270 000) and C. utilis (260 000) NADK. If the active enzyme is a dimer, the weight difference between pigeon/chicken liver and rabbit liver (136 000) NADK would indicate that the latter enzyme is an active monomer unit.     

Purification and properties of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach leaves.

An NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC. 1.2.1.12) has been purified from spinach leaves as a homogeneous protein of 150,000 daltons. Kinetic constants of 2.5 . 10(-4) M and 4 . 10(-4) M have been calculated for NAD+ and glyceraldehyde-3-phosphate, respectively. The amino acid composition is characterized by a cysteine content higher than that found in analogous enzymes. On sodium dodecyl sulphate gel electrophoresis, the native enzyme dissociates into two subunits of 37,000 and 14,000 daltons. The two subunits have been isolated in equimolar amounts by gel filtration; end-group analysis shows that alanine is the N-terminal residue of the large subunit, while serine is found at the N-terminus of the small subunit. Comparison of amino acid analysies and peptide maps shows that the two subunits have a different amino acid sequence. These results indicate that the NAD+-dependent glyceraldehyde-3-phosphate, dehydrogenase, isolated from spinach leaves has an atypical oligomeric structure, the protomer being formed by two different subunits.     

Purification and properties of a thiol protease from rat liver nuclei

A thiol protease was purified about 800-fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G-100 gel filtration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H-labelled ribosomal proteins was 4.5. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30-40% of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2-4 mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol proteases ever known.     

Amino acid-specific ADP-ribosylation. Evidence for two distinct NAD:arginine ADP-ribosyltransferases in turkey erythrocytes

An NAD:arginine mono-ADP-ribosyltransferase has been purified 270,000-fold from turkey erythrocytes through a five-step chromatographic procedure to apparent electrophoretic homogeneity. Molecular weight determinations of the enzyme by sodium dodecyl sulfate-gel electrophoresis, Ultrogel AcA 54, and TSK 3000 SW gel filtration were consistent with Mr = 32,000. The purified enzyme utilized arginine, other low molecular weight guanidino compounds, and various proteins as ADP-ribose acceptors. The Km values for NAD and arginine methyl ester were 36 and 3,000 microM, respectively. Unlike another transferase purified from turkey erythrocytes (Moss, J., and Stanley, S.J. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4809-4812), this enzyme was not activated by chaotropic salts or micromolar concentrations of histone. Thus, two distinct soluble ADP-ribosyltransferases may be present in turkey erythrocytes.     

Purification of terminal riboadenylate transferase from calf thymus gland.

A poly(A) polymerase has been purified from the soluble protein fraction of calf thymus gland. The activity is cytoplasmic and nonparticulate. Mn-2+ATP is the preferred substrate. On the basis of disc gel electrophoresis in sodium dodecyl sulfate-acrylamide gels, gel filtration, and sedimentation velocity in sucrose gradients, the enzyme has a molecular weight of 62,000 and appears to consist of one polypeptide chain. The enzyme preparation is shown to be nearly homogeneous by disc gel electrophoresis and isoelectric-focusing. The activity has a pI of about 7.4. The specific activity of the enzyme is about 1700 mumol per hour per mg of protein, giving a turnover number of about 1800 mol of substrate per mol of enzyme min- minus 1. The activity is highly specific for ATP and is inhibited by other ribonucleoside triphosphates. It is sensitive to high levels of RNA-polymerase inhibitors. Km for oligoadenylate is 50 muM in the presence of Mn-2+ and 200 muM in Mg-2+ and equivalent Vmax is achieved with either metal ion. The initiator function may be filled by a variety of oligoribonucleotides having a free 3'-OH.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.