Potato tuber succinate semialdehyde dehydrogenase: purification and characterization

Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass (Mr) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of Mr 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mM sodium pyrophosphate buffer. In 100 mM Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD+ as cofactor and NADP+ and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a Km of 4.6 microM for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 microM. The Km for NAD+ was found to be 31 microM. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethyl-maleimide, by heavy metal ions Hg2+, Cu2+, Cd2+, and Zn2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Purification and some properties of a novel heat-stable cis-toluene dihydrodiol dehydrogenase.

cis-Toluene dihydrodiol dehydrogenase was purified 200-fold from cells of a thermotolerant Bacillus species grown with toluene as the sole source of carbon and energy. The purified enzyme preparation was remarkably heat-stable and exhibited a half-life of 100 min at 80 degrees C, the temperature optimum. The activation energy of the reaction was 36 kJ.mol-1. Isoelectric focusing indicated that the pI of the native enzyme was 6.4 and that of the denatured enzyme 6.5. Although the pH optimum was 9.8, the enzyme was most stable at pH 8. The Mr of the enzyme was approx. 172,000 as determined by gel filtration and 166,000 by polyacrylamide-gel electrophoresis. The enzyme was composed of six apparently identical subunits with Mr values of 29,500. Kinetic analysis revealed that the Km for cis-toluene dihydrodiol was 92 microM and for NAD+ was 80 microM. The apparent Km values for cis-benzene dihydrodiol and cis-naphthalene dihydrodiol were 330 microM and 51 microM respectively. The enzyme was inhibited by mercurials but was unaffected by metal-ion chelators. Steady-state kinetics and product-inhibition patterns suggested that the enzyme mechanism was ordered Bi Bi.     

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Substrate specificities and inhibition studies.

The apparent Km and maximum velocity values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus were determined for a range of alcohols and aldehydes and the corresponding turnover numbers and specificity constants were calculated. Benzyl alcohol was the most effective alcohol substrate for benzyl alcohol dehydrogenase. Perillyl alcohol was the second most effective substrate, and was the only non-aromatic alcohol oxidized. The other substrates of benzyl alcohol dehydrogenase were all aromatic in nature, with para-substituted derivatives of benzyl alcohol being better substrates than other derivatives. Coniferyl alcohol and cinnamyl alcohol were also substrates. Benzaldehyde was much the most effective substrate for benzaldehyde dehydrogenase II. Benzaldehydes with a single small substituent group in the meta or para position were better substrates than any other benzaldehyde derivatives. Benzaldehyde dehydrogenase II could also oxidize the aliphatic aldehydes hexan-1-al and octan-1-al, although poorly. Benzaldehyde dehydrogenase II was substrate-inhibited by benzaldehyde when the assay concentration exceeded approx. 10 microM. Benzaldehyde dehydrogenase II, but not benzyl alcohol dehydrogenase, exhibited esterase activity with 4-nitrophenyl acetate as substrate. Both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were inhibited by the thiol-blocking reagents iodoacetate, iodoacetamide, 4-chloromercuribenzoate and N-ethylmaleimide. Benzyl alcohol or benzaldehyde respectively protected against these inhibitions. NAD+ also gave some protection. Neither benzyl alcohol dehydrogenase nor benzaldehyde dehydrogenase II was inhibited by the metal-ion-chelating agents EDTA, 2,2'-bipyridyl, pyrazole or 2-phenanthroline. Neither enzyme was inhibited by a range of plausible metabolic inhibitors such as mandelate, phenylglyoxylate, benzoate, succinate, acetyl-CoA, ATP or ADP. Benzaldehyde dehydrogenase II was sensitive to inhibition by several aromatic aldehydes; in particular, ortho-substituted benzaldehydes such as 2-bromo-, 2-chloro- and 2-fluoro-benzaldehydes were potent inhibitors of the enzyme.     

Purification and characterisation of TOL plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of Pseudomonas putida

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two enzymes of the xylene degradative pathway encoded by the plasmid TOL of a Gram-negative bacterium Pseudomonas putida, were purified and characterized. Benzyl alcohol dehydrogenase catalyses the oxidation of benzyl alcohol to benzaldehyde with the concomitant reduction of NAD+; the reaction is reversible. Benzaldehyde dehydrogenase catalyses the oxidation of benzaldehyde to benzoic acid with the concomitant reduction of NAD+; the reaction is irreversible. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase also catalyse the oxidation of many substituted benzyl alcohols and benzaldehydes, respectively, though they were not capable of oxidizing aliphatic alcohols and aldehydes. The apparent Km value of benzyl alcohol dehydrogenase for benzyl alcohol was 220 microM, while that of benzaldehyde dehydrogenase for benzaldehyde was 460 microM. Neither enzyme contained a prosthetic group such as FAD or FMN, and both enzymes were inactivated by SH-blocking agents such as N-ethylmaleimide. Both enzymes were dimers of identical subunits; the monomer of benzyl alcohol dehydrogenase has a mass of 42 kDa whereas that of the monomer of benzaldehyde dehydrogenase was 57 kDa. Both enzymes transfer hydride to the pro-R side of the prochiral C4 of the pyridine ring of NAD+.     

Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions.

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).     

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Purification and preliminary characterization.

A quick, reliable, purification procedure was developed for purifying both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from a single batch of Acinetobacter calcoaceticus N.C.I.B. 8250. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernatant, followed by chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CL-6B and Matrex Gel Red A, and finally gel filtration through a Superose 12 fast-protein-liquid-chromatography column. The enzymes co-purified as far as the Blue Sepharose CL-6B step were separated on the Matrex Gel Red A column. The final preparations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II gave single bands on electrophoresis under non-denaturing conditions or on SDS/polyacrylamide-gel electrophoresis. The enzymes are tetramers, as judged by comparison of their subunit (benzyl alcohol dehydrogenase, 39,700; benzaldehyde dehydrogenase II, 55,000) and native (benzyl alcohol dehydrogenase, 155,000; benzaldehyde dehydrogenase II, 222,500) Mr values, estimated by SDS/polyacrylamide-gel electrophoresis and gel filtration respectively. The optimum pH values for the oxidation reactions were 9.2 for benzyl alcohol dehydrogenase and 9.5 for benzaldehyde dehydrogenase II. The pH optimum for the reduction reaction for benzyl alcohol dehydrogenase was 8.9. The equilibrium constant for oxidation of benzyl alcohol to benzaldehyde by benzyl alcohol dehydrogenase was determined to be 3.08 x 10(-11) M; the ready reversibility of the reaction catalysed by benzyl alcohol dehydrogenase necessitated the development of an assay procedure in which hydrazine was used to trap the benzaldehyde formed by the NAD+-dependent oxidation of benzyl alcohol. The oxidation reaction catalysed by benzaldehyde dehydrogenase II was essentially irreversible. The maximum velocities for the oxidation reactions catalysed by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were 231 and 76 mumol/min per mg of protein respectively; the maximum velocity of the reduction reaction of benzyl alcohol dehydrogenase was 366 mumol/min per mg of protein. The pI values were 5.0 for benzyl alcohol dehydrogenase and 4.6 for benzaldehyde dehydrogenase II. Neither enzyme activity was affected when assayed in the presence of a range of salts. Absorption spectra of the two enzymes showed no evidence that they contain any cofactors such as cytochrome, flavin, or pyrroloquinoline quinone. The kinetic coefficients of the purified enzymes with benzyl alcohol, benzaldehyde, NAD+ and NADH are also presented.     

Purification and characterization of a new NAD(+)-dependent enzyme, L-tartrate decarboxylase, from Pseudomonas sp. group Ve-2.

A new enzyme, L-tartrate decarboxylase, was found in cells of Pseudomonas sp. group Ve-2. The enzyme was purified to homogeneity and characterized. The enzyme requires K+, Mg2+, and NAD+ for L-tartrate decarboxylation. The dependence of the enzymatic decarboxylation on NAD+ suggests that the decarboxylation involves redox reactions of the substrate. The enzyme catalyzes NAD(+)-linked oxidative decarboxylation of D-malate as well. The enzyme is composed of four subunits with identical molecular weight (Mr 40,000). The apparent Michaelis constants for L-tartrate and NAD+ are 1.1 mM, respectively. The cofactor requirements and the physical properties of the enzyme were similar to those of L-tartrate dehydrogenase-D-malate dehydrogenase from Rhodopseudomonas sphaeroides, and tartrate dehydrogenase from P. putida.     

Formaldehyde dehydrogenase from Pseudomonas putida. Purification and some properties

Formaldehyde dehydrogenase was isolated and purified in an overall yield of 12% from cell-free extract of Pseudomonas putida C-83 by chromatographies on columns of DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite. The purified enzyme was homogeneous as judged by disc gel electrophoresis and was most active at pH 7.8 using formaldehyde as a substrate. The enzyme was also active toward acetaldehyde, propionaldehyde, glyoxal, and pyruvaldehyde, though the reaction rates were low. The enzyme was NAD+-linked but did not require the external addition of glutathione, in contrast with the usual formaldehyde dehydrogenase from liver mitochondria, baker's yeast, and some bacteria. The enzyme was markedly inhibited by Ni2+, Pd2+, Hg2+, p-chloromercuribenzoate, and phenylmethanesulfonyl fluoride. The molecular weight of the enzyme was estimated to be 150,000 by the gel filtration method, and analysis by SDS-polyacrylamide gel electrophoresis indicated that the enzyme was composed of two subunit monomers. Kinetic analysis gave Km values of 67 microM for formaldehyde and 56 microM for NAD+, and suggested that the reaction proceeds by a "Ping-pong" mechanism. The enzyme catalyzed the oxidation of formaldehyde accompanied by the stoichiometric reduction of NAD+, but no reverse reaction was observed.     

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 catalytic properties of L-valine dehydrogenase from Streptomyces cinnamonensis.

NAD+-dependent L-valine dehydrogenase was purified 180-fold from Streptomyces cinnamonensis, and to homogeneity, as judged by gel electrophoresis. The enzyme has an Mr of 88,000, and appears to be composed of subunits of Mr 41,200. The enzyme catalyses the oxidative deamination of L-valine, L-leucine, L-2-aminobutyric acid, L-norvaline and L-isoleucine, as well as the reductive amination of their 2-oxo analogues. The enzyme requires NAD+ as the only cofactor, which cannot be replaced by NADP+. The enzyme activity is significantly decreased by thiol-reactive reagents, although purine and pyrimidine bases, and nucleotides, do not affect activity. Initial-velocity and product-inhibition studies show that the reductive amination proceeds through a sequential ordered ternary-binary mechanism; NADH binds to the enzyme first, followed by 2-oxoisovalerate and NH3, and valine is released first, followed by NAD+. The Michaelis constants are as follows; L-valine, 1.3 mM; NAD+, 0.18 mM; NADH, 74 microM; 2-oxoisovalerate, 0.81 mM; and NH3, 55 mM. The pro-S hydrogen at C-4' of NADH is transferred to the substrate; the enzyme is B-stereospecific. It is proposed that the enzyme catalyses the first step of valine catabolism in this organism.     

Calmodulin-dependent NAD kinase of human neutrophils

NAD kinase from human neutrophils has been partially purified by sequential application of Red Agarose, ion-exchange, and gel-filtration chromatography. The enzyme has a broad pH optimum, 7.0-9.5, is strictly dependent upon the presence of Mg2+, and in the absence of calcium exhibits Km values of 0.6 and 0.9 mM for NAD and ATP, respectively. NAD kinase activity is extremely sensitive to free calcium concentration, with half-maximal activity observed at free calcium concentrations of approximately 0.4 microM. In cellular extracts calcium-dependent activation of NAD kinase increases the maximum velocity of the reaction from 2- to 5-fold while not affecting Km values for NAD and ATP. The activity of the partially purified NAD kinase is stimulated 3.5-fold by the addition of calmodulin in the presence of calcium. This stimulation is inhibited by the addition of 20 microM trifluoperazine to the incubation. These data are interpreted as implicating calmodulin in NAD kinase regulation. The total concentration of NADP + NADPH in the human neutrophil used increased 2.2-fold in response to activation by phorbol myristic acetate. Finally, neutrophil NAD kinase has a Mr, based upon gel filtration, of 169,000.     

Identification of a human stomach alcohol dehydrogenase with distinctive kinetic properties

A new form of alcohol dehydrogenase, designated mu-alcohol dehydrogenase, was identified in surgical human stomach mucosa by isoelectric focusing and kinetic determinations. This enzyme was anodic to class I (alpha, beta, gamma) and class II (pi) alcohol dehydrogenases on agarose isoelectric focusing gels. The partially purified mu-alcohol dehydrogenase, specifically using NAD+ as cofactor, catalyzed the oxidation of aliphatic and aromatic alcohols with long chain alcohols being better substrates, indicating a barrel-shape hydrophobic binding pocket for substrate. mu-Alcohol dehydrogenase stood out in high Km values for both ethanol (18 mM) and NAD+ (340 microM) as well as in high Ki value (320 microM) for 4-methylpyrazole, a competitive inhibitor for ethanol. mu-Alcohol dehydrogenase may account for up to 50% of total stomach alcohol dehydrogenase activity and appeared to play a significant role in first-pass metabolism of ethanol in human.     

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of chicken erythrocyte ferrochelatase.

Ferrochelatase (EC 4.99.1.1) was purified 2000-fold to apparent homogeneity from isolated chicken erythrocyte mitochondria. The purified enzyme yields a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 42 000. The enzyme utilizes proto-, meso- and deutero-porphyrin with Km values of 37, 51 and 80 microM respectively. The disubstituted porphyrins 2,4-bisglycol deutero-porphyrin and 2,4-disulphonic deuteroporphyrin were not substrates. Mn2+, Hg2+, Pb2+ and Co2+ were strong inhibitors of the purified enzyme. Palmitic acid and oleic acid stimulated activity, whereas linoleic acid inhibited and phospholipids had variable effects. Chicken ferrochelatase was inhibited by N-ethylmaleimide and iodoacetamide. Inhibition by iodoacetamide was pseudo-first-order, but inhibition by N-ethylmaleimide appeared to be biphasic in nature with an initial high rate followed by a much lower rate of inactivation. The characteristics of the chicken erythrocyte enzyme are compared with those previously reported for mammalian liver ferrochelatase.     

The isolation, purification, and some properties of NAD-dependent isocitrate dehydrogenase from the organic acid-producing yeast Yarrowia lipolytica

The NAD+-dependent isocitrate dehydrogenase of the organic acid-producing yeast Yarrowia lipolytica was isolated, purified, and partially characterized. The purification procedure included four steps: ammonium sulfate precipitation, acid precipitation, hydrophobic chromatography, and gel-filtration chromatography. The enzyme was purified 129-fold with a yield of 31% and had a specific activity of 22 U/mg protein. The molecular mass of the enzyme was found to be 412 kDa. The enzyme consists of eight identical subunits with a molecular mass of about 52 kDa. The Km for NAD+ is 136 microM, and that for isocitrate is 581 microM. The effect of some intermediates of the citric acid cycle and nucleotides on the enzyme activity was studied. The role of isocitrate dehydrogenase (NAD+) in the overproduction of citric and keto acids is discussed.     

Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes

Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis.     

Purification and properties of uroporphyrinogen III synthase (co-synthetase) from Euglena gracilis.

Uroporphyrinogen III synthase (co-synthetase) purified from Euglena gracilis is a monomer of Mr 38 500 by gel-filtration studies and 31 000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The pI is apparently in the range 4.8-5.1. No evidence for any cofactors was found, and folate derivatives were shown to be absent; no metal ions appear to be present in the enzyme. The Km for hydroxymethylbilane is in the range 12-40 microM, and the product, uroporphyrinogen III, is an inhibitor. Modification studies suggest that arginine residues are essential for the activity of co-synthetase; lysine residues may also be essential, but histidine, cysteine and tyrosine residues are not.     

Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland.

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.     

Mannitol-1-phosphate dehydrogenase of Escherichia coli. Chemical properties and binding of substrates.

Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5'-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5'-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.