NADPH production from NADP+ using malic enzyme of Achromobacter parvulus IFO-13182

A sonicate of Achromobacter parvulus IFO-13182 produced NADPH from NADP⁺by an NADP⁺-linked malic enzyme [l-malate: NAD(P)⁺oxidoreductase, EC 1.1.1.39–40] reaction in the presence of l-malic acid and divalent metal ions. Malic enzyme of A. parvulus was stabilized by 5% l-malic acid, and activity was maintained at 60°C for 1 h. Contaminating phosphatase (orthophosphoricmonoester phosphohydrolase, EC 3.1.3.1–2) was completely inactivated by this treatment. Among the conditions tested, the optimum NADPH production was done using 36 μmol NADP⁺, 67 μmol l-malic acid, 63 μmol MgCl₂ and 1 unit of the malic enzyme in 3 ml of 55 mm phosphate buffer (pH 7.8). Conversion ratio of NADPH from NADP⁺ reached 100% after 4 h incubation at 30°C and the amount of NADPH accumulated was ～12 μmol ml^?1of the reaction mixture. No dephosphorylation of NADP⁺to NAD⁺or of NADPH to NADH was found by high performance liquid chromatography. The NADPH produced by such enzymatic reduction was purified by ethanol precipitation and dried in vacuo in powdered form with 97% purity, judged from the ratio of the absorbances at 340 and 260 nm. The purity of the NADPH produced was determined to be 95% from its coenzyme activity with NAD(P)⁺-linked glutathione reductase [NAD(P)H: oxidized-glutathione oxidoreductase, EC 1.6.4.2].     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Reduction of purothionin by the wheat seed thioredoxin system

Thioredoxin h, the thioredoxin characteristic of heterotrophic plant tissues, was purified to homogeneity from wheat endosperm (flour) and found to resemble its counterpart from carrot cell cultures. In the presence of NADPH, homogeneous thioredoxin h and partially purified wheat endosperm thioredoxin reductase (NADPH), (EC 1.6.4.5), purothionin promoted the activation of chloroplast fructose-1,6-bisphosphatase (EC 3.1.3.11). Under these conditions, NADPH provided the reducing equivalents for a series of thiol reactions in which (a) thioredoxin reductase reduced thioredoxin h thereby converting it from disulfide (S-S) to sulfhydryl (SH) form; (b) the sulfhydryl form of thioredoxin h reduced the disulfide form of purothionin—a 5 kilodalton seed storage protein with 4 S-S bridges; and (c) the sulfhydryl form of purothionin reductively activated fructose-1,6-bisphosphatase. The results show that, since thioredoxin h does not react effectively with fructose-1,6-bisphosphatase, the thioredoxin system can activate an enzyme through purothionin by secondary thiol redox control. In a related type reaction, purothionin, inhibited the activity of either Escherichia coli or calf thymus ribonucleotide reductase with reduced thioredoxin as hydrogen donor. The results suggest that purothionin competes with ribonucleotide reductase for reducing equivalents from thioredoxin. Thus, inhibition of deoxyribonucleotide synthesis should be considered a possible mechanism when examining the toxic effects of purothionin on mammalian cells in S-phase.     

Comparative studies of sheep lung and liver nitrofurantoin reductase

1. Nitrofurantoin reductase which catalyzes the bioactivation of nitrofurantoin was purified to electrophoretic homogenity from sheep liver and lung microsomes, with a yield of 15% and 35%, respectively. The specific activity of both reductases was found to be similar (140 nmol/min/mg protein).2. The effects of nitrofurantoin and NADPH concentrations, pH, ionic strength, amount of enzyme and reaction period, on the enzyme activity were studied and the optimum conditions for maximum activity of purified liver and lung nitrofurantoin reductases were determined.3. The enzyme concentration was found proportional with the square root of the rate of nitrofurantoin reduction up to approximately 15 μg protein/ml and 25 μg protein/ml incubation mixture for liver and lung nitrofurantoin reductases, respectively.4. The plots of inverse of the nitrofurantoin concentration against the inverse of the square root of the velocity for the reduction of nitrofurantoin by liver and lung enzymes gave K_m values as 27.78 μM and 32.25 μM, respectively.5. The purified liver and lung enzymes were also saturated by NADPH at similar concentrations and the K_m values were calculated as 29.4 μM and 35.5 μM, respectively.6. The effects of magnesium, nickel, cadmium and copper ions on the nitrofurantoin reductase activity were examined. Magnesium ion was found to have almost no effect, whereas the other ions inhibited the activity of both liver and lung reductases.     

Microsomal electron transport. I. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity

An electron transport system that catalyzes the oxidation of NADPH by organic, hydroperoxides has been discovered in microsomal fractions. A tissue distribution study revealed that the microsomal fraction of rat liver was particularly effective in catalyzing the NADPH-peroxidase reaction whereas microsomes from adrenal cortex, lung, kidney, and testis were weakly active. The properties of the hepatic microsomal NADPH-peroxidase enzyme system were next examined in detail.The rate of NADPH oxidation by hydroperoxides was first-order with respect to microsomal protein concentration and a K_m value for NADPH of less than 3 μm was obtained. Examination of the hydroperoxide specificity revealed that cumene hydroperoxide and various steroid hydroperoxides were effective substrates for the enzyme system. Using cumene hydroperoxide as substrate, the reaction rate showed saturation kinetics with increasing concentrations of hydroperoxide and an apparent K_m of about 0.4 mm was obtained. The NADPH-peroxidase reaction was inhibited by potassium cyanide, half-maximal inhibition occurring at a cyanide concentration of 2.2 mm. NADH was able to support the NADPH-dependent peroxidase activity synergistically.Evidence compiled for the involvement of NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) in the NADPH-peroxidase reaction included: (1) an identical pH optimum for both activities; (2) stimulation of NADPH-peroxidase activity by increasing ionic strength; (3) inhibition by 0.05 mm, p-hydroxymercuribenzoate with partial protection by NADPH; (4) inhibition by NADP⁺; and (5) inactivation by antiserum to NADPH-cytochrome c reductase. In contrast, antibody to cytochrome b₅ did not inhibit the NADPH-peroxidase activity. Evidence for the participation of cytochrome P-450 in the NADPH-peroxidase reaction included inhibition by compounds forming type I, type II, and modified type II difference spectra with cytochrome P-450; inhibition by reagents converting cytochrome P-450 to cytochrome P-420; and marked stimulation by in vivo phenobarbital administration. The NADPH-reduced form of cytochrome P-450 was oxidized very rapidly by cumene hydroperoxide under a CO atmosphere.It was concluded that the NADPH-peroxidase enzyme system of liver microsomes is composed of the same electron transport components which function in substrate hydroxylation reactions.     

Purification and immunochemical characteristics of NADPH-cytochrome P-450 oxidoreductase from tobacco cultured cells

NADPH-cytochrome P-450 oxidoreductase (EC 1.6.2.4) was purified from the microsomal fraction of tobacco (Nicotiana tabacum) BY2 cells by chromatography on two anion-exchange columns and 2′,5′ ADP-Sepharose 4B column. The purified enzyme showed a single protein band with a molecular weight of 79 kDa on SDS-PAGE and exhibited a typical flavoprotein redox spectrum, indicating the presence of an equimolar quantity of FAD and FMN. This enzyme followed Michaelis-Menten Kinetics with K_m values of 24 μM for NADPH and 16 μM for cytochrome c. An in vitro reconstituted system of the purified reductase with a partially purified tobacco cytochrome P-450 preparation showed the cinnamic acid 4-hydroxylase activity at the rate of 14 pmol min ⁻¹nmol⁻¹ P-450 protein and with a purified rabbit P-450_2C14 catalyzed N-demethylation of aminopyrine at the rate of 6 pmol min⁻¹ lnmo⁻¹ P-450 protein. Polyclonal antibodies raised against the purified reductase reacted with tobacco reductase but not with yeast reductase on Western blot analysis. Anti-yeast reductase antibodies did not react with the tobacco reductase. This result indicate that the tobacco reductase was immunochemically different from the yeast reductase. The anti-tobacco reductase antibodies totally inhibited the tobacco reductase activity, but not the yeast reductase. Also, Western blot analyses using the anti-tobacco reductase antibodies revealed that leaves, roots and shoots of Nicotiana tabacum plants contained an equal amount of the reductase protein. From these results, it was suggested that there are different antibody binding sites, which certainly participate in enzyme activity, between tobacco and yeast reductase.     

The crystal structure of the GCY1 protein from S. cerevisiae suggests a divergent aldo-keto reductase catalytic mechanism

The crystal structure of the GCY1 gene product from Saccharomyces cerevisiae has been determined to 2.5 Å and is being refined. The model includes two protein molecules, one apo and one holo, per asymmetric unit. Examination of the model reveals that the active site surface is somewhat flat when compared with the other aldo-keto reductase structures, possibly accommodating larger substrates. The K_m for NADPH (28.5 μM) is higher than that seen for other family members. This can be explained structurally by the lack of the ‘safety belt’ of residues seen in other aldo-keto reductases with higher affinity for NADPH. Catalysis also differs from the other aldo-keto reductases. The tyrosine that acts as an acid in the reduction reaction is flipped out of the catalytic pocket. This implies that the protein must either undergo a conformational change before catalysis can take place or that there is an alternate acid moiety.     

Yeast glutathione reductase. Steady-state kinetic studies of its transhydrogenase activity.

Yeast glutathione reductase catalyzes a pyridine nucleotide transhydrogenase reaction using either NADPH or NADH as the electron donor and thionicotinamideadenine dinucleotide phosphate as the electron acceptor. Competitive substrate inhibition of the transhydrogenase reaction by NADPH (K_i = 11 μM) is observed when NADPH is the electron donor. Competitive substrate inhibition by thionicotinamide-adenine dinucleotide phosphate (K_i = 58 μM) is observed with NADH as the electron donor. The turnover numbers of the two transhydrogenase reactions are similar and are equal to about 1% of the turnover number for the NADPH-dependent reduction of oxidized glutathione catalyzed by the enzyme. The transhydrogenase kinetics are analyzed in terms of a pingpong mechanism. It is concluded that the substrate inhibition results from formation of abortive complexes of NADPH with the reduced form of the enzyme and of thionicotinamide-adenine dinucleotide phosphate with the oxidized form of the enzyme. With NADPH as the electron donor, the apparent Michaelis constant for thionicotinamide-adenine dinucleotide phosphate is sensitive to the ionic composition of the assay medium. The data are interpreted to support the existence of a general pyridine nucleotide-binding site at the active site of the enzyme and separate from the binding site for oxidized glutathione.     

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability I. Dihydrofolate reductase,thymidylate synthethase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP⁺ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent K_m values for dihydrofolate in enzymes from the three strains were in the range of 4.8–7.2 μM and for NADPH 6.5–8.0 μM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10–20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthethase and 10-formyltetra-hydrofolate synthetase (formate: tetrahydrofolate ligase (ADP)-forming), EC 6.3.4.3) were similar in the three strains studied.     

Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography

Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP⁺, but GSH-dependent NADP⁺ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn²⁺. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 M and that for NADPH was about 3 M. NADP⁺ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP⁺-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H₂O₂. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.Abbreviations GSH reduced form of the tripeptide glutathione - GSSG oxidised form of glutathione     

Purification and characterization of sepiapterin reductase from rat erythrocytes

Sepiapterin reductase from rat erythrocyte hemolysate was purified 2000-fold to apparent homogeneity with 30% yield. The specific activity of the purified enzyme was 18 units/mg protein, and its molecular weight was 55 000. The enzyme consists of two identical subunits, each of which has a molecular weight of 27 500. The enzyme showed a single peak by isoelectric focusing with a pI of 4.9 and partial specific volume of 0.73 cm³/g. The amino acid composition was determined. pH optimum of the enzyme was 5.5. The equilibrium constant of 2.2·10⁹ of the enzyme showed that the equilibrium lies much in favor of dihydrobiopterin formation from sepiapterin in rat erythrocytes. From steady-state kinetic measurements, ordered bi-bi mechanism was proposed to the reaction of sepiapterin reductase in which NADPH binds to free enzyme and sepiapterin binds next. NADP⁺ is released after the release of dihydrobiopterin. The K_m values for sepiapterin and NADPH were 15.4 μM and 1.7 μM, respectively, and the V_max value was 21.7 μmol/min per mg.     

Oxygenase properties of crystallized fraction 1 protein from tobacco.

Ribulose 1,5-diphosphate-dependent oxygenase activity was demonstrated for crystallized Fraction 1 protein (RuDP² carboxylase EC 4.1.1.39) from tobacco. The kinetic properties of this oxygenase function were examined polarographically in air-equilibrated medium. Optimum activity was obtained at pH 8.4–8.6, and required 4–8 mm MgCl₂. Higher Mg²⁺ concentrations decreased activity and slightly shifted the pH optimum to 8.2–8.3. The apparent K_m (RuDP) and K_m (Mg²⁺) were 22 μm and 0.5 mm, respectively. Oxygenase activity was inhibited by bicarbonate and indirectly by KCN. Kinetic studies suggest that the active inhibitory substance is the cyanohydrin derivative formed from the reaction of KCN with RuDP.Changes in oxygenase kinetics were observed upon addition of RuDP, as previously reported for the carboxylase function of this enzyme. Oxygenase activity required preincubation of the enzyme with both Mg²⁺ and low concentrations of bicarbonate. Activities were enhanced about 20 and 70% when FDP (0.1 mm) and NADPH (0.5 mm), respectively, were included during preincubation.     

Kinetics of anthracycline antibiotic free radical formation and reductive glycosidase activity

Adriamycin free radical anion concentrations have been correlated with production of 7-deoxyadriamycin aglycone in a reaction catalyzed by NADPH-cytochrome c reductase. The free radical species is detected by electron spin resonance (ESR) spectroscopy and quantified by double integrations. The 7-deoxyaglycone product is isolated by thin-layer chromatography (TLC) and quantified by fluorometry. As the concentration of adriamycin increases, a concomitant increase in aglycone and free radical levels occurs. These results as well as those with inhibitors Vitamin K₃, Vitamin E, and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) point to an obligatory free radical intermediate in the metabolism of adriamycin. DMPO inhibits the reaction under aerobic conditions only, and shows no effect under anaerobiosis at the concentrations studied here. Vitamin E and aerobic DMPO act as free radical scavangers, while Vitamin K₃ competes for the reducing power of NADPH in the NADPH-cytochrome c reductase system.     

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr₁-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c₁NR, and the form eluted with NADH was designated c₂NR. Nitrate-grown nr₁ mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c₁NR and c₂NR had similar pH optima of 6.5, sedimentation behavior (s_20,w of 5.5-6.0), and electrophoretic mobility. However, c₁NR was more active with NADPH than with NADH, while c₂NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c₁NR) and 0.19 millimolar (c₂NR). The iNR from the mutant had a pH optimum of 7.5, s_20,w of 7.6, and was less mobile on polyacrylamide gels than c₁NR and c₂NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

     

Properties of an adrenal cytochrome P-450 (P-45011β) for the hydroxylations of corticosteroids

A highly purified preparation of cytochrome P-450, designated as P-450_11β, has been obtained from bovine adrenal cortex mitochondria. The P-450_11β exhibits remarkably high steroid hydroxylase activity in the reconstituted adrenal electron-donating system from NADPH via NADPH:adrenal ferredoxin oxidoreductase (EC 1.6.7.1) and adrenal ferredoxin. The turnover numbers (moles of hydroxylated product formed per minute per mole of P-450-heme) are 110 and 18 for respective 11β- and 18-hydroxylase activity when deoxycorticosterone is the substrate. The apparent K_m value is 6 μm for both reactions. The ratio, about 6:1 between the two activities, is constant under various experimental conditions including those in the presence of competitive inhibitors of hydroxylation. In addition to deoxycorticosterone, other steroids such as 11-deoxycortisol, 4-androstene-3,17-dione and testosterone are the hydroxylatable substrates. In cases in which 4-androstene-3,17-dione, a C₁₉-steroid, is the substrate, the hydroxylatable sites appear to be its respective 11β- and 19-position. The ratio between the two activities is about 4:1. In view of these results, it is concluded that one hemoprotein species, the P-450_11β, is responsible for the hydroxylase reactions of various Corticosteroids. 2-Methyl-1,2-di-3-pyridyl-1-propanone (metyrapone) inhibits the P-450_11β-catalyzed steroid hydroxylase reactions of either deoxycorticosterone at 11β- and 18-position or 4-androstene-3,17-dione at 11β- and 19-position (K_i = 0.1-0.2 μM). The P-450_scc-catalyzed cholesterol desmolase reaction is also inhibited, although weakly (K_i = 160 μM). In addition, both adrenal cytochromes appeared to differ from each other in spectral response to metyrapone.     

Purification and properties of two 2,5-diketo-d-gluconate reductases from a mutant strain derived from Corynebacterium sp.

Two 2,5-diketo-d-gluconate reductases, I and II, were purified respectively 918-fold and 28-fold from a mutant strain derived from Corynebacterium sp. SHS 0007. The enzymes appeared to be homogeneous on polyacrylamide gel electrophoresis. Both reductases converted 2,5-diketo-d-gluconate to 2-keto-l-gulonate in the presence of NADPH and seemed to be active only for reduction. The molecular weights of reductases I and II were estimated to be 29,000 and 34,000, respectively; and both were monomeric. Their isoelectric points were respectively pH 4.3 and pH 4.1. The optimum pH was 6.0 to 7.0 for reductase I, and 6.0 to 7.5 for reductase II. The K_m values (pH 7.0, 30°C) of reductase I for 2,5-diketo-d-gluconate and for NADPH were 1.8 mM and 12 μM, respectively; and the corresponding values of reductase II were 13.5 mM and 13 μM. Both reductases converted 5-keto-d-fructose to l-sorbose in the presence of NADPH.     

Role of NADPH and the NADPH-dependent methemoglobin reductase in the hydroxylase activity of human erythrocytes

Human erythrocytes were shown previously to catalyze the oxyhemoglobin-requiring hydroxylation of aniline, and the reaction was stimulated apparently preferentially by NADPH in the presence of methylene blue (K. S. Blisard and J. J. Mieyal,J. Biol. Chem.254, 5104, 1979). The current study provides a further characterization of the involvement of the NADPH-dependent electron transport system in this reaction. In accordance with the role of NADPH, the hydroxylase activity of erythrocytes or hemolysates from individuals with glucose-6-phosphate dehydrogenase deficiency (i.e., with diminished capacity to form NADPH) displayed decreased responses to glucose or glucose 6-phosphate, respectively, in the presence of methylene blue in comparison to samples from normal adults; maximal activity could be restored by direct addition of NADPH to the deficient hemolysates. Kinetic studies of the methylene blue-stimulated aniline hydroxylase activity of normal hemolysates revealed a biphasic dependence on NADPH concentrations: a plateau was observed at relatively low concentrations (K_m^NADPH ~ 20 μm), whereas saturation was not achieved at the higher concentrations of NADPH. The latter low efficiency phase (i.e., at the higher concentrations of NADPH) could be ascribed to a direct transfer of electrons from NADPH to methylene blue to hemoglobin. The high efficiency phase suggested involvement of the NADPH-dependent methemoglobin reductase; accordingly 2′-AMP, an analog of NADP⁺, effectively inhibited this reaction, but the pattern was noncompetitive. This behavior is suggestive of a mechanism by which both NADPH and methylene blue are substrates for the reductase and interact with it in a sequential fashion. The kinetic patterns observed for variation in NADPH concentration at several fixed concentrations of methylene blue, and vice versa, are consistent with this interpretation.     

An oxygen-18 tracer investigation of the mechanism of myo-inositol oxygenase

In the conversion of $\text{myo}$-inositol to D-glucuronic acid catalyzed by $\text{myo}$-inositol oxygenase only one atom of ¹⁸O from ¹⁸O₂ is incorporated into the product, and it is found exclusively in the carboxyl group. Control experiments indicate that under the reaction conditions no exchange of solvent oxygens with D-glucuronate occurs. To avoid exchange during isolation and analysis the oxygenase product was enzymically reduced to L-gulonate and isolated in that form. The results eliminate one possible mechanism for the oxygenase reaction, but are consistent with two others which seem chemically reasonable.     

Biosynthesis of alkane-2,3-diols: enzymatic reduction of 3-hydroxyoctadecane-2-one to octadecane-2,3-diol by a NADPH (B-side) specific microsomal reductase from the uropygial glands of ring-necked pheasants (Phasianus colchicus).

Cell-free preparations from the uropygial gland of ring-necked pheasant catalyzed the reduction of a synthetic R,S-mixture of 3-hydroxyl[3-¹⁴C]octadecane-2-one (acyloin) to a mixture of threo- and erythro-[3-¹⁴C]octadecane-2,3-diol, the final step in the postulated pathway for the biosynthesis of alkane-2,3-diols. The product of enzymatic reduction was identified by Chromatographic techniques and chemical degradation studies. The acyloin reductase showed a pH optimum near 4.0 and specificity for NADPH. With stereospecifically labeled [³H]NADPH, it was shown that acyloin reductase preferentially transferred hydride from the B-side of the nicotinamide ring to the acyloin. A typical Michaelis-Menten substrate saturation was observed for the acyloin and an apparent K_m of 70 μm was calculated from linear double reciprocal plots. Acyloin reductase was inhibited by thioldirected reagents such as p-chloromercuribenzoate and N-ethylmaleimide. Subcellular fractionation of the gland homogenates using sucrose density gradient centrifugation showed that acyloin reductase activity coincided with NADPH:cytochrome c reductase activity, strongly suggesting that acyloin reductase is localized in the microsomal membranes.     

Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei

Pattanakitsakul S. and Ruenwongsa P. 1984. Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei. International Journal for Parasitology14: 513–520. Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) from Plasmodium berghei were copurified by Sephacryl S-300 and Sephadex G-200 column chromatography and found to have an apparent mol. wt of 132,000. Electrophoresis of the partially purified enzyme under non-denaturing conditions showed the comigration of TS and DHFR. The mol. wt of TS was estimated to be 65,000 on SDS-gel electrophoresis. Both enzymes exhibit a broad pH optimum in the range of 6.5–8.0. Urea, NaCl and KC1 inhibit TS but activate DHFR. For TS, the apparent K_m for dUMP and methylene-tetrahydrofolate have been found to be 71.4 and 312.5 μM, respectively. For DHFR, the apparent K_m for dihydrofolate and NADPH have been found to be 4.4 and 12.5 μM, respectively. Inhibition of DHFR by pyrimethamine, methotrexate and trimethoprim are competitive with dihydrofolate with K_is of 0.63, 0.5 and 1.88 nM, respectively. FdUMP inhibition of TS is competitive with dUMP with K_is of 0.05 μM, but inhibition by methotrexate is uncompetitive with dUMP and MTHF with K_ii of 103 and 23 μM, respectively.