Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

Isolation and characterization of a cellobiose dehydrogenase formed by a asporogenic mycelial fungus INBI 2-26(-)

A nonsporulating fungus isolated from dioxine-containing tropical soils forms cellobiose dehydrogenase, when grown in media supplemented by a source of cellulose. The enzyme purified to homogeneity by SDS-PAGE (yield, 43%) had an M(r) of 95 kDa; its pH optimum was in the range 5.5-7.0; more than 50% activity was retained at pH 4.0-8.0 (citrate-phosphate buffer). The absorption spectrum of the enzyme in the visible range had the characteristic appearance of flavocytochrome proteins. Cellobiose dehydrogenase oxidized cellobiose and lactose (the respective K(M) values at pH 6.0 equaled 4.5 +/- 1.5 and 56 microM) in the presence of dichlorophenolindophenol (K(M) app = 15 +/- 3 microM at pH 6.0) taken as an electron acceptor. Other sugars were barely if at all oxidized by the enzyme. Neither ethyl-beta-D-cellobioside, heptobiose, nor chitotriose inhibited the enzymatic oxidation of lactose, even under the conditions of 100-fold molar excess. The enzyme was weakly inhibited by sodium azide dichlorophenolindophenol reduction and exhibited affinity to amorphous cellulose. At 55 degrees C and pH 6.0 (optimum stability), time to half-maximum inactivation equaled 99 min. The enzyme reduced by cellobiose was more stable than the nonreduced form. Conversely, the presence of an oxidizer (dichlorophenolindophenol) decreased the stability eight times at pH 6.0. In addition, the enzyme acted as a potent reducer of the single-electron acceptor cytochrome c3+ (K(M) app = 15 microM at pH 6.0).     

Entrapment of horseradish peroxidase in sugar-modified silica monoliths: toward the development of a biocatalytic sensor

A miniaturized HRP-entrapped bioreactor was prepared by a one-step enzyme immobilization method using a biocompatible sol-gel processing method employing either diglycerylsilane (DGS) or sodium silicate (SS) as precursors and a covalently tethered sugar, N-(3-triethoxysilylpropyl)gluconamide (GLS) as a silica modifier. Factors such as leaching, catalytic efficiency and long-term stability were examined to assess the role of the precursor and modifier in influencing enzyme performance. The results showed that sodium silicate derived materials modified with covalently bound sugars at a level of 10 mol% were optically transparent and provided the highest catalytic turnover rate for entrapped HRP. The stability and reusability of the entrapped HRP was found to be satisfactory for at least 1 month in the GLS-doped SS materials, and the entrapped HRP was able to respond linearly to the presence of peroxide over the concentration range of 0-750 microM with a detection limit of 6 microM, demonstrating the potential of this material for the development of a reusable optical biosensor.     

Properties of a highly purified mitochondrial deoxyguanosine kinase

Deoxyguanosine kinase, purified over 6000-fold from beef liver mitochondria by means of deoxyguanosine-3'-(4-aminophenyl phosphate)-Sepharose affinity chromatography, was nearly homogeneous. It phosphorylates only deoxyguanosine and deoxyinosine among the natural nucleosides, with apparent Km values of 4.7 and 21 microM, respectively. Among nucleoside analogs tested, only arabinosylguanine (Ki = 125 microM) and 8-aza-deoxyguanosine (Ki = 450 microM) competed with deoxyguanosine. The relative molecular mass of the enzyme is 56,000, as determined by equilibrium sedimentation, and sodium dodecyl sulfate-gel electrophoresis suggests two subunits of Mr 28,000. The pH optimum for enzyme activity is 5.5, but optimum enzyme stability is seen at pH 7.0. Triton X-100 increased the stability of the enzyme markedly. ATP is the best phosphate donor at pH 5.5, but pyrimidine triphosphates such as dTTP and UTP are more efficient donors at pH 7.4. The activation energy, at pH 5.5, was estimated to be 10.9 kcal/mol. Amino acid modification experiments suggest the involvement of arginine, cysteine, and probably histidine. The inactivation of the enzyme by modification of these amino acid residues was time and pH dependent. Both substrates protected the enzyme from inactivation in every case but that of photooxidation by Rose Bengal, where only deoxyguanosine prevented inactivation.     

Species-dependent differences of the biochemical properties of diamine oxidase

Diamine oxidase (DAO) from tissues of mice, rats and humans showed different properties with respect to stability and kinetic parameters. DAO-activities in homogenates of rat or human tissues, but not of mouse tissues, rapidly decreased upon storage at -20 degrees C. The Km-value for putrescine was 90 microM in mouse kidney or intestine. In rats different Km-values were observed before (272 microM) and after freezing (102 microM). A similar effect was observed with DAO in human kidney (321 and 39 microM, respectively). Treatment of rats with heparin resulted in a depletion of intestinal DAO and the concomitant appearance of DAO in blood. The enzyme remaining in the intestine showed the lower Km-value.     

Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings

3-Hydroxy-3-methylglutaryl-CoA reductase (NADPH) was solubilized with polyoxyethylene ether (Brij) W-1 from a heavy-membrane fraction, sedimented at 16000 X g from a cell-free homogenate of four-day-old, dark-grown radish seedlings (Raphanus sativus L.). Approximately 350-fold purification of the solubilized enzyme activity was achieved by (NH4)2SO4 precipitation followed by column chromatography on DEAE-Sephadex A-50, blue-dextran-agarose and HMG-CoA-hexane-agarose. The presence of detergent, which was required at all times to maintain activity, did not interfere with the chromatographic procedures used. Sucrose density centrifugation suggested an apparent molecular mass of 180 kDa with subunits of 45 kDa (polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate). The enzyme was stable at 67.5 degrees C for 30 min in the presence of glycerol, dithioerythritol and detergent. Studies of enzyme stability and activation indicate that the enzyme is a hydrophobic protein with free thiol groups that are essential for full activity. The activation energy was estimated to be 92 kJ (Arrhenius plot). Antibodies raised against rat liver and yeast hydroxymethylglutaryl-CoA (HMG-CoA) reductase failed to bind or inactivate the radish enzyme. When both HMG-CoA and NADPH concentrations were varied, intersecting patterns were obtained with double-reciprocal plots. The apparent Km values determined in this way are 1.5 microM [(S)-HMG-CoA], and 27 microM (NADPH). Concentrations of NADPH greater than 150 microM caused substrate inhibition at low HMG-CoA concentrations resulting in deviations from linearity in secondary plots. Analysis of these data and the product inhibition pattern suggest a sequential mechanism for the reduction of HMG-CoA to mevalonic acid with HMG-CoA being the first substrate binding to the enzyme, followed by NADPH.     

Purification and characterization of NADP+-linked isocitrate dehydrogenase from an alkalophilic Bacillus

We have succeeded in purifying to homogeneity a very labile NADP+-linked isocitrate dehydrogenase (isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42) from a strain of alkalophilic Bacillus, by a simple method, with an overall yield over 76% of the original activity. The molecular weight on Sephadex G-200 was around 90,000; and that by electrophoresis on SDS-polyacrylamide gels was about 44,000. The sedimentation coefficient (s020,w) and isoelectric point of the enzyme were determined to be 3.22 S and pH 4.7, respectively. The enzyme required Mn2+ for the reaction and for stability. The optimum pH for the reaction was in the range 7.8-8.4 at 30 degrees C; the optimum temperature at pH 8.0 was 75 degrees C; the activation energy of the reaction was 6.2 kcal/mol. The Km values for threo-Ds-isocitrate, DL-isocitrate, and NADP+ were 5.4 microM, 9.9 microM, and 7.3 microM, respectively. This enzyme was inhibited by NADPH, glyceraldehyde 3-phosphate, 3-phosphoglycerate, phosphoenol pyruvate, cis-aconitate, alpha-ketoglutarate, and oxaloacetate. In addition, it was subject to a concerted inhibition by a combination of glyoxylate and oxaloacetate, and also to a cumulative inhibition by nucleoside triphosphates.     

Purification of the NADP+:F420 oxidoreductase of Methanosphaera stadtmanae

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2 and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420 oxidoreductase activity. The NADP+:F420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP+ and F420 in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420 reduction was 6.0, and 8.5 for NADP+ reduction. During the purification process, it was noted that precipitation with (NH4)2SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Km values were 370 microM for NADP+, 142 microM for NADPH, 62.5 microM for F420, and 7.7 microM for F420H2. These values were different from the Km values observed in the cell-free extract.     

Purification and characterization of a cathepsin L-like enzyme from the body wall of the sea cucumber Stichopus japonicus

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS-PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine7-amido-4-methylcoumarin with K(m) (69.92 microM) and k(cat) (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 degrees C. It showed thermal stability below 40 degrees C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.     

Phosphatidylinositol synthase from canine pancreas: solubilization by n-octyl glucopyranoside and stabilization by manganese

Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase) is active in mammalian pancreas, where it plays a role in the resynthesis of phosphatidylinositol (PI) during agonist-stimulated inositol-phospholipid metabolism. The enzyme was found to be present in relatively high specific activity [30 nmol of PI formed min-1 (mg of protein)-1] in dog pancreas microsomal membranes, and its activity in these membranes was partially characterized. The Km for myo-inositol was 0.76 mM, and the apparent Km for cytidine(5')diphospho-1,2-diacylglycerol (CDP-diacylglycerol) was 18 microM. The apparent Ka values for activation by Mn2+ and Mg2+ were respectively 42 microM and 2.5 mM. The pH optimum was 8.5-9.0. The enzyme was solubilized in stable form and in nearly quantitative yield with 40 mM n-octyl glucopyranoside (OG), with 4-6 mg of OG/mg of microsomal protein. In the presence of solubilizing levels of OG, the enzyme exhibited less than maximal activity, but full activity was restored by dilution of the OG to below its critical micelle concentration of 20-25 mM. The presence of Mn2+ was essential for stabilization of the OG-solubilized enzyme, with half-maximal stabilization at 40 microM Mn2+. The stability of the OG-solubilized enzyme was sufficient to facilitate purification of the enzyme in the presence of this detergent, with 67% of the activity remaining after 3 days at 4 degrees C. The enzyme was partially purified by OG extraction and DEAE-cellulose chromatography, in 98% yield, to a specific activity of 290 nmol of PI formed min-1 (mg of protein)-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills

A soluble F(1)-ATPase was isolated from the mitochondria of crayfish (Orconectes virilis) gill tissue. The maximal mitochondrial disruption rate (95%) was obtained by sonicating for 4 min at pH 8.6. A 15-fold purification was estimated. The properties for both soluble and membrane-bound enzyme were studied. Both enzyme forms were stable at 4 to -70 degrees C when kept in 20% glycerol. Soluble F(1)-ATPase was more stable at room temperature than membrane-bound enzyme. It displayed a narrower pH profile (pK(1) =6.58, pK(2)=7.68) and more acid pH optimum (7.13) than membrane-bound enzyme (pK(1)=6.42, pK(2)=8.55, optimum pH 7.49). The anion-stimulated activities were in the order HCO(3)(-)>SO(4)(2-)>Cl(-). The apparent K(a) values for soluble enzyme were 11.4, 11.2, and 10.9 mM, respectively, but the K(a) of HCO(3)(-) for membrane-bound enzyme (14.9 mM) was higher than for soluble enzyme. Oligomycin and DCCD inhibited membrane-bound F(1)-ATPase with I(50) of 18.6 ng/ml and 2.2 microM, respectively, but were ineffective in inhibiting soluble enzyme. Both enzyme forms shared identical sensitivity to DIDS (I(50)=12.5 microM) and vanadate (I(50)=9.0 mM). Soluble ATPase was significantly more sensitive to pCMB (I(50)=0.15 microM) and NO(3)(-) (I(50)=28.6 mM) than membrane-bound enzyme (I(50)=1.04 microM pCMB and 81.5 mM NO(3)(-)). In addition, soluble F(1)-ATPase was slightly more sensitive to azide (I(50)=91.8 microM) and NBD-Cl (I(50)=9.18 microM) than membrane-bound enzyme (I(50)=111.6 microM azide and 12.88 microM NBD-Cl). These data suggest a conformational change transmission between F(0) and F(1) sectors and slight conformational differences between soluble F(1) and membrane-bound F(1). In addition, an unmodified F(0) stabilizes F(1) and decreases F(1) sensitivities to inhibitors and modulators.     

cDNA sequence and kinetic properties of human lung fructose(1, 6)bisphosphatase

A cDNA encoding fructose(1,6)bisphosphatase was isolated from total human lung RNA. The cDNA contained an open reading frame encoding 337 amino acids. The determined nucleotide sequence of the lung cDNA was significantly different from muscle cDNA and slightly differed from human liver cDNA in a single mutation (Gly-336 for Ala-336) and a T for C substitution in position 648. The human lung fructose(1, 6)bisphosphatase [Fru(1,6)Pase] was isolated and its kinetic parameters were compared with liver and muscle isoenzymes. Values of kcat for the lung Fru(1,6)Pase were lower than for the liver and muscle enzyme. Like the liver isoenzyme, lung Fru(1,6)Pase is significantly less inhibited by AMP than the muscle enzyme. The values of I0.5 were 9.5, 9.8, and 0.3 microM for the liver, lung, and muscle enzyme, respectively. The lung enzyme was slightly more sensitive to fructose(2,6)bisphosphate [Fru(2,6)P2] inhibition than the liver enzyme. Ki was 75 microM for the lung and 96 microM for the liver enzyme. The synergistic effect of AMP and Fru(2,6)P2 on the lung and liver Fru(1,6)Pase was also observed. In the presence of AMP the corresponding values of Ki for Fru(2,6)P2 were 16 microM for the lung and 10 microM for the liver enzyme.     

D-glyceraldehyde-3-phosphate dehydrogenase subunit cooperativity studied using immobilized enzyme forms

Tetrameric D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) isolated from rabbit skeletal muscle was covalently bound to CNBr-activated Sepharose 4B via a single subunit. Catalytically active immobilized dimer and monomeric forms of the enzyme were prepared after urea-induced dissociation of the tetramer. A study of the coenzyme-binding properties of matrix-bound tetrameric, dimeric and monomeric species has shown that: (1) an immobilized tetramer binds NAD+ with negative cooperativity, the dissociation constants being 0.085 microM for the first two coenzyme molecules and 1.3 microM for the third and the fourth one; (2) coenzyme binding to the dimeric enzyme form also displays negative cooperativity with Kd values of 0.032 microM and 1.1 microM for the first and second sites, respectively; (3) the binding of NAD+ to a monomer can occur with a dissociation constant of 1.6 microM which is close to the Kd value for low-affinity coenzyme binding sites of the tetrameric or dimeric enzyme forms. In the presence of NAD+ an immobilized monomer acquires a stability which is not inferior to that of a holotetramer. The catalytic properties of monomeric and tetrameric enzyme forms were compared and found to be different under certain conditions. Thus, the monomers of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase displayed a hyperbolic kinetic saturation curve for NAD+, whereas the tetramers exhibited an intermediary plateau region corresponding to half-saturating concentrations of NAD+. At coenzyme concentrations below half-saturating a monomer is more active than a tetramer. This difference disappears at saturating concentrations of NAD+. Immobilized monomeric and tetrameric forms of D-glyceraldehyde-3-phosphate dehydrogenase from baker's yeast were also used to investigate subunit interactions in catalysis. The rate constant of inactivation due to modification of essential arginine residues in the holoenzyme decreased in the presence of glyceraldehyde 3-phosphate, probably as a result of conformational changes accompanying catalysis. This effect was similar for monomeric and tetrameric enzyme forms at saturating substrate concentrations, but different for the two enzyme species under conditions in which about one-half of the active centers remained unsaturated. Taken together, the results indicate that association of D-glyceraldehyde-3-phosphate dehydrogenase monomers into a tetramer imposes some constraints on the functioning of the active centers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hyperforin and its analogues inhibit CYP3A4 enzyme activity

Literature indicates that herb-drug interaction of St. John's wort is largely due to increased metabolism of the co-administered drugs that are the substrates of cytochrome P450 (CYP) 3A4 enzyme, alteration of the activity and/or expression of the enzyme. The major St. John's wort constituents, acylphloroglucinols, were evaluated for their effects on CYP3A4 enzyme activity to investigate their roles in herb-drug interaction. Hyperforin and four oxidized analogues were isolated from the plant and fully characterized by mass spectral and NMR analysis. These acylphloroglucinols inhibited activity of CYP3A4 enzyme potently in the fluorometric assay using the recombinant enzyme. Furoadhyperforin (IC(50) 0.072 microM) was found to be the most potent inhibitor of CYP3A4 enzyme activity, followed by furohyperforin isomer 1 (IC(50) 0.079 microM), furohyperforin isomer 2 (IC(50) 0.23 microM), hyperforin (IC(50) 0.63 microM) and furohyperforin (IC(50) 1.3 microM). As the acylphloroglucinols are potent inhibitors of the CYP3A4 enzyme, their modulation of the enzyme activity is unlikely to be involved in increased drug metabolism by St. John's wort.     

Determination of L-carnitine by flow injection analysis with NADH fluorescence detection

A flow injection analysis method for determining L-carnitine is reported. The system uses the enzyme L-carnitine dehydrogenase covalently immobilized to Eupergit C. The NADH produced by the action of the enzyme, which is proportional to the L-carnitine concentration, is quantified using fluorescence detection. The system response was rapid and had a wide range of linearity. At a flow rate of 0.2 ml/min, a detection limit of 1 microM (20 pmol) was obtained for L-carnitine, peak areas were linear up to 100 microM, and samples could be injected every 4 min. The method performed well as a routine assay, showing high sensitivity (54,000 AU/microM), a precision of 0.96%, and the ability to carry out 144 consecutive assays with an RSD of 1.47% (good stability). Comparisons were made with other known methods for L-carnitine determination. Presence of D-carnitine had no effect on L-carnitine assay. The analysis was valid for determining L-carnitine concentrations in commercial pharmaceutical preparations.     

Purification and characterization of a bacterial nitrophenol oxygenase which converts ortho-nitrophenol to catechol and nitrite.

A nitrophenol oxygenase which stoichiometrically converted ortho-nitrophenol (ONP) to catechol and nitrite was isolated from Pseudomonas putida B2 and purified. The substrate specificity of the enzyme was broad and included several halogen- and alkyl-substituted ONPs. The oxygenase consisted of a single polypeptide chain with a molecular weight of 58,000 (determined by gel filtration) or 65,000 (determined on a sodium dodecyl sulfate-polyacrylamide gel). The enzymatic reaction was NADPH dependent, and one molecule of oxygen was consumed per molecule of ONP converted. Enzymatic activity was stimulated by magnesium or manganese ions, whereas the addition of flavin adenine dinucleotide, flavin mononucleotide, or reducing agents had no effect. The apparent Kms for ONP and NADPH were 8 and 140 microM, respectively. 2,4-Dinitrophenol competitively (Ki = 0.5 microM) inhibited ONP turnover. The optimal pH for enzyme stability and activity was in the range of 7.5 to 8.0. At 40 degrees C, the enzyme was totally inactivated within 2 min; however, in the presence of 1 mM ONP, 40% of the activity was recovered, even after 10 min. Enzymatic activity was best preserved at -20 degrees C in the presence of 50% glycerol.     

Human liver folylpolyglutamate synthetase: biochemical characterization and interactions with folates and folate antagonists

Folylpolyglutamate synthetase (FPGS) was isolated from human liver cytosol by 0-30% (w/v) ammonium sulfate fractionation and characterized biochemically. Using aminopterin (AMT), L-[3H]glutamate and MgATP as cosubstrates, maximal gamma-L-glutamylation activity was observed in the presence of the activators KCl and NaHCO3. ATP and 2-mercaptoethanol were each required for enzyme activity and stability. In the absence of ATP, human liver FPGS rapidly inactivated at 37 degrees C (t1/2 approximately 8 min), whereas FPGS isolated from rabbit liver was significantly more stable (t1/2 = 68 min). Both folates and antifolates were effectively polyglutamylated by the isolated human liver enzyme. Km parameters determined for AMT (Km = 4.3 microM) were similar to those determined for several reduced folates (tetrahydrofolic acid, dihydrofolic acid, and folinic acid; Km = 3-7 microM), while significantly higher Km values were observed for methotrexate (MTX) and 5-methyltetrahydrofolic acid (Km = 50-60 microM) and for folic acid (Km = 100 microM). All of the substrates examined exhibited Vmax values ranging from 30 to 90% of the AMT value (Vmax = 935 pmol product/mg/h). The order of reactivity for these substrates differed from that determined in parallel studies for FPGS isolated from rat and rabbit liver. In the case of AMT and several reduced folates, inhibition of human liver FPGS was observed at substrate concentrations at or above 50-250 microM. FPGS isolated from six individual human livers exhibited highly similar biochemical and kinetic properties, suggesting the presence of the same or at least highly similar enzyme species in each individual, with a five-fold interindividual range in specific activities observed. Comparison of MTX with its higher polyglutamates (MTX-Glu2 to MTX-Glu6) as FPGS substrates indicated a significant decrease in Vmax values with increasing glutamate chain length which was partially compensated for by a corresponding decrease in Km. Consistent with these observations, the isolated enzyme was unable to synthesize polyglutamates higher than MTX-Glu3 when MTX was supplied as substrate, raising the question as to how MTX polyglutamates containing up to five or six gamma-L-glutamate residues are formed in vivo.     

Activation of carbonyl reductase from pig lung by fatty acids.

The NADPH-linked reductase activity of pig lung carbonyl reductase was activated two- to fivefold by fatty acids with a carbon chain length greater than nine at pH 7.0. cis-Unsaturated fatty acids of C:18 and C:20 were potent activators, showing Ka values of 2-14 microM which were lower than the values of 21-125 microM for saturated fatty acids (C:9 to C:16). Of the fatty acids arachidonic acid (C20:4) gave the highest activation. No significant stimulatory effect was observed with acyl CoAs, fatty alcohols, phospholipids, and nonionic detergents. Anionic detergents (sodium dodecyl sulfate and sarkosyl) stimulated the enzyme activity more than ninefold, but the Ka values for them were much higher than those for the cis-unsaturated fatty acids. Although no change in molecular weight or in subunit composition was observed in the enzyme activated by C20:4, the activation led to a decrease in thermal stability of the enzyme. The binding of C20:4 to the enzyme was instantaneous and reversible, shifted the pH optimum of the activity from 5.8 to 6.5, and changed the inhibitor sensitivity. In addition, C20:4 acted as an allosteric effector abolishing the negative interaction of the enzyme with carbonyl substrates which was seen without the fatty acid, but the activation increased both Vmax and [S]0.5 values for the substrates. Kinetic analysis with respect to NADPH concentration, in which no cooperativity was detected with or without C20:4, indicated that C20:4 was a nonessential activator of mixed type showing a binding constant of 10 microM. These results suggest that cis-unsaturated fatty acids may be potential modulators of pulmonary carbonyl reductase.     

Comparison of properties of the cellular and extracellular phosphodiesterases induced by cyclic adenosine 3',5'-monophosphate in Dictyostelium discoideum

The kinetic properties and susceptibilities to various agents of intracellular (particulate and soluble) and extracellular phosphodiesterases [EC 3.1.4.17] of Dictyostelium discoideum induced by cyclic adenosine 3',5'-monophosphate (cyclic AMP) were studied and compared. Intracellular particulate phosphodiesterase was obtained by solubilization of the light mitochondrial fraction with Emulgen. The Michaelis constants of this enzyme were 4.5 +/- 0.7 and 10 +/- 0.7 microM, while those of the intracellular soluble phosphodiesterase were 4.6 +/- 0.3 and 13 +/- 2.8 microM. However, the Michaelis constant of the extracellular phosphodiesterase was 6.8 +/- 0.9 microM, differing from the values of the two intracellular enzymes. Susceptibilities of the enzyme activity to various agents (theophylline, caffeine, dithiothreitol, glutathione, etc.) were essentially the same among these three phosphodiesterases. In the presence of 10 mM ethylenediaminetetraacetate, the activities of the particulate and the soluble enzymes were both decreased to about 60%, while that of the extracellular enzyme remained at 90%. The inhibition constants of cyclic inosine monophosphate for the cellular enzymes (35 and 100 microM for the particulate enzyme, and 37 and 90 microM for the soluble one) were considerably different from the value for the extracellular enzyme (48 microM). These results suggest that the characteristics of these three phosphodiesterases are substantially similar, but that the affinity of the intracellular (particulate and soluble) enzymes for the substrate is somewhat different from that of the extracellular enzyme.     

Purification and characterization of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney

A NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) from porcine kidney was purified to homogeneity by acid precipitation, blue agarose affinity chromatography, hydroxyapatite-ultrogel adsorption chromatography, DEAE-Sephadex ion-exchange chromatography and NAD(+)-agarose affinity chromatography. The specific activity of the homogeneous enzyme was 31.2 U/mg. The molecular mass of the native enzyme was estimated to be 55,000 Da, whereas that of SDS-treated enzyme was 29,000 Da indicating that the native enzyme was dimeric. Compared to human placental 15-OH-PGDH, porcine kidney enzyme gave a similar general amino acid residue distribution. Chemical modification of the enzyme with N-ethyl maleimide (3 microM), N-chlorosuccinimide (20 microM) or 2,4,6-trinitrobenzenesulfonic acid (2.5 microM) followed pseudo-first-order inactivation kinetics, and inactivation could be prevented by the presence of NAD+ (1 mM) but not of prostaglandin E1 (140 microM) indicating the involvement of cysteine, methionine and lysine residues in the coenzyme binding site. Inactivation by diethyl pyrocarbonate (1.25 mM) or phenylglyoxal (10 mM) also showed pseudo-first-order kinetics suggesting that histidine and arginine residues were catalytically or structurally important in the native enzyme. These studies provide new insights into the structure and function of 15-OH-PGDH.