Determination of nucleoside triphosphates by use of combined reactions of hexokinase and glucose-6-phosphate dehydrogenase.

ATP is known to be easily determined fluorometrically after it is utilized to produce the corresponding amount of NADPH by combined reactions of hexokinase and glucose-6-phosphate dehydrogenase. We studied further whether nucleoside triphosphates other than ATP can be also determined in a similar manner if they were incubated for a longer period with an increased amount of hexokinase. It was shown that CTP, GTP, ITP, and UTP can be utilized to produce the corresponding amount of NADPH after an incubation of at least 60 min and that 0 to 50 nmols of these nucleotides were able to be determined fluorometrically.     

Modulation of cytochrome oxidase activity by inorganic and organic phosphate.

The activity of cytochrome oxidase reconstituted into phospholipid vesicles has been studied as a function of orthophosphate, ATP and inositol hexakisphosphate concentrations. The respiratory-control ratio was found to be quite sensitive to these compounds and was inversely related to the anion concentration. This effect is related to a phosphate-dependent decrease in the rate constant for ferrocytochrome c oxidation observed in the presence of ionophores. The data cannot be interpreted simply on the basis of ionic strength, which is known to limit cytochrome c binding to cytochrome oxidase, since cytochrome oxidase-containing vesicles responded differently to phosphate depending on the energization state of the phospholipid membrane.     

A robotics-based automated assay for inorganic and organic phosphates.

Phosphate analyses are fundamental to a broad range of biochemical applications involving inorganic phosphate and organic phosphoesters such as phospholipids, phosphorylated proteins, and nucleic acids. A practical automated method utilizing robotics is described in this report. Five colorimetric methods of phosphate analyses based on formation of a phosphomolybdate complex and compatible with the automated assay were tested, and the fundamental chemistry is discussed. The relative sensitivities are malachite green > crystal violet > quinaldine red > ascorbate reduction > antimony-modified ascorbate reduction, although only a fourfold improvement was observed in going from the modified ascorbate procedure to malachite green. Malachite green was selected to optimize the assay because this dye provided the highest sensitivity. However, where color stability and low blanks are more important than sensitivity, the ascorbate reduction and quinaldine red methods were found to be better choices than malachite green. Automation using a robotic liquid-handling system substantially reduces the labor required to process large arrays of samples. The result is a sensitive, nonradioactive assay of inorganic phosphate with high throughput. A digestion step in an acid-resistant 96-well plate was developed to extend the assay to phosphate esters. The robotic-based assay was demonstrated with inorganic phosphate and a common phospholipid, phosphatidylcholine.     

Bass liver 6-phosphogluconate dehydrogenase: inhibition by nucleoside phosphates and by fructose 1,6 bisphosphate

Nucleoside 5'-triphosphates, 5'-diphosphates and 5'-monophosphates are inhibitors of the 6-phosphogluconate dehydrogenase enzyme from bass liver. The 2'- and 3'-monophosphates of adenosine and guanosine are also inhibitory, the 2'-isomers being especially potent inhibitors. The catalytic activity of 6-phosphogluconate dehydrogenase has been found to be markedly inhibited by fructose 1, 6 bisphosphate. As the Km for 6-phosphogluconate, the Ki for fructose 1,6 bisphosphate and the concentration of both compounds in bass liver are all comparable, it appears that the inhibition of 6-phosphogluconate dehydrogenase by fructose 1,6 bisphosphate may be of significance in the regulation of carbohydrate metabolism in bass liver.     

Solubilization of organic and inorganic phosphates by three highly efficient soil bacterial isolates

Screening soil samples collected from a diverse range of slightly alkaline soil types, we have isolated 22 competent phosphate solubilizing bacteria (PSB). Three isolates identified as Pantoea agglomerans strain P5, Microbacterium laevaniformans strain P7 and Pseudomonas putida strain P13 hydrolyzed inorganic and organic phosphate compounds effectively. Bacterial growth rates and phosphate solubilization activities were measured quantitatively under various environmental conditions. In general, a close association was evident between phosphate solubilizing ability and growth rate which is an indicator of active metabolism. All three PSB were able to withstand temperature as high as 42°C, high concentration of NaCl upto 5% and a wide range of initial pH from 5 to 11 while hydrolyzing phosphate compounds actively. Such criteria make these isolates superior candidates for biofertilizers that are capable of utilizing both organic and mineral phosphate substrates to release absorbable phosphate ion for plants.     

Enzymic cis-trans isomerization of nitrofuran derivatives: isomerizing activity of xanthine oxidase, lipoyl dehydrogenase, DT-diaphorase and liver microsomes.

Xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) supplemented with an electron donor could catalyze the cis-trans isomerization of 3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide, 3-(5-nitro-2-furyl)-2-phenylacrylamide and 3-(5-nitro-2-furyl)-2-(2-furyl)acrylonitrile. The direction of isomerization (cis leads to trans, cis in equilibrium trans or trans leads to cis) is dependent on the chemical structure of these nitrofuran derivatives. Lipoyl dehydrogenase (NADH:lipoamide oxidereductase, EC 1.6.4.3), DT-diaphorase (NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2) and liver microsomes could also catalyze the conversion of cis-3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide to its trans isomer in the presence of an appropriate electron donor. Such isomerizing activity of these enzymes is much higher than their nitro-reducing activity. In addition, the cis-trans isomerization of some nitrofuran derivatives was demonstrated with the liver slices and the small intestines of rats. A new cis-trans isomerization mechanism which is based on transfer of a single electron by an enzyme system to a nitrofuran derivative to give the radical-anion was proposed. This postulated mechanism was supported by the preliminary experiments using pulse radiolysis technique.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.     

Multiple molecular forms of xanthine dehydrogenase and related enzymes. IV. The relationship of aldehyde oxidase to xanthine dehydrogenase

Variants of the enzyme aldehyde oxidase in Drosophila melanogaster are described. In addition to electrophoretic variants, a mutant that causes low levels of the enzyme has been found by screening more than 80 strains for aldehyde oxidase levels. The locus of the mutation maps on the third chromosome near lpo and aldox. The existence of the ry, lpo, and aldox mutants and of the new mutant indicates that xanthine dehydrogenase, pyridoxal oxidase, and aldehyde oxidase are under a separate genetic control, in addition to a common genetic control by ma-l and lxd. The genetic separation is shown to be accompanied by physical separation of the enzymes with DEAE-cellulose column chromatography and (NH ₄)₂SO₄fractionation. Further data on the metabolism of aldehydes by xanthine dehydrogenase and aldehyde oxidase are presented. Although xanthine dehydrogenase requires NAD or a similar cofactor to metabolize purine and pteridine substrates, aldehyde oxidase oxidizes salicylaldehyde to salicylic acid without dissociable cofactors and with the uptake of oxygen.This work was supported in part by Research Grant GM-08202, by a Predoctoral Fellowship (J.C.) and a Genetics Training Grant (J.C. and E.D.), and by a Research Career Development Award (E.G.), all from the National Institutes of Health. Part of this work was submitted by J.C. to the University of North Carolina at Chapel Hill in partial fulfillment of the degree of Doctor of Philosophy.     

Coupled reactions for the determination of analytes and enzymes based on the use of luminescence

Immobilized enzymes are widely used in the clinical laboratory in the assay of several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable and re-usable, and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow methods based on the use of nylon tube coil or epoxy methacrylate column as solid support. All the NAD(P)/NAD(P)H-dependent dehydrogenases (bacterial luciferase), ATP-dependent enzymes (firefly luciferase) and oxidases producing H₂O₂ (peroxidase) can be immobilized and a large variety of analytes have been sensitively measured. As an alternative format we also reported a dry chemistry method in which all the enzymes, substrates and cofactors are ready to use, supported on dry cellulose disks. Methodological problems such as flow conditions, stability, pH, ionic strength and analytical performances are also reported.     

Milk xanthine oxidase type D (dehydrogenase) and type O (oxidase). Purification, interconversion and some properties

1. The xanthine oxidase of cow's milk, crude or purified, appears as an oxidase (type O), and can be converted almost completely into a NAD(+)-dependent dehydrogenase (type D) by treatment with dithioerythritol or dihydrolipoic acid, but only to a small extent by other thiols. 2. The D form of the enzyme is inhibited by NADH, which competes with NAD(+). 3. The kinetic constants of the two forms of the enzyme are similar to those of the corresponding forms of rat liver xanthine oxidase. 4. Milk xanthine oxidase is converted into an irreversible O form by pretreatment with chymotrypsin, papain or subtilisin, but only partially with trypsin. 5. The enzyme as purified shows a major faster band and a minor slower band on gel electrophoresis. The slower band is greatly reinforced after xanthine oxidase is converted into the irreversible O form by chymotrypsin.     

Protease-catalyzed synthetic reactions and immobilization-activation of the enzymes in hydrophilic organic solvents.

The catalytic feature of serine proteases for synthetic reactions in hydrophilic organic solvents and effects of immobilization by complexation with polysaccharides are described. Free alpha-chymotrypsin and subtilisin Carlsberg catalyze esterification, transesterification, and peptide synthesis in hydro-organic cosolvents with less than 10% water. Subtilisin BPN' is catalytically less active. The medium effects on the reaction kinetics and product yield were investigated in terms of the nature of solvent and water content in the reaction systems. The substrate- and stereo-specificities of the enzymes suggest that the enzymes maintain their native conformations in these low-water organic solvents. The catalytic activities of the proteases markedly increase by immobilization or complexation with polysaccharides, such as chitin or chitosan. The results of the rate measurements suggest that the primary role of the support materials is the activation of the enzymes and the increase in substrate concentration at reaction sites.