Comparative Study of the Efficacy of Seven Paper-Reagent Strips and Conventional Biochemical Tests in Identifying Gram-negative Organisms

Gram-nagative organisms were tested with commercially available reagentimpregnated strips (PATHO-TEC). Of the 291 strains, all were tested by using seven paper tests and their conventional counterparts. Excellent correlation was obtained with the oxidase, phenylalanine-deaminase, and Voges-Proskauer tests. Indole tests made on liquid medium cultures also gave complete correlation, but some false-negative results with indole-positive Proteus strains were obtained when growth from solid medium was tested by the strip method. Paper strip urease tests were positive within 2 hr with all Klebsiella and some Serratia, Herellea, and Citrobacter strains as well as with Proteus strains. Approximately 15% of citrate strip test results differed from those of the conventional tests, and reproducibility was poor on retest. The lysine decarboxylase strip test showed a number of discrepancies and posed problems of interpretation and readability. Paper reagent strip methods are simple and convenient and merit further development to increase the specificity of those which depend on pH change up to that achieved with the Voges-Proskauer, oxidase, phenylalanine, and indole methods.     

The use of unfixed cryostat sections for electron microscopic study of D-amino acid oxidase activity in rat liver.

Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisomes, whereas the crystalline core remained unstained. Control incubations were performed in the absence of substrate; the lack of final reaction product in peroxisomes indicated the specificity of the reaction. We conclude that the semipermeable membrane technique opens new perspectives for localization of enzyme activities at the ultrastructural level without prior tissue fixation, thus enabling localization of the activity of soluble and/or labile enzymes.     

The cytochrome c binding site on cytochrome c oxidase.

Cytochrome $\text{c}$ was chemically coupled to cytochrome $\text{c}$ oxidase using the reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) which couples amine groups to carboxyl residues. The products of this reaction were analyzed on 2.5–27% polyacrylamide gradient gels electrophoretically. Since cytochrome $\text{c}$ binds to cytochrome oxidase electrostatically in an attraction between certain of its lysine residues and carboxyl residues on the oxidase surface, EDC is an especially appropriate reagent probe for binding-subunit studies. Coupling of polylysine to cytochrome oxidase using EDC was also performed, and the products of this reaction indicate that polylysine, an inhibitor of the cytochrome $\text{c}$ reaction with oxidase, binds to the same oxidase subunit as does cytochrome $\text{c}$, subunit IV in the gel system used.     

Microbial metabolism of aryl sulphonates A re-assessment of colorimetric methods for the determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzene sulphonates

The reaction of 2,4-dinitroanilinomaleimide with sulphite which has been claimed as the basis of a suitable colorimetric assay for the anion was carefully re-examined. The sulphite-imide addition product provides a suitable and specific qualitative test for sulphite after separation by paper chromatography but the method as previously used is probably measuring the hydrolysis of the imide to 2,4-dinitroanilinomaleamic acid and cannot be used for sulphite determination either colorimetrically or in kinetic assays. A new colorimetric method for the determination of sulphite based on its reaction with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoic acid) is described and compared for sensitivity with thep-rosaniline-HCHO method. Both methods were used to show the formation of sulphite as the initial product of arylsulphonate metabolism by bacteria. The failure to find sulphite in similar cultures of a third organism was attributed to the very high activities of sulphite oxidase found in extracts. The Ellman reagent was examined as the basis of an indicator medium for the detection of sulphite-excreting colonies.     

A rapid and sensitive colorimetric assay of amine oxidase

A sensitive colorimetric assay for amine oxidase with Δ¹-pyrroline as the product is described based on the formation of a red-colored complex with ninhydrin reagent in acidic medium.     

Effect of Amphotericin B on Dopachrome Tautomerase Activity and Other Melanogenic Parameters in Cultured B16/F10 Melanoma Cells

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 μg/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173–175) for tyrosine hydroxylase.     

Monoclonal antibody to human cytochrome c: Effect on electron-transfer reactions

A monoclonal antibody has been produced to an antigenic site on human cytochrome c which includes amino acid number 58 (isoleucine). This area is on the bottom back of the cytochrome, removed from the postulated binding/reaction sites for oxidase and reductase, but in the area of the molecule where an appreciable change in conformation is seen on oxidation-reduction. In spectrophotometric assays, where binding of cytochrome c to the oxidase or reductase is rate-limiting, the antibody gave stimulation of the reductase reaction under some conditions, where the oxidase reaction was inhibited. Also variation of the pH of the reaction medium resulted in differential effects on the oxidase and reductase reactions. Different effects of the antibody were seen when the oxidase was assayed polarographically, as compared to the spectrophotometric measurements. The data show that the binding/reaction sites on cytochrome c for the oxidase and reductase must be different. They suggest that binding of antibody may affect conformational changes in the whole molecule, distorting the binding/reaction sites. Conformational changes may be involved as a control mechanism in cytochrome c-mediated electron-transfer reactions.     

Coupling of glucose oxidase and Fenton's reaction for a simple and inexpensive assay of β-glucosidase

A simple and inexpensive assay for β-glucosidase, based on the coupling of glucose oxidase and Fenton's reagent has been described. Hydrogen peroxide formed as a result of the action of glucose oxidase on glucose (derived from the action of β-glucosidase on cellobiose) oxidizes ferrous sulphate, resulting in an increase in absorbance. The oxidation products produced a peak of maximum absorbance at 340 nm. Using this assay system, a linear relationship between glucose concentration in the range 5.55–27.78 mmol l^?1(100–500 mg dl^?1) and absorbance was obtained, indicating conformity to Beer's law. The preciseness of the glucose oxidase/Fenton's reagent for the assay of glucose was shown to be satisfactory. β-Glucosidase was assayed using the hexokinase assay reagent and the glucose oxidase/ferrous sulphate reagent. The values obtained using both reagents did not differ significantly. Although 2.6 times less sensitive than the hexokinase reagent when absorbance is measured at 340 nm, the glucose oxidase/Fenton's reagent is 10 times cheaper and could be used satisfactorily for routine assays of β-glucosidase and other carbohydrases including cellulase and amylase. In this respect, fructose, mannose, xylose, sucrose and cellobiose did not affect the sensitivity of the reagent. Of several metals tested, only aluminium interfered with the reagent, decreasing its sensitivity.     

The development of an immobilized lactate oxidase system for lactic acid analysis

An enzyme designated as lactate oxidase was purified from Acetobacter peroxydans by using the partition methods of separation. A DE-52 cellulose column was used for the primary purification of lactate oxidase, and the purified enzyme was covalently bound to a porous cellulose bead matrix in which benzoquinone was used as the coupling reagent. The physicochemical properties of the native and immobilized enzymes were determined including molecular weight, cofactor requirements, and optimal reaction conditions. Lactate oxidase was shown not to be subject to product inhibition, and to require Mg(2+) as a metal cofactor. Analysis of an immobilized lactate oxidase packed-bed reactor indicated that this system may not be subject to internal diffusional limitations. Molecular oxygen appeared to be a cosubstrate of the enzyme, and a reaction mechanism was postulated to predict the kinetic behavior of the immobilized reactor system. Applications of the immobilized lactate oxidase reactor for the pulse-flow analysis of lactic acid in whole milk and in a yeast fermentation system were considered.     

New Methods for Chemo-Enzymatic Galactosidation of 2S Rapeseed Protein

Two chemo-enzymatic methodologies to synthesize neoglycoproteins from rapeseed 2S protein (napin) were developed. In the first approach, glycosidases were used to catalyse 1-O-glycosylation of serine residues, whereas in the second one, 6-N-galactosylation was examined using an amino-reduction reaction between the epsilon-NH2 of lysine residues and 6-oxogalactosides (readily available by means of the oxidation reaction of the corresponding galactosides mediated by galactose oxidase). Our results indicated that glycosidases were unable to glycosylate native proteins. Conversely, this reaction was possible, although in low yields (10%), after the introduction of a hydroxyethylene spacer. The latter modified proteins were obtained via the condensation of epsilon-NH2 of lysines with ethylene carbonate in basic medium (40% yield). The second approach was much more efficient, as 61% of the lysine residues were shown to be 6-N-galactosylated using sodium cyanoborohydride as a reduction reagent.     

The use of diaminobenzidine (DAB) for the histochemical demonstration of cytochrome oxidase activity in unfixed plant tissues

Summary Cytochrome oxidase activity was demonstrated in unfixed root segments from Lupinus albus at the ultrastructural level using the osmiophilic reagent 3,3-diaminobenzidine (DAB). Precipitate, the formation of which was completely inhibited by 0.01 M KCN, and observed almost entirely on mitochondrial cristae, is considered to be produced by cytochrome oxidase activity. Heterogeneity of mitochondria as to the intensity of the reaction in the same cell could not be established with certainity. However, mitochondria of the root tip cells and cells belonging to the plerome consistently did not show histochemically demonstrable cytochrome oxidase activity.     

Effect of amphotericin B on dopachrome tautomerase activity and other melanogenic parameters in cultured B16/F10 melanoma cells.

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173-175) for tyrosine hydroxylase.     

Mechanisms of H2O2 formation by leukocytes. Evidence for a plasma membrane location.

It is postulated that the increase in H₂O₂ formation following phagocytosis in guinea pig polymorphonuclear leukocytes is due to the activation of a plasma-membrane-located NAD(P)H oxidase. The cyanide-resistant oxidase activity of intact leukocytes was markedly stimulated when the leukocytes were suspended in a hypotonic medium. Hydrogen peroxide was the principal product of the oxidase reaction. Evidence that the oxidase activity was located on the outside surface of the plasma membrane was the finding that added NAD(P)H was rapidly oxidized and the plasma membrane was impermeable to NADH or NADPH. Further evidence was the marked inhibition of the oxidase by p-CMB which also did not penetrate the plasma membrane. The oxidase was also inhibited on disruption of the plasma membrane. In addition, the enhanced oxidase activity under hypotonic conditions decreased to normal values when the medium was made isotonic and suggested that a reversible conformational change in the plasma membrane was responsible for the activation of oxidase activities.     

Arrangement of the subunits in solubilized and membrane-bound cytochrome c oxidase from bovine heart.

The arrangement of the six cytochrome c oxidase subunits in the inner membrane of bovine heart mitochondria was investigated. The experiments were carried out in three steps. In the first step, exposed subunits were coupled to the membrane-impermeant reagent p-diazonium benzene [32S]sulfonate. In the second step, the membranes were lysed with cholate anc cytochrome c oxidase was isolated by immunoprecipitation. In the third step, the six cytochrome c oxidase subunits were separated from each other by dodecyl sulfate-acrylamide gel electrophoresis and scanned for radioactivity. Exposed subunits on the outer side of the mitochondrial inner membrane were identified by labeling intact mitochondria. Exposed subunits on the matrix side of the inner membrane were identified by labeling sonically prepared submitochondrial particles in which the matrix side of the inner membrane is exposed to the suspending medium. Since sonic irradiation leads to a rearrangement of cytochrome c oxidase in a large fraction of the resulting submitochondrial particles, an immunochemical procedure was developed for isolating particles with a low content of displaced cytochrome c oxidase. With mitochondria, subunits II, V, and VI were labeled, whereas in purified submitochondrial particles most of the label was in subunit III. The arrangement of cytochrome c oxidase in the mitochondrial inner membrane is thus transmembraneous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. In a study of purified cytochrome c oxidase labeled with p-diazonium benzene [32S]sulfonate, the results were similar to those obtained with the membrane-bound enzyme. Subunits I and IV were inaccessible to the reagent, whereas the other four subunits were accessible. In contrast, all six subunits became labeled if the enzyme was dissociated with dodecyl sulfate before being exposed to the labeling reagent.     

Localization of Hydrogen Peroxide Production in Pisum sativum L. Using Epi-Polarization Microscopy to Follow Cerium Perhydroxide Deposition

Cerium is becoming an increasingly popular reagent for histochemical localization of oxidases and phosphatases because it combines directly with reaction products to form fine precipitates of electron-dense materials that can be easily detected using transmission electron microscopy or laser confocal scanning microscopy. We used epi-polarization microscopy to detect cerium perhydroxide deposits formed when H2O2 was produced by diamine oxidase in pea (Pisum sativum L.) epicotyls exposed to exogenous putrescine. Diamine oxidase activity was abundant in cortical cell walls but showed little, if any, association with vascular tissues. Maps of cerium deposition generated using scanning electron microscopy/x-ray microanalysis verified these observations. This study demonstrates the use of epi-polarization microscopy to follow cerium deposition, and the ready accessibility of this microscopy technique should facilitate more widespread use of cerium for plant histochemistry and cytochemistry.     

Reaction of cimetidine with Fenton reagent. A biomimetic model for the mixed-function oxidase drug metabolism

Cimetidine sulfoxide, N-desmethylcimetidine, N-desmethylcimetidine sulfoxide, cimetidine guanylurea, and the 5-hydroxymethylimidazole derivative of cimetidine sulfoxide were isolated from the reaction of cimetidine with Fenton reagent. The product distribution from the reaction of cimetidine with Fenton reagent clearly simulates the metabolism of cimetidine by the mixed-function oxidase.     

Chemical modifications of D-amino acid oxidase. Evidence for active site histidine, tyrosine, and arginine residues

1. D-amino acid oxidase is inactivated by reaction with a low molar excess of dansyl chloride at pH 6.6, with complete inactivation accompanied by incorporation of 1.7 dansyl residues per mol of enzyme-bound flavin. The presence of benzoate, a potent competitive inhibitor, protects substantially against inactivation. Evidence is presented that the inactivation is due to dansylation of an active site histidine residue. Reactivation may be obtained by incubation with hydroxylamine. Diethylpyrocarbonate also inactivates the enzyme and modifies the labeling pattern with dansyl chloride. 2. Butanedione in the presence of borate reacts rapidly to inactivate D-amino acid oxidase. Reactivation is obtained spontaneously on removal of borate, implicating reaction of butanedione with an active site arginine residue. 3. Fluorodinitrobenzene appears to behave as an active site-directed reagent when mixed with D-amino acid oxidase at pH 7.4. Complete inactivation is obtained with incorporation of 2.0 dinitrophenyl residues per mol of enzyme-bound flavin. Again benzoate protects against inactivation; only one dinitrophenyl residue is incorporated in the presence of benzoate. The active site residue attacked by fluorodinitrobenzene has been identified as tyrosine.     

A suitable method for localization of IAA oxidase

The method for histochemical detection of IAA oxidase proposed by Sagi was tested, based on colour reaction of natural products of IAA oxidation with Ehrlich-like reagent.     

Microbial metabolism of aryl sulphonates a re-assessment of colorimetric methods for the determination of sulphite and their use in measuring desulphonation of aryl and alkylbenzene sulphonates.

The reaction of 2,4-dinitroanilinomaleimide with sulphite which has been claimed as the basis of a suitable colorimetric assay for the anion was carefully re-examined. The sulphite-imide addition product provides a suitable and specific qualitative test for sulphite after separation by paper chromatography but the method as previously used is probably measuring the hydrolysis of the imide to 2,4-dinitroanilinomaleamic acid and cannot be used for sulphite determination either colorimetrically or in kinetic assays. A new colorimetric method for the determination of sulphite based on its reaction with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) is described and compared for sensitivity with the p-rosaniline-HCHO method. Both methods were used to show the formation of sulphite as the initial product of arylsulphonate metabolism by bacteria. The failure to find sulphite in similar cultures of a third organism was attributed to the very high activities of sulphite oxidase found in extracts. The Ellman reagent was examined as the basis of an indicator medium for the detection of sulphite-excreting colonies.     

Effects of hypoxia and reoxygenation on the conversion of xanthine dehydrogenase to oxidase in Chinese hamster V79 cells

The effects of hypoxia and reoxygenation on the conversion of xanthine dehydrogenase to the free radical-producing xanthine oxidase in Chinese hamster V79 cells have been investigated using a newly developed fluorimetric enzyme assay. Hypoxia caused an increase in xanthine oxidase activity from 25% to 80% of the total activity of xanthine oxidase and dehydrogenase. The ratio returned to normal levels within 24 h of aerobic incubation. Hypoxia caused the release of xanthine oxidase in the medium of V79 cells and an increase in total protein concentration in the medium. There was an early change induced in lipid peroxidation markers and this was inhibited by allopurinol. The effects of glucose deprivation and calcium blockers were also investigated. Fura-2 AM was found to interact with V79 cells, making it impossible to determine intracellular calcium levels in V79 cells by this reagent.