Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications.

A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose Cl 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose.

Monoclonal antibodies block murine IL-4 receptor function.

IL-4 is a cytokine which can induce B-lymphocyte proliferation, increase cell-surface Ia expression, and induce some activated B cells to differentiate and begin to secrete IgE. IL-4 binds specifically to a cell-surface receptor (IL-4R) on cells from a variety of lineages including T and B cells. In general both primary cells and in vitro cell lines express less than 5000 receptors per cell. Utilizing a subclone of the cytotoxic T cell line CTLL-2 expressing a high level of IL-4R, mAb against the murine IL-4R were prepared. Two mAb have been identified which have different properties. These antibodies, designated M1 and M2, recognize sequences specific to the murine IL-4R. Immunoprecipitation studies with M1 and M2 on CTLL-2 cells have identified the receptor as a Mr = 145,000 cell-surface protein. Similar results have been obtained with the recently isolated full length murine IL-4R cDNA expressed in COS-7 cells. In addition the antibodies are capable of inhibiting IL-4 binding. One antibody, M1, is also a potent inhibitor of IL-4-induced proliferation. These antibodies will be useful in dissecting a wide array of activities attributed to IL-4.

Bioluminescent assay of D-3-hydroxybutyrate in serum.

An enzymatic assay of D-3-hydroxybutyrate in which the hydroxybutyrate dehydrogenase reaction is coupled to the bacterial oxidoreductase-luciferase system is described. The bioluminescent assay is based on either, end-point, or on initial velocity measurements. This simple and rapid assay requires a single serum sample of 10 microliters. Its linear range covers two orders of magnitude from 10(-6) mol/l upwards. This assay is suitable for the routine determination of D-3-hydroxybutyrate in human blood with good accuracy.

Bioluminescent assay of NADPH-dependent isocitrate dehydrogenase and its substrates and cofactors.

A bioluminescent assay for NADPH-dependent isocitrate dehydrogenase and for its substrates and cofactors was developed. The method is based on continuous NADPH monitoring in the reaction. The linear range of the assay for the enzyme activity is from 0.05 U/liter to 30 U/liter. It is about 300 times more sensitive than the corresponding spectrophotometric assay at 340 nm. Good correlation exists between both assays. Isocitrate, NADP, manganese, and magnesium can be measured at picomole levels. The applicability of the assays to serum analysis is discussed.

Effects of ligands and pH on the reactions of aspartate aminotransferase with aminooxyacetate and hydroxylamine.

The bimolecular association and first-order dissociation rate constants for the reactions of aminooxyacetate and hydroxylamine with the cytosolic aspartate aminotransferase (EC 2.6.1.1) of pig heart were estimated from pH 4.8 to pH 9.5. The acidic form of the enzyme (pK = 6.3) was more reactive than the unprotonated enzyme, but the rates of breakdown of the complexes were not affected by pH. The equilibrium dissociation constants, which were of the order of magnitude of 10(-7) M, were thus lowest at acidic pH values. Aminooxyacetate and hydroxylamine reacted similarly, with the former being more reactive. Glutarate and acetate inhibited the rates of formation and dissociation but had no effect on the overall equilibrium constants between enzyme and inhibitor. On the other hand, the substrate analog erythro-beta-hydroxy-L-aspartate, which forms a complex with the enzyme prosthetic group, acted competitively by preventing the inhibitors from reacting. The rate constants for formation of complexes with free pyridoxal phosphate were much less than those for the enzyme and, unlike the enzyme, hydroxylamine was more reactive than aminooxyacetate.

The effect of luciferase and NADH:FMN oxidoreductase concentrations on the light kinetics of bacterial bioluminescence.

The effects of NADH:FMN oxidoreductase and luciferase concentrations on the light kinetics of the bacterial bioluminescent reaction were investigated. Light emission with low decay rates was obtained by regulating the conversion of NADH to NAD+ by controlling oxidoreductase activity. Constant light emission can be obtained when the oxidoreductase activity is below 2.5 U/1 in the assay system. The luciferase concentration affects the light intensity but it has no effect on the decay rate of light emission. The substrate decanal and the end-products NAD+ and capric acid had no effect on the light kinetics. The Michaelis constants of bacterial luciferase for FMNH2 and decanal were 3 X 10(-6) M and 8 X 10(-7) M, respectively, and those of oxidoreductase for FMN and NADH were 6.1 X 10(-6) M and 1.6 X 10(-5) M, respectively.

Simultaneous extraction and combined bioluminescent assay of NAD+ and NADH.

A new method for extracting pyridine nucleotides from tissue samples at room temperature that allows the simultaneous extraction of both the oxidized and reduced nucleotide when using a 70% buffered ethanol solution as the extractant has been developed. The extraction efficiencies for NAD+ and NADH were 91 and 102%, respectively. The extraction method was followed by a combined bioluminescent assay of both nucleotides. A bacterial bioluminescent system, which included luciferase and low levels of a NADH-specific oxidoreductase, was used to produce a constant light intensity directly proportional to the amount of NADH in the tissue extract sample. When the NADH had been measured, the NAD+ present in the extract was enzymatically converted to NADH by the addition of alcohol dehydrogenase, after which the second increase in light level was recorded. The sensitivity of the bioluminescent assay presented here is 5 X 10(-14) mol NADH or NAD+ per assay.

Metabolic effects of a low-magnesium diet in pigs.

1. Pigs were fed on semi-purified food. The magnesium content of the experimental diet was 1.0 and that of the control diet 1.9 g/kg. 2. In the low-Mg group serum triglycerides and blood lactate values were increased and base excess and standard bicarbonate values were decreased, indicating metabolic acidosis. 3. A significant positive dependence was found between blood pH and serum Mg:Ca value as well as between blood pH and serum Mg, and also between body temperature and blood lactate values in the low-Mg group. None of these dependences was significant in the control group with magesium acetate supplementation in the food. 4. No specific histopathological changes were found in heart, liver or kidney of the experimental animals. 5. The results indicate that an increase in serum triglycerides does not need to depend on the amount of quality of food fat. 6. The present study shows that, in pigs, a low-Mg diet may cause metabolic disturbances in instances with the food Mg content is distinctly higher than the normal values recommended by the (UK) Agricultural Research Council (1966) and (US) National Research Council (1968).

D-Amino acids of the amino acid pool and occurrence of racemase and D-amino acid oxidase activities in Escherichia coli B.

Less than 20% of the amino acid content of the amino acid pool of Escherichia coli B exists in the D-form. Alanine, glutamic acid, and valine were shown by gas- chromatography to be partially in the D-form. Only D-alanine was formed by racemization in the crude extract of this organism. Alanine racemase was easily released from the membranes or vesicles but D-alanine oxidase activity remained firmly bound to the membrane. Most protein amino acids stimulated proline uptake into the vesicles, and the oxidative deamination activities were verified by the proline uptake stimulating amino acids. It is concluded that the obligatory pathway of L-amino acid--D-amino acid--oxo acid which exists in the oxidation of L-alanine does not exist with other L-amino acids. It is likely that other D-amino acids in the pool are formed in the presence of D-amino acid oxidase or D-amino acid aminotransferase.

The effect of culture age, chloramphenicol and B6 inhibitors on intra- and extracellular keto and amino acids of Escherichia coli B.

Keto acids and free amino acids were assayed in the cells and the medium of Escherichia coli B growing in the presence of chloramphenicol, cycloserines, aminooxyacetate, and limiting nitrogen source. Under these growth-limiting conditions the cells accumulated ketoglutarate and 'ketovaline' but no other keto acids. In all experiments only ketoglutarate, pyruvate, and 'ketovaline' were found in the medium. Amino acids are released into the medium in the early phases of growth and the composition of the extracellular amino acids is similar to that of the amino acid pool. The concentrations of free amino acids were 10-3-10-4 times higher in the cell than in the medium. The internal pool composition is fixed under all growth-limiting conditions. In the presence of the drugs the cells release amino acids into the medium.

D-alanine oxidase form Escherichia coli: localization and induction by L-alanine.

Dialyzed membranes of Escherichia coli prepared by an ethylenediaminetetraacetic acid-lysozyme method catalyze the oxidation of both l-alanine and d-alanine. The specific activities for the oxidations of both d-alanine and l-alanine are increased fivefold when the cells are grown in the presence of either l-alanine or dl-alanine, but are increased only slightly when grown in the presence of d-alanine. In the dl-alanine-induced system, the specific activities for the oxidations of some other d-amino acids are also raised. dl-alanine also induces two other alanine catabolizing enzymes, alanine dehydrogenase and alanine-glutamate aminotransferase which are found in the "soluble" fraction of lysozyme-treated cells. The oxidations of both l-alanine and d-alanine were associated with the membranes of induced cells. After the membranes were disintegrated by sonic treatment, both l-alanine and d-alanine oxidation catalysts sedimented in a sucrose density gradient together with d-lactate and l-lactate dehydrogenases, apparently as a single multienzyme complex.

D-alanine oxidase from Escherichia coli: participation in the oxidation of L-alanine.

Cell wall-membrane preparations of Escherichia coli, prepared by the ethylenediaminetetraacetic acid-lysozyme method, contain enzymes which catalyze the oxidation of d-alanine and, to a lesser extent, l-alanine into pyruvate and ammonia without the formation of hydrogen peroxide. The kinetic parameters were (i) pH optima of 8.3 to 8.4 for l- and d-alanine and (ii) a K(m) value of 6.6 +/- 0.2 mM for d-alanine. Several coenzymes were without effect when added to the reaction mixture. The participation of d-alanine oxidase in the oxidation of l-alanine was demonstrated. The evidence is based on (i) results of cellular fractionation; (ii) labeling experiments; (iii) inhibition studies with aminooxyacetate and cycloserine; (iv) denaturation experiments; and (v) demonstration of the presence of an active racemase.