A simple radiometric assay for estradiol 2-hydroxylase activity

[2-³H]Estradiol was synthesized from 2-iodoestradiol by reduction with sodium borotritiride in the presence of palladium chloride as a catalyst. When the labeled substrate was incubated with the rat liver and kidney microsomes, the tritium label was liberated quantitatively depending upon the 2-hydroxylase activity. Tritiated water produced in the incubation medium was recovered as a satisfactory rate by passage through a column of Amberlite XAD-2 resin without any interference due to the labeled substrate. The present method for the assay of estradiol 2-hydroxylase activity was found to be simple, reliable, and sensitive (detection limit:29 pmol of 2-hydroxyestradiol).     

A rapid radioenzymatic assay for dopamine and N-acetyldopamine.

Dopamine (DA) was measured in various tissue extracts as [³H]methoxy-N-acetyldopamine after incubation with two partially purified enzymes, catechol-O-methyl transferase (EC 2.1.1.1) and N-acetyltransferase (EC 2.3.1.5), in the presence of [³H]adenosylmethionine and acetyl-CoA. This product can be separated quantitatively from labeled products of norepinephrine and epinephrine by solvent extraction. N-Acetyl-DA can be assayed by omitting the acetylating system from the incubation mixture. The procedure is rapid, convenient for processing large numbers of samples, and has a sensitivity of approximately 0.1 pmol. It has been used to measure DA in ganglia and in individual neurons from gastropod mollusks.     

A new,fast, and sensitive assay for NADH-ferredoxin oxidoreductase detection in clostridia

A new, simple and very sensitive assay for NADH-ferredoxin or flavodoxin reductase activity is described. The assay is based on the nonenzymatic reduction of the metronidazole by ferredoxin or flavodoxin. In the presence of NADH, ferredoxin or flavodoxin and cell-free extract of clostridia, no metronidazole reduction is observed; the reaction occurs only if acetyl-CoA is added to the reaction mixture. Metronidazole reduction is quantitated by the spectrophotometric measurement at 320 nm. In this assay the change in absorbance is linearly related to the amount of clostridial extract for concentration of 0.1 to 0.8 mg/ml and to the flavodoxin or ferredoxin for concentrations of 0.5 to 8 nmol/ml.     

A new method for the detection and assay of α-1,3-glucanases

A colorimetric method that is specific for the assay of α-1,3-glucanases is presented. The enzyme substrate consists of Cibacron Blue F3GA complexed with a dextranase-treated streptococcal glucan. The method is especially convenient for tests involving large numbers of samples, and can be adapted to quantitative as well as qualitative applications. The assay is sufficiently sensitive for screening bacterial samples as potential sources of α-1,3-glucanase.     

A quantitative assay for ciliate chemotaxis

A quantitative bioassay for ciliate chemotaxis based on the capillary principle is described using Tetrahymena thermophila as test organism. The attractant-containing assay tube designed for the bioassay attracts up to 4 X 10(4) cells in 2 h which makes electronic cell counting of the chemotactic response feasible. The attractants used are solutions of proteose peptone and yeast extract which also are growth media for this organism.     

A sensitive fluorimetric rate assay for biotinidase using a new derivative of biotin, biotinyl-6-aminoquinoline

The previously reported method for the estimation of biotinidase (EC 3.5.1.12) is an endpoint colorimetric assay based on the hydrolysis of biotinyl-4-aminobenzoate, followed by diazotization, and is not suitable for our studies of biotinidase. A fluorimetric rate assay of biotinidase which uses a newly synthesized derivative biotinyl-6-aminoquinoline is described here.     

Microsomal oxidation of thiobenzamide. A photometric assay for the flavin-containing monooxygenase

The hepatotoxin thiobenzamide is S-oxidized by the microsomal flavin-containing monooxygenase (MFMO)¹ in liver, lung, and kidney of rabbit, mouse and rat. Its oxidation is accompanied by a large spectral shift which can be used as the basis of a simple convenient photometric assay for the MFMO system.     

Superoxide dismutase: a photochemical augmentation assay.

Cell envelope vesicles containing bacteriorhodopsin, prepared from Halobacterium halobium, have previously been shown to accumulate glutamate to high concentration gradients when illuminated. This active transport is energized by a sodium gradient (Na_out⁺ ? Na_in⁺), which arises from Na⁺-efflux coupled to the light-induced H⁺-gradient. The oxidation of dimethyl phenylenediamine (DPD) by the vesicles also can drive uphill glutamate transport, and such transport is inhibited by KCN, azide, ionophores, or uncouplers. K_T for glutamate is 1.4 × 10^?7m under these conditions, as compared to 1.3 × 10^?7m for light-induced transport. The respiration-induced transport of glutamate is dependent on high Na⁺ concentrations on the vesicle exterior and requires low Na⁺ concentrations in the interior. When Na⁺ of increasing concentrations is included in the vesicles, transport proceeds with increasing lags, similarly to the case of light-driven transport. In vesicles to which DPD is added first, and then KCN at increasing time intervals (5 to 15 min), glutamate transport occurs after the addition of KCN, with increasing rates, even though respiration is inhibited. This indicates that the energy generated by DPD-oxidation is conserved over several minutes. These results suggest that in the case of respiration-dependent glutamate transport the translocation is also driven by a Na⁺-gradient; thus, there is a single glutamate transport system independent of the source of energy. The generation of such an Na⁺-gradient during DPD-oxidation implies that the respiration component involved, cytochrome oxidase, is functionally equivalent to bacteriorhodopsin, which acts as a proton pump.     

A unique activity assay for carboxypeptidase A in human serum

Measurement of carboxypeptidase A, one of the pancreatic proteolytic enzymes, in human serum is made possible by a combination of affinity chromatography to isolate and concentrate the enzyme followed by monitoring activity spectrophotometrically with a high-turnover peptide substrate. Concentrations of enzyme in the nanogram-per-milliliter range can be determined with high precision and reliability. Initial clinical application of this method demonstrates no detectable activity in serum from normal individuals, but the enzyme is present in the sera of individuals with pancreatitis.     

Homoserine kinase of Escherichia coli: kinetic mechanism and inhibition by L-aspartate semialdehyde

The kinetic mechanism of homoserine kinase, purified to homogeneity from Escherichia coli, was examined by initial velocity techniques at pH 7.6. Whereas ATP displayed normal Michaelis-Menten saturation kinetics (Km = 0.2 mM), L-homoserine showed hyperbolic saturation kinetics only up to a concentration of 0.75 mM (Km = 0.15 mM). Above this concentration, L-homoserine caused marked but partial inhibition (Ki approximately 2 mM). The kinetic data indicated that the addition of substrates to homoserine kinase occurs by a preferred order random mechanism, with ATP preferentially binding before L-homoserine. When the ATP concentration was varied at several fixed inhibitory concentrations of L-homoserine, the resulting inhibition pattern indicated hyperbolic mixed inhibition. This suggested a second binding site for L-homoserine. L-Aspartate semialdehyde, an amino acid analog of L-homoserine, proved to be an alternative substrate of homoserine kinase (Km = 0.68 mM), and was subsequently used as a probe of its kinetic mechanism. In aqueous solution, at pH 7.5, this analog was found to exist predominantly (ca 85%) as its hydrated species. When examined as an inhibitor of the physiological reaction, L-aspartate semialdehyde showed mixed inhibition versus both L-homoserine and ATP. Although the pH profiles for the binding of L-homoserine as a substrate (Km) and as an inhibitor (Ki) were identical, the kinetic data were best fit to a two-site model, with separate catalytic and inhibitory sites for L-homoserine.     

A spectrophotometric method for the assay of phospholipase D activity

Phosphatidylcholine phosphatidohydrolase (EC 3.1.4.4, phospholipase D) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. We have developed a spectrophotometric assay for phospholipase D using choline kinase, pyruvate kinase, and lactate dehydrogenase to couple the release of choline with the oxidation of NADH. The assay was linear both with time and with enzyme concentration. The assay should prove useful for continuous monitoring of enzyme activity, determination of initial rates of reaction, and detailed kinetic studies of phospholipase D. The method is limited to analysis of purified preparations of phospholipase D lacking competing activities to the coupled system.     

A sensitive cyclic nucleotide phosphodiesterase assay for transient enzyme kinetics

A rapid and sensitive method was developed for the quantitative determination of alpha-tocopherol in tissues and plasma of rats and mice. Tissue and plasma were extracted in acetone and chromatographed on a reverse-phase C18 column with 2% water in methanol. Fluorescence and ultraviolet detection were used for tissue and plasma alpha-tocopherol levels, respectively. Extraction of tissues and plasma was found to be more complete in acetone than in other solvent systems analyzed. The average recovery of alpha-tocopherol added to tissue samples was 97%. As little as 0.1 g of tissue or 0.1 ml plasma can be accurately used for analysis. The method is sensitive to 0.05 micrograms alpha-tocopherol/g tissue.     

A direct fluorometric assay of benzo[a]pyrene hydroxylase.

A technique to measure the activity of pyruvate carboxylase spectrophotometrically in crude liver homogenates is described. The assay is based on the transformation of oxaloacetate, which is formed during the carboxylation reaction, into citrate in the presence of excess acetyl CoA and citrate synthase. After removal of pyruvate with KBH₄ and of protein with HClO₄, citrate is cleaved with citrate lyase into oxaloacetate and acetate, and oxaloacetate then is measured spectrophotometrically. Optimal concentrations of pyruvate, Mg²⁺, ATP, and KHCO₃ for the carboxylation reaction and the V_max were in good correlation with the data found by others using [¹⁴C]pyruvate.     

A radiometric assay for methyl red azoreductase DT-diaphorase [EC 1.6.99.2]

A rapid radiometric assay for measuring oxygen insensitive methyl red azoreductase has been developed. [¹⁴C]Methyl red was synthesized from [U-¹⁴C]aniline; the final product had a radiochemical purity of ≥98%. [¹⁴C]Methyl red was then used as the substrate to assay azoreductase activity using a modification of a previously published procedure. Results obtained by the radiometric assay were comparable to those obtained using the fluorescent procedure. The radiometric assay is quick, simple, and reliable. Methyl red azoreductase has been shown to be identical with DT-diaphorase [EC 1.6.99.2]. The assay described can therefore be used to assay DT-diaphorase activity and does not suffer from the limitations, such as lack of specificity and low sensitivity, usually associated with DT-diaphorase assays.     

The effect of halides on the equilibrium and reactivity of aspartate aminotransferase.

For the pork heart, extramitochondrial aspartate aminotransferase (EC 2.6.1.1), the “half-reaction” equilibrium, amino acid + phosphopyridoxal enzyme ? keto acid + phosphopyridoxamine enzyme, is displaced in favor of the phosphopyridoxamine enzyme by the addition of halide ions. The order of effectiveness is I^? > Br^? > Cl^? > F^?. A kinetic analysis of this equilibrium with alanine and pyruvate as substrates showed that halide ions (0.01–0.1 m) both increase the rate of the forward reaction and decrease the rate of the reverse reaction. Chloride ions decrease the rate of the reverse reaction by competitively inhibiting the formation of an intermediate enzyme-pyruvate complex. The rate of the forward reaction is proportional to the alanine concentration up to 0.5 m alanine, indicating that the initial combination of alanine with the enzyme is the rate-limiting step in this direction. The activation by anions must therefore involve the initial binding of the substrates to the enzyme. Chloride ions also cause a marked activation of the enzyme in the presence of glutarate by displacing the inhibitory glutarate from the enzyme. These results indicate that some enzyme activations may be due to relieving a preexisting inhibition by ligand substitution reactions. The finding that aspartate aminotransferase has an anion-sensitive “half-reaction” equilibrium, or redox potential, suggests that transaminases may function in both active and passive transport of anions across membranes.     

A simple method for quantitative, semiquantitative, and qualitative assay of protein.

Aqueous cesium trichloroacetate permits the buoyant resolution of various RNAs and also of DNA at room temperature and neutral pH. Precipitate formation does not occur, under either native or denaturing conditions. The compositional buoyant density gradient was determined, and the buoyant densities of a variety of RNAs are presented. The buoyant densities increase in the order protein < DNA ? duplex RNA ? single-stranded RNA.     

Immunocapillarymigration--a new method for immunochemical quantitation.

A new simple and rapid method for immunochemical quantitation called immunocapillarymigration is described. It is based upon the attachment of antibodies to a porous insoluble support and the subsequent capillarymigration of the antigen-containing solution in the porous support. The migration of the antigen solute is specifically delayed in comparison to the migration of the solvent and other solutes in the process and the relative delay decreases with increasing antigen concentration. When applied to the quantitation of transferrin in human plasma, immunocapillarymigration gave results which agreed with those obtained by single radial immunodiffusion.     

The hemolytic plaque assay: theory for finite layers.

We extend the mathematical theory of hemolytic plaque growth to include plaques produced by cells secreting antibodies in layers of finite thickness. Previous theories have assumed that the layer was either two-dimensional or of infinite thickness. By using the method of images we derive an equation for the plaque radius as a function of time for layers of any thickness. We show that at short times and at long times the equation reduces to the appropriate infinite three-dimensional and two-dimensional limiting forms, and obtain expressions for estimating the range of times for which these limiting results are valid. For the liquid monolayer technique we obtain a new limiting result. The equation for the plaque radius is a transcendental equation which we solve numerically for a number of cases of interest. These results illustrate a variety of different features of plaque growth associated with the finite thickness of the layer. Experimental studies are usually carried out in layers whose thicknesses are not standardized. In the assays commonly used the thickness h can vary more than six hundred fold, i.e. 1 × 10^?3 cm ?h? 6.5 × 10^?1 cm. Such variation in h will cause widely different kinetics of plaque growth. For typical plaque experiments of one hour duration the two-dimensional limit is valid when h ? 3 × 10^?3 cm while the infinite thickness limit is valid when h? 10^?1 cm. For thicknesses in between these values the finite layer results must be used.