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 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 permeabilized-cell assay for transaminase B activity in Escherichia coli K-12

The assay for transaminase B (EC 2.6.1.6) activity, developed by D. E. Duggan and J. A. Wechsler (1973, Anal. Biochem.51, 67–79) has been modified to allow for the measurement of activity in Escherichia coli cells made permeable by cetyltrimethylammonium bromide (CETAB). A concentration of 10 mg% CETAB was found to be most effective in treating the cells without having a significant effect on transaminase B activity. Extraction of the dinitrophenylhydrazone of 2-oxoisovalerate by toluene was not affected by the CETAB treatment. We further report that the Na₂CO₃ extraction step is not required to measure color formed by the dinitrophenylhydrazone of 2-oxoisovalerate. This CETAB-treated cell assay is accurate to study transaminase B activity through most of the logarithmic phase of growth of Escherichia coli.     

A recommended procedure for the estimation of bovine testicular hyaluronidase in the presence of human serum

A procedure is described for the assay of bovine testicular hyaluronidase in human blood following intravenous administration of the enzyme. Inhibition of hyaluronidase by the reported nonspecific serum inhibitor is minimal. However, the presence of human serum does alter the pH profile of hyaluronidase and enhances the activity of the enzyme at low pH values. Preliminary data indicates that the effects caused by serum on the pH optimum and activity of the enzyme are largely associated with the albumin fraction and are not due to the presence of endogenous serum hyaluronidase. The activation effect is not specific for any particular blood type and is independent of whether serum or citrated plasma is used. A similar effect to that of serum on hyaluronidase activity is produced by different buffer mixtures or increased NaCl concentration. It is recommended that bovine testicular hyaluronidase be measured at pH 4.0 in 0.1 m sodium citrate buffer containing 0.15 m NaCl as under these conditions the addition of human serum or citrated plasma does not alter the pH optimum of the enzyme. These recommendations necessitate certain modifications of the reducing N-acetylhexosamine assay method of Reissig et al. (J. L. Reissig, J. L. Strominger, and L. F. Leloir, 1955, J. Biol. Chem.217, 959–966).     

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.     

New human liver alcohol dehydrogenase forms with unique kinetic characteristics

All adult and infant human liver homogenates studied thus far show two previously unreported forms of alcohol dehydrogenase on starch gel electrophoresis. Under the conditions employed, these forms migrate toward the anode and readily stain for pentanol but virtually not for ethanol oxidizing activity. In contrast, all human ADH isoenzymes identified previously are cathodic and react equally well with either substrate. These new ADH forms have been separated from the other known ones by DEAE-cellulose chromatography and are then purified on Agarose-hexane-AMP. Although the physical characteristics of the new anodic ADH forms are similar to those of the known human ADH isoenzymes, the former are not inhibited by 12 mM 4-methyl pyrazole, oxidize ethanol very poorly and appear to prefer longer chain alcohols as substrates.     

A sensitive and simple assay of starch synthase activity with pyruvate kinase and luciferase

A coupled recording assay of cyclic AMP phosphodiesterase activity at and below 1 μm is presented. The method is especially useful in preparative work where speed is more important than high accuracy, but results agree well with a more accurate radioactive assay. Attention is drawn to the fact that, because the product of the coupled reaction, ATP, is also an intermediate, its instantaneous rate of formation can exceed the rate of the first step under certain conditions.     

Inhibitory factor of phospholipase A2 in normal human serum

Normal human serum and plasma were shown to contain a factor inhibiting phospholipase A2. This factor has been separated from human serum and plasma by chromatography on a Blue-Ultrogel column and was eluted by tris-HCl buffer (pH 7.2); the proteins eluted by 1 M NaCl-tris HCl buffer exhibited phospholipase A2 activity. This activity was abolished when the inhibitory factor was added to proteins possessing such activity. The inhibitory factor was not dialysable, sensitive to both heat and trypsin treatment, suggesting that it is a protein. In vitro, the same factor inhibited phospholipase A2 rat serum.     

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 new assay for L-aspartate:2-oxoglutarate aminotransferase.

A new enzymatic assay for aspartate aminotransferase is presented. The 2-oxoglutarate formed in transamination between l-glutamate and oxalacetate was determined in a system coupled with hydroxyglutarate dehydrogenase and NADH by following a decrease in absorbance at 340 nm. The method allowed accurate determination of the initial velocity of the reaction, which was proportional to the enzyme concentration. The Michaelis constants of pig heart cytosolic aspartate aminotransferase for l-glutamate and oxalacetate and the amino acceptor specificity using l-glutamate as an amino donor were determined. The method was applicable to the determination of the enzyme activity in various materials including rat serum and bacterial crude extract.     

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 more sensitive assay for histone deacetylase

The new assay uses as substrate a peptide derived from the amino terminal domain of calf histone H4. The peptide contains all the lysines that are acetylated in H4 in vivo and these lysines are specifically labeled in vitro with acetic anhydride to a high specific activity. This substrate allows histone deacetylase activity to be measured economically and with high sensitivity either with pure enzyme or with crude extracts.     

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.     

Cimetidine assay in human plasma by liquid chromatography

An assay for the determination of cimetidine in human plasma is described. Cimetidine was extracted from alkalized plasma with ethyl acetate, washed once over hydrochloric acid, re-extracted into ethyl acetate, and the organic phase was evaporated to dryness. The residue was dissolved in ethanol and injected into a liquid chromatograph.In vitro sulphoxidation was found to occur in whole blood, for which reason the assay was performed in plasma. The accuracy of the method was found to be within 3% and the lower limit for sensitivity was demonstrated to be 0.1 mg/l using 750 μl plasma.Five volunteers received 1 g cimetidine perorally per day given in four doses with various intervals. Blood samples were drawn hourly, five dose intervals over two days. The average minimum concentration of plasma cimetidine was found to correlate significantly with the mean value of the area under the time/concentration curve over a period of three dose intervals (r = 0.96).     

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.     

DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin

Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.     

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.     

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.