A simple assay of aryl hydrocarbon hydroxylase in cultured human lymphocytes

A novel technique is described for assay of aryl hydrocarbon hydroxylase in cultured human lymphocytes. The sensitivity is equal to that of previous methods, but this method requires fewer manipulations. One million lymphocytes are incubated for one hour with 2 micrograms of benzo(a)pyrene in a glass cuvette. The reaction is stopped by addition of neutral formalin and the cell suspension is alkalinized with NaOH. Fluorescence intensity of the suspension is measured with excitation at 465 nm and emission at 520 nm.     

A simple and sensitive fluorometric assay for tyrosine hydroxylase.

A simple, rapid, and sensitive fluorometric assay for measuring the activity of tyrosine hydroxylase is described. The enzyme activity is detected by converting tyrosine to 3,4-dihydroxyphenylalanine (dopa), which is then subjected to conversion to the highly fluorescent product by the trihydroxyindole method. The assay method is very reproducible, more sensitive than a radiochemical method for the determination of tyrosine hydroxylase activity using the isolation of [³H]water commonly used, and linear from 0.2 to 12 nmol of dopa. The method should be applicable for the assay of the enzyme with a wide range of activity.     

A simple and sensitive fluorescence assay for tyrosine hydroxylase activity.

A simple and sensitive fluorescence assay for tyrosine hydroxylase (TH) activity wasdevised based on rapid isolation of enzymatically formed dopa by a double-column procedure fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminum oxide). Interfering substances were removed by the first Amberlite CG-50 column. Dopa was adsorbed on the second aluminum oxide column, then eluted with 0.5 m acetic acid, and assayed by the highly sensitive hydroxyindole method of Johnson et al. (1973, Anal. Biochem.54, 129–136). The standard incubation mixture (total volume, 0.5 ml) contained 0.3 mm l-tyrosine, 1.0 mm 6-methyl-5,6,7,8-tetrahydropterin, 100 mm mercaptoethanol, and an optimal concentration of ferrous ion. d-Tyrosine was used for the blank incubation. Recovery of dopa added to the standard incubation mixture as internal standard was about 70% and was reproducible. The fluorescence characteristics of the product were the same as those of authentic dopa. Blank fluorescence was very low even with crude enzyme preparations. The limit of sensitivity was 100 pmol of dopa formed, which is close to the sensitivity of radioassays. TH activity in homogenates of rat brain stem or human putamen could be assayed in the standard incubation system containing ferrous ion. The validity of this fluorescence assay has been shown by the agreement between the values obtained by this method and by radioassay using l-[U-¹⁴C]tyrosine as substrate. In the rapid assay procedure dopa in the eluate from aluminum oxide was assayed directly by native fluorescence. Although the sensitivity was about 1 nmol, this rapid assay procedure was found to be particularly useful for the purification of TH.     

Radioactive assay for aryl hydrocarbon hydroxylase. Improved method and biological importance

By addition of two volumes of a 1M aqueous KOH/dimethylsulfoxide ($\text{15}\text{85}$; v/v) mixture to the enzymatic incubation medium, it is possible to selectively extract the unmetabolized benzo(a)pyrene in hexane. Therefore, the radio-activity remaining in the water phase corresponds to all the in vitro synthesized metabolites. This isotopic method is very sensitive (2 × 10^?11 moles) and is almost insensitive to the room lighting. The aryl hydrocarbon hydroxylase activities found with this method are 2,3 and 10 times higher in the liver, lung and kidney respectively compared to those obtained with the fluorimetric method.     

A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB) lipoproteins. Its activity is classically measured by incubating purified MTP or cellular homogenates with donor vesicles containing radiolabeled lipids, precipitating the donor vesicles, and measuring the radioactivity transferred to acceptor vesicles. Here, we describe a simple, rapid, and sensitive fluorescence assay for MTP. In this assay, purified MTP or cellular homogenates are incubated with small unilamellar donor vesicles containing quenched fluorescent lipids (triacylglycerols, cholesteryl esters, and phospholipids) and different types of acceptor vesicles made up of phosphatidylcholine or phosphatidylcholine and triacylglycerols. Increases in fluorescence attributable to MTP-mediated lipid transfer are measured after 30 min. MTP's lipid transfer activity could be assayed using apoB lipoproteins but not with high density lipoproteins as acceptors. The assay was used to measure MTP activity in cell and tissue homogenates. Furthermore, the assay was useful in studying the inhibition of the cellular as well as purified MTP by its antagonists. This new method is amenable to automation and can be easily adopted for large-scale, high-throughput screening.     

A specific kinetic assay for phenylalanine hydroxylase

An assay procedure is given which is speedy, accurate, and specific, permitting direct recording of velocities, and obviating the use of reagents other than those necessary for the enzymatic reaction itself. The method is suitable for the study of enzyme mechanism and inhibition and also offers distinct advantages when used for other purposes, e.g., assay during purification of enzymes or for measurement of phenylalanine hydroxylase activity in the liver of hyperphenylalaninemics.The method is based on the phenylalanine-dependent change in absorbance of the tetrahydropteridine cofactor as it is oxidized to the dihydro form. The reaction rate measured by this procedure is linear over a wide range of enzyme concentration. The K_m and V for both tetrahydropteridine and for phenylalanine were the same as the values determined by the old procedure. Measurement of the stoichiometry of the reaction showed that one dihydropteridine is formed per tyrosine formed, or per DPNH consumed. The rate of reaction was identical to that measured by a coupled assay using DPNH and purified dihydropteridine reductase.     

A sensitive radioisotope assay for pectin methyl esterase activity

The preparation of polygalacturonic acid [¹⁴C]-labeled methyl ester (pectic acid ester) is described. This labeled polysaccharide is employed as the substrate in a simple, sensitive, and rapid assay procedure for measuring pectin methyl esterase activity.     

The utility of the microsomal 4-chlorobiphenyl hydroxylase enzyme assay in distinguishing between phenobarbitone- and 3-methylcholanthrene-induced microsomal monooxygenases

4-Chlorobiphenyl was used as a substrate for the in vitro determination of rat hepatic microsomal, cytochrome P-450-dependent monooxygenase activity. The 4-chlorobiphenyl hydroxylase assay was tested for its ability to distinguish between a variety of phenobarbitone- and 3-methylcholanthrene-type inducers. Two radiometric procedures were employed to investigate the metabolism of 4-chlorobiphenyl. First, the metabolite profile of 4-chlorobiphenyl was analyzed by radio-thin-layer chromatography. This procedure permitted an assessment of the effects of microsomal enzyme inducers on both the qualitative and quantitative aspects of 4-chlorobiphenyl metabolism. Second, the rate of 4-chlorobiphenyl metabolism was determined by a differential extraction procedure which separated unreacted starting material (hexane phase) from metabolites (base phase). This procedure provided a rapid measurement of the overall activity of 4-chlorobiphenyl hydroxylase. Irrespective of the animal pretreatment, the metabolite profile of 4-chlorobiphenyl was dominated by 4′-chloro-4-biphenyl. Unlike the qualitative aspects, the quantitative aspects of 4-chlorobiphenyl metabolism were markedly influenced by animal pretreatment. Specifically, 3-methylcholanthrene-type inducers (3-methylcholanthrene and 3,3′,4,4′-tetrachlorobiphenyl) enhanced the activity of 4-chlorobiphenyl hydroxylase at least 10 times more than phenobarbitone-type inducers (phenobarbitone, 2,2′,4,4′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl) enabling these two classes of inducers to be clearly distinguished. It is concluded that 4-chlorobiphenyl is preferentially metabolized by the 3-methylcholanthrene-inducible form(s) of cytochrome P-450 and that this class of microsomal enzyme inducers can be readily distinguished from phenobarbitone-type inducers by means of the 4-chlorobiphenyl hydroxylase assay.     

A rapid and simplified assay method for tyrosine hydroxylase

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.     

A sensitive,fluorometric method for the assay of microsomal hydroxylase: N-demethylation of p-chloro-N-methylaniline

A fluorometric method for the assay of microsomal hydroxylase activity is described. N-Demethylation of p-chloro-N-methylaniline yields p-chloroaniline, which is coupled with fluorescamine, extracted with ethylacetate, and measured fluorometrically. This method can determine low levels of N-demethylase activity.     

Rapid assay for lysyl-protocollagen hydroxylase activity

A method for the assay of lysyl-protocollagen hydroxylase activity is described. This method depends upon the formation of tritiated water when lysine residues of 4,5-³H-lysyl-protocollagen are hydroxylated. The labeled protocollagen was prepared from carrageenan induced guinea pig granuloma tissue. There was a linear relationship between the amount of tritiated water and tritiated hydroxylysine formed in this assay. The assay is reproducible and more rapid than previously described assays for lysyl-protocollagen hydroxylase. Use of this method will facilitate further studies on collagen hydroxylysine formation.