Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases

Various hydroxyacyl coenzyme A (CoA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-¹⁴C]d-(–)-hydroxybutyric acid, [1-¹⁴C]d-lactic acid, [1-¹⁴C]l-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, d-(–)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-¹⁴C]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-¹⁴C]lactyl-CoA was used as a substrate at a rate that was only less than 10^–4 of the rate than with [3-¹⁴C]d-(–)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-¹⁴C]d-lactyl-CoA as substrate at a relatively high rate. Correspondence to: A. Steinbüchel     

A rapid and sensitive assay for arginase

An assay for arginase is described that uses l-[guanido-¹⁴C]arginine as substrate. Unhydrolyzed arginine is removed in a batch procedure with sulfonate resin and the [¹⁴C]urea product is determined quantitatively in the resin supernatant. The assay requires 5 min and is performed in one tube. The sensitivity is approximately 0.1 munits of arginase. Arginase activities in fetal calf serum and in murine macrophage extract have been determined and the bovine liver enzyme has been used as a reference.     

Serine Transhydroxymethylase: A Simplified Radioactive Assay; Purification and Stabilization of Enzyme Activity Employing Affi-Gel Blue

An improved radioactive assay has been developed for serine transhydroxymethylase. This assay involves the direct measurement of the [¹⁴C]HCH0 which is generated when [3-¹⁴C]-serine is employed as the substrate. The new assay eliminates 14 the need for a solvent extraction of a [C]HCHO-dimedon adduct which is the basis of the assay devised by Taylor and Weissbach.

The enzyme has been purified employing Affi-Gel Blue. The purified enzyme retains full activity when bound to this affinity chromatography matrix and can be stored in this state at 4° indefinitely.     

Radiometric Method for the Detection of Coliform Organisms in Water

A new radiometric method for the detection of coliform bacteria in water has been described. The method is based on the release of ¹⁴CO₂ from [¹⁴C]lactose by bacteria suspended in growth medium and incubated at 37 C. The evolved ¹⁴CO₂ is trapped by hyamine hydroxide and counted in a liquid scintillation spectrometer. The method permits the detection of 1 to 10 organisms within 6 h of incubation. Coliform bacteria suspended in water for several days recover from starvation and may be quantitated by the proposed method. Bacteria from water samples may also be concentrated by filtration through membrane filters and detected by the radiometric assay.     

Measurement of choline acetyltransferase with [14C]acetate by a cycling procedure

A multiple enzyme and multisubstrate cycling system is described for the radiometric determination of cholineacetyltransferase (ChAT) activity in crude tissue homogenates. The methods employs [¹⁴C]acetate coupled with the enzymes acetate kinase (AK) and phosphotransacetylase (PTA) for the generation of [¹⁴C]acetyl CoA. By recycling it was possible to avoid product inhibition of ChAT by CoA, ATP was maintained constant by rephosphorylation of ADP. Kinetics of the individual enzyme reactions were studied and the parameters obtained were used to select appropriate conditions to maintain linearity of varying amounts ChAT activity over a sixty minute time course. The sensitivity of the method is limited only by the specific activity of commercially available isotope labeled acetate.Special issue dedicated to Dr. O. H. Lowry.     

Use of charcoal adsorption for the microassay of acetyl-CoA-hydrolase.

A sensitive and rapid radiochemical micromethod is described for measuring the activity of acetyl-CoA hydrolase (EC 3.1.2.1). [1-¹⁴C]Acetyl-CoA is incubated with tissue homogenates; unhydrolyzed [1-¹⁴C]acetyl-CoA is separated from the radiolabeled product, [1-¹⁴C]acetate, by adsorption to charcoal. The soluble [1-¹⁴C]acetate is measured by liquid scintillation techniques. This procedure makes it possible to measure as little as 0.2 to 0.4 nmol acetate generated per assay.     

A modified radioisotopic assay for measuring glutamine synthetase activity in tissue extracts.

A radioisotope assay for the measurement of glutamine synthetase activity has been developed in which tandemly arranged ion-exchange columns of Dowex 1-acetate and Amberlite CG-50 (H⁺) are used to separate the product, [¹⁴C]glutamine, from unreacted [U-¹⁴C]glutamate and other labeled compounds, particularly γ-aminobutyrate, that are formed by competing reactions. The technique is sensitive, reproducible, and suitable for multiple determinations. The assay has been used successfully to measure glutamine synthetase activity in neural and nonneural tissues which contain appreciable amounts of glutamate decarboxylase activity.     

An easy assay for histone acetyltransferase activity using a PhosphorImager

A simple radiometric assay for histone acetyltransferase (HAT) activity employing a PhosphorImager is described. In the proposed procedure, following incubation of [1-¹⁴C]acetyl coenzyme A (CoA), histones, and HAT enzyme, radiolabeled histones are fixed on GF/F glass microfiber filter while the excess of acetyl CoA is washed out. Afterward, the filter is exposed to a phosphor-screen and the resulting spot signals are quantified with a PhosphorImager. Given the small volumes required, the new assay reduces reagent consumption and contaminated waste. Moreover, the assay can be performed with a large number of samples simultaneously, is applicable on different protein substrates, and is adaptable to the analysis of other protein modifications.     

A rapid and sensitive radiometric assay procedure for thiamine pyrophosphokinase activity using anion-exchange paper discs

A radiochemical method for the direct measurement of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, EC 2.7.6.2) activity was described earlier (1,2). It avoided the difficulties associated with assay systems based on the coenzyme nature of thiamine pyrophosphate in TPP-dependent¹ enzyme reactions using apopyruvate decarboxylase (3) (2-oxoacid carboxylase, EC 4.1.1.1) or apotransketolase (4) (sedoheptulose-7-phosphate: d-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1). Since the chromatographic isolation of TPP is time-consuming, a procedure for the rapid determination of thiamine pyrophosphokinase activity was desirable.The simplified method described here takes advantage of the anionic character of TPP. The assay is carried out with [¹⁴C]thiamine as substrate. After incubation with the enzyme in the presence of Mg²⁺-ATP, the reaction mixture is applied to a DEAE-cellulose paper disc. The disc is extensively washed with sodium acetate resulting in the quantitative elution of [¹⁴C]thiamine and partial retention of [¹⁴C]TPP. This is quantitatively measured using the liquid scintillation counting technique.A similar procedure has been described for the determination of glycerol kinase (ATP: glycerol phosphotransferase, EC 2.7.1.30) and hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) activities (5).     

A novel direct homogeneous assay for ATP citrate lyase

ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes the synthesis of acetyl-CoA and oxaloacetate using citrate, CoA, and ATP as substrates and Mg²⁺ as a necessary cofactor. The ACL-dependent synthesis of acetyl-CoA is thought to be an essential step for the de novo synthesis of fatty acids and cholesterol. For this reason, inhibition of ACL has been pursued as a strategy to treat dyslipidemia and obesity. Traditionally, ACL enzyme activity is measured indirectly by coupling to enzymes such as malate dehydrogenase or chloramphenicol acetyl transferase. In this report, however, we describe a novel procedure to directly measure ACL enzyme activity. We first identified a convenient method to specifically detect [¹⁴C]acetyl-CoA without detecting [¹⁴C]citrate by MicroScint-O. Using this detection system, we devised a simple, direct, and homogeneous ACL assay in 384-well plate format that is suitable for high-throughput screening. The current assay consists of 1) incubation of ACL enzyme with [¹⁴C]citrate and other substrates/cofactors CoA, ATP, and Mg²⁺, 2) EDTA quench, 3) addition of MicroScint-O, the agent that specifically detects product [¹⁴C]acetyl-CoA, and 4) detection of signal by TopCount. This unique ACL assay may provide more efficient identification of new ACL inhibitors and allow detailed mechanistic characterization of ACL/inhibitor interactions.     

A solid-phase radioimmunoassay for fibrinogen.

A solid-phase radioimmunoassay for fibrinogen has been developed utilizing [¹⁴C]-methylated fibrinogen as standard antigen and fibrinogen-specific antibodies covalently linked to Sepharose. Fibrinogen was [¹⁴C]-methylated by reductive alkylation using [¹⁴C] formaldehyde and sodium borohydride. The methylated fibrinogen was unaltered in clotting ability and antigenicity.The assay, an isotope dilution assay, is quantitative for picomole amounts of fibrinogen. It is specific for fibrinogen in homologous plasma and in the presence of a variety of other proteins.     

An ultraviolet absorbance method for determining acetylsalicylic acid hydrolase activity

An ultraviolet absorbance method for quantitation of acetylsalicylic acid esterase (hydrolase) activity has been developed and validated. The sensitivity of the method was found to be 2.8 nmol/ml-min in the assay cuvette. Linearity of the reaction with enzyme concentration and time has been demonstrated. The product of the enzymatic reaction, salicylic acid, has been identified by thin-layer chromatography using acetyl-[¹⁴C]salicylic acid. The quantities of salicylic acid produced in 5, 10, and 15 min of incubation were equal when assayed by the spectrophotometric method and by the acetyl-[¹⁴C]salicylic acid thin-layer chromatographic method. The time required for assay by ultraviolet absorbance is approximately 3 min/sample.     

Histidine regulation in Salmonella typhimurium: XVI. A sensitive radiochemical assay for histidinol dehydrogenase

A sensitive radiochemical assay for measurement of histidinol dehydrogenase is presented. The method is based upon separation of the product of the reaction. [¹⁴C]histidine, from the substrate, [¹⁴C]histidinol, on small Dowex 50 columns. The assay can be performed on cell extracts or on toluenized cells and is approximately 100 times more sensitive than previously reported assays for this enzyme.[¹⁴C]histidinol is obtained in high yields through conversion of uniformly labeled ¹⁴C-glucose by a strain of Salmonella typhimurium derepressed for the histidine operon and blocked at the histidinol dehydrogenase step. Accumulated [¹⁴C]histidinol is purified from the culture supernatant by ion-exchange chromatography.This sensitive assay has facilitated measurement of reduced levels of histidine operon expression in promoter mutants, and has been adapted for study of histidine operon regulation in a cell free protein synthesizing system.     

Improvements in a Non-Proprietary Radiometric Medium to Allow the Detection of Some Pseudomonas Species and Alcaligenes faecalis

The addition of [5-¹⁴C]glutamate and [¹⁴C]formate to a non-proprietary medium containing [¹⁴C]glucose, Trypticase, yeast extract, thiotone, and salts enabled the radiometric detection of the presence of nonfermenters of glucose. It did not interfere with the rapid detection of the presence of aerobic and anaerobic sporeforemers and nonsporeformers.     

Studies on ATP

The experiments described in this paper serve as a contribution to the solution of the discrepancies which exist in the assay of ATP:thiamine diphosphate phosphotransferase activity (EC 2.7.4.15), presently in use as a tool for the diagnosis of Leigh's disease (SNE, subacute necrotizing encephalomyelopathy). The results obtained with this phosphotransferase assay can, in part, be explained by the presence of thiamine triphosphate (ThTP) in the preparation of thiamine diphosphate (ThDP) used as a substrate, by the inhibition by ATP of the ThTP phosphohydrolase activity, present in fractions of rat brain homogenates, and by the stimulation by ThDP of the ATPase activity. When [2-¹⁴C-thiazole]thiamine was used for the synthesis of [¹⁴C]ThTP in fractions of rat brain, it was found that after chromatographic separation of thiamine and its phosphates,¹⁴C radio-activity could be demonstrated in the ThTP fractions, even in the absence of an enzyme source. Probably a complex is formed between [¹⁴C]thiamine and a phosphate ester which behaves chromatographically as ThTP. It is concluded that the assay system for the measurement of ThTP synthesis in its present form is, in our hands, not suitable for diagnostic purposes.     

A microassay for mammalian histidine decarboxylase.

This report describes a microassay procedure for mammalian histidine decarboxylase (HDC) based on the measurement of [¹⁴C]O₂ formed from l-[1-¹⁴C]histidine. This assay is particularly useful for quick measurement of HDC activity both in microgram quantities of cell or tissue extract and in tissues that contain significant levels of endogenous histamine.Using this assay, we have shown that the pH optimum, K_m and thermolability of HDC are similar for extracts prepared both from normal rat peritoneal mast cells and from the Furth mouse mastocytoma. HDC activity could be detected in homogenates prepared from 10⁵ rat mast cells, and it was expressed on a per cell basis. Mast cell HDC activity varied with the strain of rat from which the cells were obtained and with the season when they were assayed.     

Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography.

A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[¹⁴C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [¹⁴C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus.     

A rapid and sensitive lipase assay using triethylaminoethyl cellulose columns

A procedure has been developed for the separation of labeled fatty acids from tri-, di-, and monoglycerides using small disposable columns of TEAE-cellulose. This procedure is used as the basis of a lipase assay which is rapid, sensitive and unaffected by wide variations in the composition of the reaction mixtures. 0.5 nmole [¹⁴C]oleic acid can be detected, and the entire procedure requires less than 3 min.     

A convenient assay for ADP reductase activity using Dowex-1-borate columns

A convenient assay for ADP reductase activity has been developed in which [¹⁴C]deoxyadenosine is separated from [¹⁴C]adenosine on Dowex-1-borate columns. The separation is complete and the recovery of deoxyadenosine is quantitative. Using disposable Pasteur pipets as the columns, a large number of ADP reductase assays can be handled at one time.