Identification of a carbon-oxygen lyase activity cleaving the ether linkage in carboxymethyloxysuccinic acid

Cell-free extracts of an organism capable of degrading the synthetic ether carboxymethyloxysuccinic acid were used to study the mechanism of degradation of this compound. The initial degradation products were acidic and could be separated by high-voltage electrophoresis. Extended incubation of [¹⁴C]carboxymethyloxysuccinate with the cell-free extracts gave rise to radioactive carbon dioxide and to glycolic, malic, fumaric, and pyruvic acids which were identified by electrophoresis. Identification of the malic and glycolic acids was confirmed by the addition of non-radioactive carrier malic and glycolic acids and recrystallization to constant specific radioactivity. The identification of pyruvate was substantiated by thin-layer chromatography of the 2,4-dinitrophenylhydrazine derivative. The presence of fumaric acid was substantiated by its ultraviolet absorbance.Shorter incubations give rise to only malic, glycolic, and fumaric acids. When the time course of the reaction was followed carefully over the first 15 min of the reaction, it was found that initially nearly equimolar quantities of glycolic and fumaric acids were produced. The early production of fumaric acid was also demonstrated by following the increase in 245-nm absorbance of the degradation mixture. The fumaric acid was subsequently hydrated to malic acid, and the quantity of fumaric acid present decreased slightly until it reached a steady-state level. The presence of fumarase in the extract was demonstrated. It was concluded that the initial attack on the carboxymethyloxysuccinic acid was a cleavage of the ether linkage by an elimination mechanism catalyzed by a carbon-oxygen lyase giving rise to glycolic and fumaric acids.     

Analysis of glucose 1-phosphate at the picomole level with [C]UTP and uridine 5′-diphosphoglucose pyrophosphorylase

A new sensitive method is described for glucose 1-phosphate analysis. The key reaction is the pyrophosphorolysis of UDP-glucose catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase. The reaction product, [¹⁴C]UDP-glucose, is separated from [¹⁴C]UTP by adsorbing [¹⁴C]UTP selectively onto polyethyleneimine cellulose or by separating both labeled compounds on one-dimensional polyethyleneimine thin-layer chromatograms. The sensitivity of the method for glucose 1-phosphate analysis is 5 pmol. The method has been successfully employed to monitor the level of glucose 1-phosphate in early germination of wheat embryos.     

A sensitive fluorimetric procedure for the determination of aliphatic epoxides under physiological conditions

Ribose 1-phosphate has been measured in rat tissues by an enzymatic radioactive assay. The sugar phosphate is converted into [¹⁴C]inosine via the two following combined reactions: ribose 1-phosphate + [¹⁴C]adenine ? [¹⁴C]adenosine + phosphate (adenosine phosphorylase); [¹⁴C]adenosine + H₂O → [¹⁴C]inosine + NH₃ (adenosine deaminase). Tissue extracts are incubated in the presence of excess [¹⁴C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of ribose 1-phosphate present in tissue extracts. Liver was found to contain the highest level of ribose 1-phosphate (ca. 800 nmol/g wet wt).     

A simplified procedure for the concentration,desalting, and thin-layer chromatography of mevalonic acid from reaction mixtures used to assay 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity

A procedure has been described for the quantitative isolation of [¹⁴C]-mevalonic acid from reaction mixtures used for the assay of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. It consists of absorbing the reaction mixtures on Whatman No. 4 filter-paper supports and concentrating the radioactive substrate and the product within a 2-mm² area of the paper by two-dimensional elution with nonpolar solvents. This procedure simultaneously results in desalting of the reaction mixture, thus facilitating an excellent thin-layer chromatographic separation of mevalonolactone uncontaminated by the radioactive substrate. Among other advantages of the method are (a) quantitative extraction of mevalonolactone, thus avoiding the necessity of using an internal standard; (b) possibility of simultaneous processing of a large number of samples with the elution being carried out overnight without frequent supervision; (c) simplicity of the technical operations involved; and (d) inexpensiveness of the materials needed for analysis.     

Analysis of metabolism of carcinogenic polycyclic hydrocarbons by position-sensing proportional counting of thin-layer chromatograms.

A procedure which combines thin-layer chromatography with position-sensing proportional counting has been developed for analyzing the metabolism of carcinogenic polycyclic aromatic hydrocarbons. The profiles of the metabolites of [³H]benzo(a)pyrene and 7,12-[12-¹⁴C]dimethylbenz(a)anthracene produced in cell culture were comparable when obtained by this procedure and by standard methods. However, position-sensing proportional counting allows simultaneous counting of all components of a sample within 10–20 min, and thereby permits the analysis of many hydrocarbon samples in a short time. In addition, the procedure eliminates the necessity of cutting or scraping carcinogen-containing thin-layer chromatograms.     

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.     

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.     

A new isotopic assay for purine nucleoside phosphorylase

We have developed a new assay for purine nucleoside phosphorylase which is based on the release of tritium when [2-3H]inosine is used as the substrate and the reaction is coupled with xanthine oxidase. After the reaction is terminated, residual [2-3H]inosine is adsorbed on charcoal and the supernatant solution is assayed for radioactivity by liquid scintillation spectrometry. The new method gave results indistinguishable from those obtained by spectrophotometric determination of uric acid produced by the phosphorylase-xanthine oxidase-coupled reaction or by radioassay of chromatographically isolated [8-14C]hypoxanthine when [8-14C]inosine was used as substrate. The new method is faster than those involving chromatographic isolation of products. In comparison with spectrophotometric methods, it not only requires less manual time, but it also has the advantage that it can be used to study inhibitors whose ultraviolet absorption might interfere with spectrophotometric determination of uric acid.     

THE [14C]DEOXYGLUCOSE METHOD FOR THE MEASUREMENT OF LOCAL CEREBRAL GLUCOSE UTILIZATION: THEORY,PROCEDURE, AND NORMAL VALUES IN THE CONSCIOUS AND ANESTHETIZED ALBINO RAT1

Abstract— A method has been developed for the simultaneous measurement of the rates of glucose consumption in the various structural and functional components of the brain in vivo. The method can be applied to most laboratory animals in the conscious state. It is based on the use of 2-deoxy-D-[¹⁴C]glucose ([¹⁴C]DG) as a tracer for the exchange of glucose between plasma and brain and its phosphorylation by hexokinase in the tissues. [¹⁴C]DG is used because the label in its product, [¹⁴C]deoxyglucose-6-phosphate, is essentially trapped in the tissue over the time course of the measurement. A model has been designed based on the assumptions of a steady state for glucose consumption, a first order equilibration of the free [¹⁴C]DG pool in the tissue with the plasma level, and relative rates of phosphorylation of [¹⁴C]DG and glucose determined by their relative concentrations in the precursor pools and their respective kinetic constants for the hexokinase reaction. An operational equation based on this model has been derived in terms of determinable variables. A pulse of [¹⁴C]DG is administered intravenously and the arterial plasma [¹⁴C]DG and glucose concentrations monitored for a preset time between 30 and 45min. At the prescribed time, the head is removed and frozen in liquid N₂-chilled Freon XII, and the brain sectioned for autoradiography. Local tissue concentrations of [¹⁴C]DG are determined by quantitative autoradiography. Local cerebral glucose consumption is calculated by the equation on the basis of these measured values. The method has been applied to normal albino rats in the conscious state and under thiopental anesthesia. The results demonstrate that the local rates of glucose consumption in the brain fall into two distinct distributions, one for gray matter and the other for white matter. In the conscious rat the values in the gray matter vary widely from structure to structure (54-197 μmol/100 g/min) with the highest values in structures related to auditory function, e.g. medial geniculate body, superior olive, inferior colliculus, and auditory cortex. The values in white matter are more uniform (i.e. 33–40 μmo1/100 g/min) at levels approximately one-fourth to one-half those of gray matter. Heterogeneous rates of glucose consumption are frequently seen within specific structures, often revealing a pattern of cytoarchitecture. Thiopental anesthesia markedly depresses the rates of glucose utilization throughout the brain, particularly in gray matter, and metabolic rate throughout gray matter becomes more uniform at a lower level.     

A microdetermination of ( 14 C)lactate in biological fluids

A simple method, based on a single-step conversion of lactate to pyruvic acid semicarbazone by lactic dehydrogenase, has been developed for the microdetermination of [¹⁴C]lactate in the biological fluids. Pyruvic semicarbazone thus formed is efficiently separated from other labelled metabolites by means of charcoal treatment followed by thin-layer chromatography on cellulose plate. The entire procedure applied to rat whole blood revealed the mean recovery of 75.2% with a good reproducibility.     

A radiometric microassay for cellulase activity

The method described measures the release of ¹⁴C products from [U-¹⁴C]cellulose. The assay is simple and utilizes very small incubation volumes. Activity is evident within the first 30 min of reaction. This method should be particularly suited to activity surveys of culture filtrates from fermentations as well as the analysis of fractions from enzyme purification studies.     

Plasmodium knowlesi: In vitro biosynthesis of methionine

Methionine biosynthesis was studied in rhesus monkey erythrocytes infected with Plasmodium knowlesi malaria which were cultured in vitro with l-[3-¹⁴C]serine, methyl-[¹⁴C]tetrahydrofolic acid, and l-[³⁵S]homocysteine. Radioactivity derived from [3-¹⁴C]serine was detected in approximately equivalent amounts in methionine and thymidylic acid by thin-layer chromatography of acid-hydrolysates of washed erythrocytes. The results with methyl-[¹⁴C]tetrahydrofolic acid were inconclusive. Radioactivity from l-[³⁵S]homocysteine also appeared in methionine but the level of homocysteine required for maximal activity was tenfold that of serine. The results indicate that the serine: 5,10-methylenetetrahydrofolic acid: 5-methyl-tetrahydrofolic acid: methionine biosynthetic pathway is present in the P. knowlesi malaria parasite.     

Isolation of cyclic AMP and cyclic GMP by thin-layer chromatography. Application to assay of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase

A procedure is described for isolation of cAMP and cGMP by thin-layer chromatography on polyethylenimine cellulose. Chromatographs are developed (descending) twice in the same direction with two different solvents. This procedure separates cAMP and cGMP from other radioactive metatolites of [³H] or [¹⁴C] ATP or GTP. Application of this isolation method to assay of adenylate cyclase, (EC 4.6.1.1), guanylate cyclase (EC 4.6.1.2), and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) has proven convenient and provides results of unusual quality.     

Determination of tissue UDP-glucuronic acid levels by high-pressure liquid chromatography

A new and sensitive method to measure UDP-glucuronic acid extracted from as little as 25 mg wet weight tissue has been developed. This procedure employs high-pressure liquid chromatography and liquid scintillation spectrophotometry to measure p-[¹⁴C]nitrophenylglucuronide generated enzymatically from p-[¹⁴C]nitrophenol and UDP-glucuronic acid. The reaction was catalyzed by UDP-glucuronyltransferase obtained from rat liver microsomes. The tissue levels of UDP-glucuronic acid assayed were 2 to 20 μmol/100 g wet wt, which are well below the levels detectable by the widely used spectrophotometric method.     

A radioisotopic method for the assay of aspartate carbamoyltransferase and carbamoyl phosphate

The use of [¹⁴C]aspartate of high specific activity and thin-layer chromatography on polyethyleneimine cellulose for the separation of carbamoyl aspartate from aspartate has enabled the measurement of aspartate carbamoyltransferase and carbamoyl phosphate synthase activities and carbamoyl phosphate concentrations in extracts from Escherichia coli. The assay method described is sensitive to the formation of about 1 pmol of carbamoyl aspartate.     

A procedure for the synthesis of p-fluorosulfonyl[14C]-benzoyl-5′-adenosine with [14C] in the benzoyl moiety

Carboxy-p-fluorosulfonyl[¹⁴C]benzoyl-5′-adenosine has been synthesized with the radiolabel ultimately derived from carboxy-p-amino[¹⁴C]benzoic acid by a synthetic route employing four reaction steps. Starting with 1 mmol of p-amino[¹⁴C]benzoic acid, p-fluorosulfonyl[¹⁴C]benzoyl-5′-adenosine is obtained with an overall yield of 25–30%.     

Specificity of an HPETE peroxidase from rat PMN

The 15,000xg supernatant of sonicated rat PMN contains 5-lipoxygenase that converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A₄ and an HPETE peroxidase that catalyzes reduction of the 5-HPETE. The specificity of this HPETE peroxidase for peroxides, reducing agents, and inhibitors has been characterized to distinguish this enzyme from other peroxidase activities. In addition to 5-HPETE, the HPETE peroxidase will catalyze reduction of 15-hydroperoxyeicosatetraenoic acid, 13-hydroperoxyoctadecadienoic acid, and 15-hydroperoxy-8,11,13-eicosatrienoic acid, but not cumene or t-butylhydroperoxides. The HPETE peroxidase accepted 5 of 11 thiols tested as reducing agents. However, glutathione is >15 times more effective than any other thiol tested. Other reducing agents, ascorbate, NADH, NADPH, phenol, p-cresol, and homovanillic acid, were not accepted by HPETE peroxidase. This enzyme is not inhibited by 10 mM KCN, 2 mM aspirin, 2 mM salicylic acid, or 0.5 mM indomethacin. When 5-[¹⁴C]HPETE is generated from [¹⁴C]arachidonic acid in the presence of unlabeled 5-HPETE and the HPETE peroxidase, the 5-[¹⁴C]HETE produced is of much lower specific activity than the [¹⁴C]arachidonic acid. This indicates that the 5-[¹⁴C]HPETE leaves the active site of 5-lipoxygenase and mixes with the unlabeled 5-HPETE in solution prior to reduction and is a kinetic demonstration that 5-lipoxygenase has no peroxidase activity. Specificity for peroxides, reducing agents, and inhibitors differentiates HPETE peroxidase from glutathione peroxidase, phospholipid-hydroperoxide glutathione peroxidase, a 12-HPETE peroxidase, and heme peroxidases. The HPETE peroxidase could be a glutathione S-transferase selective for fatty acid hydroperoxides.     

Translocation of cytidine 5′-monophosphosialic acid across golgi apparatus membranes

Golgi apparatus, isolated from rat liver, incorporate [¹⁴C]sialic acid from CMP[¹⁴C]sialic acid into endogenous glycolipid and glycoprotein acceptors. Incorporation of [¹⁴C]sialic acid into endogenous glycoprotein acceptors was stimulated an average of 3-fold by Triton X-100 at an optimal concentration of 0.05% and was inhibited at higher concentrations. Incorporation of [¹⁴C]sialic acid into endogenous glycolipid acceptors was not stimulated by detergent. The major glycolipid product was identified by thin-layer chromatography as the ganglioside G_d3. SDS-polyacrylamide gel electrophoresis of the glycoprotein products demonstrated incorporation of [¹⁴C]sialic acid into 6–7 major bands. Neuraminidase studies determined that approximately 60% of the [¹⁴C]sialic acid incorporated into endogenous acceptors in the absence of detergent had a luminal orientation. Furthermore, electron microscopy studies showed that the isolated Golgi apparatus fraction consisted of intact membrane cisternae. Our results demonstrate that sialylation of cisternal acceptors located on the inside of the membrane occurs in the absence of detergent. They are consistent with carrier-mediated transport as a mechanism to allow CMPsialic acid to traverse the Golgi apparatus membrane and to be used to glycosylate endogenous glycoprotein and glycolipid acceptors.     

H(+)-linked transport of salicylic acid, an NSAID, in the human trophoblast cell line BeWo

We investigated thetransport of salicylic acid and L-lactic acid across theplacenta using the human trophoblast cell line BeWo. We performeduptake experiments and measured the change in intracellular pH(pH_i). The uptakes of [¹⁴C]salicylic acid andL-[¹⁴C]lactic acid were temperature- andextracellular pH-dependent and saturable at higher concentrations. Bothuptakes were also reduced by FCCP, nigericin, and NaN₃.Various nonsteroidal anti-inflammatory drugs (NSAIDs) stronglyinhibited the uptake of L-[¹⁴C]lactic acid.Salicylic acid and ibuprofen noncompetitively inhibited the uptake ofL-[¹⁴C]lactic acid.-Cyano-4-hydroxycinnamate (CHC), a monocarboxylate transporterinhibitor, suppressed the uptake ofL-[¹⁴C]lactic acid but not that of[¹⁴C]salicylic acid. CHC also suppressed the decrease ofpH_i induced by L-lactic acid but had littleeffect on that induced by salicylic acid or diclofenac. These resultssuggest that NSAIDs are potent inhibitors of lactate transporters,although they are transported mainly by a transport system distinctfrom that for L-lactic acid.

     

Single step thin-layer chromatographic method for quantitation of enzymatic formation of fatty acid anilides

The activity of the enzyme involved in catalyzing the formation of fatty acid anilides can be measured by quantitating the fatty acid anilides formed. We have shown earlier that oleic acid is the most preferred substrate among other fatty acids studied for the conjugation with aniline. The reaction product (oleyl anilide) could be separated by thin-layer chromatography (TLC) and then quantified by reversed-phase high-performance liquid chromatography (HPLC). Using [1-¹⁴C]oleic acid as substrate, the fatty acid anilide forming activity can be determined in a single step by TLC analysis. The conventional TLC methods used for the separation of the fatty acid esters, however, could not resolve oleyl anilide from the residual [1-¹⁴C]oleic acid. Therefore, a simple and reliable TLC method was developed for the separation of oleyl anilide from oleic acid using a freshly prepared solvent consisting of petroleum ether–ethyl acetate–ammonium hydroxide (80:20:1, v/v). Using this solvent system the relative flow (R_f) values were found to be 0.54 for oleyl anilide and 0.34 for aniline, whereas oleic acid remained at the origin. The TLC procedure developed in the present study could be used to determine the fatty acid anilide forming activity using [1-¹⁴C]oleic or other fatty acids as substrate and was also found suitable for the analysis of fatty acid anilides from the biological samples.