Determination in plasma of angiotensin-converting enzyme inhibitor by inhibitor-binding assay

A method of determining a new angiotensin-converting enzyme inhibitor (CS-622) and its active metabolite (RS-5139) in plasma by inhibitor-binding assay has been developed using high-performance liquid chromatography. The assay is based on the principle that the amount of inhibitor bound to the enzyme is inversely related to the amount of hippuric acid liberated on hydrolysis from the artificial substrate (hippuryl- -hystidyl- -leucine). Plasma was heated at 60°C for 15 min, to inactivate endogenous enzyme, and preincubated with rabbit-lung angiotensin-converting enzyme at 37°C for 3 min. The artificial substrate (5.75 mg/ml in pH 8.3 phosphate buffer containing sodium chloride) was added to the resulting solution, and the mixture was incubated for 30 min. The reaction was terminated by the addition of 2 M hydrochloric acid. The hippuric acid liberated on hydrolysis was extracted with ethyl acetate and determined by reversed-phase chromatography using methylparaben as an internal standard. The total concentration of the inhibitor and its metabolite were determined by this method after de-esterification by rat-plasma esterase. The standard curve was obtained by the regression analysis of log concentration against logit response. The within-day and day-to-day precision were satisfactory. The proposed method is simple, rapid and sensitive enough to determine angiotensin-converting enzyme inhibitor in plasma.     

Hydrolysis of substance P and its analogs by angiotensin-converting enzyme from rat lung. Characterization of endopeptidase activity of the enzyme

Hydrolysis of substance P and nine kinds of substance P analogs by angiotensin-converting enzyme highly purified from rat lung was examined by using amino-group fluorometry and high-performance liquid chromatography. The enzyme hydrolyzed substance P and several analogs, notwithstanding that they did not contain free C-terminal residues. The analyses of cleavage products separated by high-performance liquid chromatography indicated that the enzyme hydrolyzed substance P and its analogs mainly at the bond between Phe8-Gly9 and also at another bond, possibly between Gly9-Leu10, to a lesser extent by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action. The analogs that had D-amino acid residues substituted at the presumed cleavage sites were scarcely hydrolyzed. It was further found that (Pyr6)-fragment (6-11) was hydrolyzed by the enzyme more efficiently than the other fragment-type analogs and was cleaved at a single bond by the endopeptidase activity of the enzyme. Therefore, this fragment was used as a substrate in order to characterized the endopeptidase activity of the enzyme by employing fluorometry. The activity was dependent on chloride ion, and was inhibited by captopril, MK-421, and EDTA. Thus, the endopeptidase activity of the enzyme showed properties similar to those of the dipeptidyl carboxypeptidase activity of the enzyme.     

Simplified cysteine dioxygenase activity assay allows simultaneous quantitation of both substrate and product

A high-performance liquid chromatography (HPLC) method for enzyme activity assays using a hydrophilic interaction liquid chromatography (HILIC) column in combination with an evaporative light scattering detector was developed. The method was used to measure the activity of the non-heme mono-iron enzyme cysteine dioxygenase. The substrate cysteine and the product cysteine sulfinic acid are very weak chromophores, making direct ultraviolet (UV) detection without derivatization rather insensitive; moreover, derivatization of cysteine is often not efficient. Using the system described, underivatized substrate and product in samples from cysteine dioxygenase activity assays could be separated and analyzed. Furthermore, it was possible to quantify cysteic acid, the noncatalytic oxidation product of cysteine sulfinic acid. Acetone was used both to stop the enzymatic reaction by protein precipitation and as an organic mobile phase, making sample preparation very easy and the assay highly reproducible.     

Assay method for Escherichia coli photolyase activity using single-strand cis-syn cyclobutane pyrimidine dimer DNA as substrate

A high-performance liquid chromatography method for the assay of Escherichia coli photolyase activity was developed. When cis-syn cyclobutane pyrimidine dimer was used as substrate, the Michaelis constant (K(m)) value for the photolyase activity was 100 nM. The linear range of the calibration curve of the photolyase activity was 0.026-6.64 microU/assay tube. The correlation coefficient for this linearity was 0.998. The limit of detection (S/N = 3) was 26 nU/assay tube. The photolyase activity was increased 1.6-fold in the presence of 5,10-methenyltetrahydrofolic acid in the enzyme reaction mixture.     

A new feature of angiotensin-converting enzyme in the brain: hydrolysis of substance P

Highly purified rat brain angiotensin-converting enzyme hydrolyzes substance P which contains a C-terminal amino acid with an amidated carboxyl group. The hydrolysis of substance P verified by amino-group fluorometry and by high-performance liquid chromatography is inhibited by captopril, but not by phosphoramidon. The presence of sodium chloride is essential for the hydrolysis. The analyses of cleavage products indicate that the enzyme hydrolyzes substance P between Phe7-Phe8 and Phe8-Gly9 by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action.     

Visual detection of peptidase activity using fluorogenic substrates in a microtiter plate assay

A simple, inexpensive, and sensitive assay for peptidase activity has been devised. The assay was performed in a microtiter plate and was based on fluorogenic peptide substrates, many of which are commercially available. 7-Amino-4-methyl coumarin the fluorescent product liberated during an incubation period of between 1 and 16 h, was detected by inspection of the plate under ultraviolet light of wavelength 356 nm. A fluorometer was not required. Using alpha-chymotrypsin as a model enzyme, with succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine 4-methyl-coumaryl-7-amide as substrate, it was shown that as little as 4 fmol of enzyme could be detected. The method was non-quantitative and was particularly suited to location of enzyme activity in fractions during a purification procedure. The validity of the assay was demonstrated by detection of activity of a known enzyme, alpha-chymotrypsin, after its purification by size-exclusion high-performance liquid chromatography. The method was used to locate two forms of aminopeptidase activity, in fractions from size-exclusion chromatography of an extract from reproductive tissue of Helix aspersa, using L-leucine 4-methyl-coumaryl-7-amide as substrate.     

IV. Determination of aromatic L-amino acid decarboxylase in serum of various animals by high-performance liquid chromatography with electrochemical detection

This paper describes the distribution of aromatic L-amino acid decarboxylase (AADC), using both L-DOPA and L-5-hydroxytryptophan (L-5-HTP) as substrates, in serum of various animals. The ratio of the activities of the enzyme towards both substrates was also determined in the same serum. AADC activity was discovered in serum using our new and highly sensitive assay for AADC activity by high-performance liquid chromatography (HPLC) with electrochemical detection (ED) with L-DOPA and L-5-HTP as substrates. It was found that among the species used, guinea pig serum had the highest activity, and we made a systematic study on guinea pig serum AADC.     

Electrochemical determination of arylsulfatase activity using high-performance liquid chromatography

A highly sensitive assay for arylsulfatase A was developed using high-performance liquid chromatography with electrochemical detection. The retention time of the enzymatic product, p-nitrocatechol, on a reverse phase column was approximately 2.0 min. The assay was able to detect 0.43 pmol p-nitrocatechol in a 20-microliter ethanol extract of the reaction mixture. The coefficients of variation for seven determinations of the intra- and interassays were 9 and 8%, respectively. The levels of arylsulfatase A activity in human saliva samples and leukocyte and platelet lysates determined by this assay were within 6 and 11%, respectively, of the levels determined by a spectrophotometric assay. The high-performance liquid chromatography assay may have utility in measuring arylsulfatase A activity in biological samples with low activity or specific activity or in samples with compounds which interfere with the spectrophotometric assay.     

Successive glycosyltransfer activity and enzymatic characterization of pectic polygalacturonate 4-alpha-galacturonosyltransferase solubilized from pollen tubes of Petunia axillaris using pyridylaminated oligogalacturonates as substrates

Polygalacturonate 4-alpha-galacturonosyltransferase (pectin synthase) was solubilized from pollen tubes of Petunia axillaris and characterized. To accomplish this, an assay method using fluorogenic pyridylaminated-oligogalacturonic acids (PA-OGAs) as acceptor substrates was developed. When the pollen tube enzyme was solubilized with 0.5% (v/v) Triton X-100 and was incubated with PA-OGA and UDP-galacturonic acid (UDP-GalUA), successive transfer activity of more than 10 GalUAs from UDP-GalUA to the nonreducing end of PA-OGA was observed by diethylaminoethyl high-performance liquid chromatography. This activity was time- and enzyme concentration-dependent. The optimum enzyme activity was observed at pH 7.0 and 30 degrees C. Among the PA-OGAs investigated, those with a degree of polymerization of more than 10 were preferred as substrates. The crude pollen tube enzyme had an apparent K(m) value of 13 microM for the PA-OGA with a degree of polymerization 11 and 170 microM for UDP-GalUA. The characteristics of the P. axillaris pollen tube enzyme and the usefulness of fluorogenic PA-OGAs for the assay of this enzyme are discussed.     

High-performance liquid chromatographic determination of d-amino acid oxidase activity

A new procedure for the assay of d-amino acid oxidase activity has been developed. α-Ketoisovaleric acid, derived from d-valine, was estimated by high-performance liquid chromatography after reaction with o-phenylenediamine to give the corresponding quinoxalinol derivative. α-Ketovaleric acid was used as an internal standard to ensure the reproducibility of the method. As an example of application, kidney cortex homogenates were analyzed for their d-amino acid oxidase activity. The advantages of the presented procedure for the determination of the enzymatic activity in biological samples compared with previously reported procedures are discussed.     

A spectrophotometric assay for dehydroascorbate reductase

A simple spectrophotometric assay for dehydroascorbate reductase based on the change in absorbance associated with the formation of ascorbic acid is described. Using a partially purified preparation from spinach leaves, the reaction was found to be linear with time and enzyme concentration. The reaction rate determined by this assay correlated well with that obtained by a high-performance liquid chromatography method. Possible advantages over currently available assays as well as potential applications are discussed.     

棕点湍蛙皮肤分泌液中促胰岛素释放肽的分离纯化、基因克隆及活性分析

通过分离纯化棕点湍蛙(Amolops loloensis)皮肤分泌液中的生物活性物质,得到有促胰岛素释放活性的分离峰,并鉴定其结构.采用葡聚糖Sephadex G-50凝胶层析和反相高效液相(RP-HPLC)等手段对棕点湍蛙皮肤分泌液进行分离纯化,利用胰岛素释放实验进行活性检测,Edman降解法测定活性峰的氨基酸序列,反转录法构建cDNA文库并克隆其基因.得到一个具有显著的促胰岛素释放活性的十六肽,测得其氨基酸序列为:FMPIvGKsMSGLSGKL-NH2,命名为amolopin-1.由cDNA(开放阅读框为192bp)推导的氨基酸一级结构显示,其前体由64个氨基酸残基(aa)组成,包括高度保守的信号肽(22aa),酸性肽以及成熟肽.经过数据库序列比对,从棕点湍蛙皮肤中得到一个新的促胰岛素释放肽,进一步分析其作用机理和药代动力学,极有可能得到一个新的治疗糖尿病的降糖药物.     

Purification of rat liver mitochondrial aspartate aminotransferase and separation of its isoforms utilizing high-performance liquid chromatography

A rapid method for purification of mitochondrial aspartate aminotransferase from rat liver employing high-performance liquid chromatography is reported. The product is purified 80-fold with a recovery greater than or equal to 50% in a single day. The amino acid composition, N-terminal amino acid sequence, specific activity, and spectral characteristics of the isolated enzyme are similar to those previously reported for this protein. The protein is homogeneous by standard electrophoretic and chromatographic criteria, but can be resolved into at least five isoforms by a carboxymethylated resin column using high-performance liquid chromatography. The principal isoform initially isolated is converted into two additional isoforms with lower specific activity upon storage at 4 degrees C.     

Improved determination of bovine glutaminyl cyclase activity using precolumn derivatization and reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive, rapid and reproducible assay for the determination of glutaminyl cyclase activity is reported. This method is based on the monitoring of the absorption of l-pyroglutamic acid beta-naphthylamide at 235 nm, enzymatically formed from the substrate l-glutaminyl-beta-naphthylamide, after separation by high-performance liquid chromatography using a C-18 reversed-phase column by isocratic elution. The detection limit of this method is at a level as low as 0.08 nmol/ml and, the time consumed for analysis is <6.5 min per sample for separation and quantification. The optimum pH for glutaminyl cyclase activity was 8.0-8.5. The K(m) and V(max) values were 100.2+/-2.9 microM and 332 +/-21.7 pmol/(h microg protein), respectively, with the use of enzyme extract obtained from bovine pituitary. Glutaminyl cyclase activity was strongly inhibited by zinc(II) ion and 1,10-phenanthroline. By using this assay, the stimulatory effect of bacterial lipopolysaccharide on this enzyme activity was observed in macrophage cell line RAW 264.7. Our newly developed assay would be useful for clarification of the physiological role of this enzyme.     

Production of lipolyzed butteroil by a calf pregastric esterase immobilized in a hollow fiber reactor: III. Effect of glycerol

Lipolysis of butter oil in a hollow fiber reactor containing an immobilized calf pregastric esterase was studied at 40 degrees C, a pH of 6.0, and glycerol concentrations of 0, 150, and 500 g/L in the buffer solution. The concentrations of 10 fatty acid species in the lipolyzed product were determined using high-performance liquid chromatography. The rate of loss of enzyme activity and the relative selectivities of this esterase depended on the glycerol concentration. By contrast, the overall rate of release of fatty acids was not affected by the glycerol concentration. Loss of enzyme activity was modeled using first-order kinetics. The models for deactivation and reaction kinetics were fit simultaneously to the data. The model was successful in describing the rates of release of all 10 fatty acid species for a range of space times from 0 to 25 h. The parameters of the model were tested for dependence on glycerol concentration.     

Comparative quantitation of abscisic acid in plant extracts by gas-liquid chromatography and an enzyme-linked immunosorbent assay using the avidin-biotin system

In this report we describe an enzyme-linked immunosorbent assay (ELISA) for the quantitation of abscisic acid (ABA) in plant extracts. A microtitration plate is coated with an ABA-protein complex. The ABA, standard or sample, is then added to each well with a limiting quantity of rabbit anti-ABA antibodies. During the following incubation period, antibodies bind either to free or to bound ABA on the plates. After washing, bound antibodies are indirectly labelled in two steps by the means of biotinylated goat antirabbit immunoglobulin-G antibodies which act as a link between rabbit anti-ABA antibodies and an avidin-alkaline phosphatase complex. The relative enzyme activity bound is measured spectrophotometrically. The detection limit of this method is 5 pg ABA and the measuring range extends to 10 ng. Gas-liquid-chromatography controls, with an electron capture detector, show a good correlation with ELISA results obtained using extracts of Lycopersicon esculentum, Nicotiana tabacum and Pseudotsuga menziesii samples purified by high-performance liquid chromatography. This provides a good argument for the accuracy of the immunoenzymatic method. The indirect labelling of antibodies, with the avidin-biotin amplifying system, should make this technique suitable for the quantitation of other plant growth substances against which specific antibodies are available.Abbreviations ABA abscisic acid - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - GLC gas liquid chromatography - HPLC high-performance liquid chromatography - IgG Immunoglobulin G - PBS phosphate-buffered saline     

Some properties and subcellular distribution of acyl-coenzyme A: retinol acyltransferase activity in rat testes

The present study was conducted to examine esterification of retinol by testicular microsomes. The microsomes were isolated from rat testes and were incubated under varying assay conditions with [3H]retinol. [3H]Retinylpalmitate was identified by reversed-phase high-performance liquid chromatography as an esterified product. The rate of esterification was increased by the addition of a fatty acyl-CoA. Coenzyme A esters of oleic, palmitic and stearic acids were equally effective substrates for retinol esterification. A 17-fold increase was observed in the presence of palmitoyl-CoA when microsomes had been pretreated with hydroxylamine, a reagent that reacts with coenzyme A thioesters to form hydroxamic acids. The esterifying activity was stimulated by the addition of dithiothreitol (4 mM) and fatty acid-free bovine serum albumin (1 mg/ml). The optimal concentrations for retinol and palmitoyl-CoA were 40 microM and 30-40 microM, respectively. The enzyme activity was inhibited by p-hydroxymercuribenzoate, sodium taurocholate and 5,5'-dithiobis-(2-nitrobenzoic acid), but not by EDTA. The enzyme activity was highest in microsomes (36%). However, some activity was present in mitochondria (29%). These results clearly show the presence of a fatty acyl-CoA: retinol acyltransferase that catalyzes the esterification of retinol in rat testes.     

In VitroMeasurement of β-Lactamase-Catalyzed Ampicillin Hydrolysis by RecombinantEscherichia coliExtracts Using Quantitative High-Performance Liquid Chromatography

We report a rapid and simple protocol for measuring the β-lactamase activity from recombinantEscherichia colicells transformed with any of the common plasmid vectors that provide ampicillin resistance through constitutive expression and periplasmic localization of the β-lactamase TEM-1. The hydrolytic enzyme was extracted from theE. coliperiplasm and the β-lactamase activity determined by measuring conversion of ampicillin to aminobenzyl-penicilloic acid using quantitative high-performance liquid chromatography. Under saturating conditions thein vitroassay was linear as a function of both incubation time and enzyme concentration. Application of this assay to investigate TEM-1 expression, from two different protein expression vector systems, demonstrated the potential importance of this assay in studies of recombinant protein expression and translocation.     

Enkephalin-degrading dipeptidyl carboxypeptidase in guinea pig serum: its properties and action on bioactive peptides

A dipeptidyl carboxypeptidase, which cleaved the Gly3-Phe4 bond of enkephalins, was purified from guinea pig serum 420-fold. The optimum pH of the enzyme was in the neutral range (pH 7.25), and the molecular weight was estimated to be approx. 280,000. The enzyme hydrolyzed Met- and Leu-enkephalin with Km values of 0.30 and 0.50 mM, respectively. The enzyme was inhibited by metal chelators and p-chloro-mercuribenzoate. Captopril showed high inhibitory potency, while phosphoramidon and Phe-Ala showed no effect on the enzyme activity. Therefore, the obtained enzyme can be classified as an angiotensin-converting enzyme (EC 3.4.15.1). Among the bioactive peptides examined, bradykinin and angiotensin I were hydrolyzed by the enzyme. Angiotensin III showed a stronger inhibitory effect than that of angiotensin II. Substance P, gastrin I, and secretin were also inhibitory toward the enzyme activity. On high-performance liquid chromatography analysis, Met-enkephalin-Arg6-Phe7 and Leu-enkephalin-Arg6 were cleaved sequentially at the second peptide bond of the C terminus. Thus, the dipeptidyl carboxypeptidase in guinea pig serum may play a role not only in the angiotensin-bradykinin system but also in the metabolism of circulating enkephalins and other bioactive peptides.     

Measurement of 5-aminolevulinic acid synthase activity in whole and fractionated human bone marrow: effect of myeloid cell lysis by monoclonal antibody

A sensitive radiochemical assay for the measurement of bone marrow and erythroblast 5-aminolevulinic acid (ALA) synthase (EC 2.3.1.37) was developed and optimized with respect to sample preparation and reagent concentration. Succinylacetone (4,6-dioxoheptanoic acid) was used to prevent ALA utilization during the incubation period. Sample purification on a Sep-Pak cartridge (Waters Associates) followed by reverse-phase high-performance liquid chromatography (HPLC) allowed rapid isolation of pure ALA-pyrrole, free from radioactive succinate and other contaminants. ALA synthase activity was measured in unfractionated bone marrow and in samples from which myeloid cells had been removed by monoclonal antibody-mediated cell lysis. Myeloid-derived ALA synthase was calculated and found to contribute approximately half of the total unfractionated marrow enzyme activity. This suggests that results from previous studies using unfractionated bone marrow which have assumed that myeloid cells are an insignificant source of ALA synthase require reappraisal.