Analysis of human synovial fluid phospholipase A2 on short chain phosphatidylcholine-mixed micelles: development of a spectrophotometric assay suitable for a microtiterplate reader.

The development of a reliable assay for human synovial fluid phospholipase A2 (HSF PLA2) is important for the kinetic characterization of the enzyme and for the identification of enzyme inhibitors. This enzyme behaves differently from other extracellular PLA2s in many standard phospholipase assays and is generally assayed using radiolabeled, autoclaved Escherichia coli as a substrate. We have now developed a nonradioactive, continuous, spectrophotometric assay for this enzyme that is adaptable for use with a microtiterplate reader and is suitable for screening enzyme inhibitors. The assay uses a thioester derivative of diheptanoyl phosphatidylcholine as a substrate, with which the enzyme displays a specific activity of about 25 mumol min-1 mg-1. The substrate concentration curve fits a Hill equation with an apparent Km of 500 microM and a Hill coefficient of two. The enzyme has a pH optimum of 7.5 in this assay and requires about 10 mM Ca2+ for maximal activity. The presence of 0.3 mM Triton X-100 was necessary to solubilize the substrate; however, higher concentrations of the detergent inhibited enzyme activity. Using this spectrophotometric assay, inhibition of HSF PLA2 by a thioether phosphonate phosphatidylethanolamine analog was observed with an IC50 of 18 microM.     

A direct, highly sensitive fluorometric assay for a microsomal cytochrome P450-mediated O-demethylation using a novel coumarin analog as substrate

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (lambdaA = 380 nm, lambdaF = 480 nm) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2 and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 380 nm and a fluorescence/emission wavelength of 480 nm, the fluorescence of this substance (lambdaA = 338 nm, lambdaF = 422 nm) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrome P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.     

Crayfish abdominal muscle adenylate cyclase. Studies on the stimulation by a Ca2+-binding protein.

A plasma-membrane preparation of crayfish muscle showed an adenylate cyclase activity which is inhibited to about 80% of its original activity by 100 microM-EGTA. Measurements of the enzyme activity in the presence of 100 microM-EGTA and various concentrations of Ca2+ revealed an increase in enzyme activity of about 400%, indicating an adenylate cyclase which is dependent on Ca2+ for activity. Fluphenazine (1 mM), a blocker of the Ca2+-binding protein calmodulin, decreased enzyme activity to zero. The enzyme can be re-activated by the addition of certain concentrations of calmodulin to the assay medium. This suggests that crayfish muscle adenylate cyclase is dependent on Ca2+ and calmodulin for activity.     

MTT比色法测定促肝细胞生长物质对肝细胞生长的刺激活性

本实验建立了用简便的ＭＴＴ比色法对促肝细胞生长物质的促肝细胞增殖作用的测定方法,确定了实验的最适条件。与传统的３Ｈ ＴｄＲ掺入法进行比较的结果显示,ＭＴＴ比色法与３Ｈ ＴｄＲ掺入法测定结果基本相符,灵敏度相近,但消除了同位素的污染,是一个测定促肝细胞生长物质刺激肝细胞增殖活性的简便方法。     

A continuous fluorescence displacement assay for the measurement of phospholipase A2 and other lipases that release long-chain fatty acids.

1. A new continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino)undecanoic acid from rat liver fatty-acid-binding protein by long-chain fatty acids released as a result of phospholipase A2-catalysed hydrolysis of phospholipids. The initial rate of decrease in fluorescence is linearly related to enzyme activity. 2. The assay will detect enzyme activity down to about 10 pmol/min per ml and gives a linear response up to about 10 nmol/min per ml. 3. The assay will work with all phospholipids that have been tested including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Substrates carrying a net negative charge showed the highest rates of hydrolysis. 4. The assay will work, in principle, with an enzyme catalysing the release of long-chain fatty acids from a fatty-acylated substrate. This has been confirmed with pancreatic lipase and cholesterol esterase.     

Determination of acrosin activity of boar spermatozoa by the clinical method: optimization of the assay and changes during short-term storage of semen

A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen.     

Oestrogenic activity of CPRG (chlorophenol red-β-D-galactopyranoside), a β-galactosidase substrate commonly used in recombinant yeast oestrogenic assays

The finding that a variety of chemicals display oestrogenic activity has resulted in the development of in vitro and in vivo assays to assess oestrogenic activity. One such assay, the yeast oestrogen assay (YES) makes use of recombinant yeast cells that harbour an oestrogen receptor expression cassette and a reporter construct, coding for bgalactosidase. The induction mechanism starts with the binding of oestrogenic compounds to the oestrogen receptor. This complex activates the production of β-galactosidase. The β-galactosidase activity is thus a measure of the oestrogenic activity of chemical compounds. In the YES assay, the β-galactosidase activity may be quantified with the chromogenic substrate chlorophenol red-β-d-galactopyranoside (CPRG). In the present study it is reported that CPRG or its β-galactosidase degradation product chlorophenol red act in the YES as an oestrogenic compound itself. The implications of this finding are described. It is especially argued that chlorophenol red production after prolonged incubation of the assay might be misinterpreted as an oestrogenic effect of the test compound.     

Measurement of microsomal ATPase activities: a comparison between the inorganic phosphate-release assay and the NADH-coupled enzyme assay

The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted.     

A nonradioactive,high throughput assay for chitin synthase activity

Wheat germ agglutinin (WGA) binds with high affinity and specificity to several sites on chitin polymers. Based on these properties we have modified and adapted a previously patented (U.S. patent 5,888,757) nonradioactive, high throughput screening assay for antimicrobial agents, making it suitable as a quantitative enzymatic assay for the activity of individual chitin synthase isozymes in yeast. The procedure involves binding of synthesized chitin to a WGA-coated surface followed by detection of the polymer with a horseradish peroxidase-WGA conjugate. Horseradish peroxidase activity is then determined as an increment in absorbance at 600 nm. Absorbance values are converted to amounts of chitin using acid-solubilized chitin as a standard. The high sensitivity (lower limit of detection about 50 ng chitin), low dispersion (lower than 10%), and high throughput (96-well microtiter plate format) make this assay an excellent substitute for the conventional radioactive chitin synthase assay in cell-free extracts. We have applied this method to the differential assay of chitin synthase activities (Chs1, Chs2, and Chs3) in cell-free extracts of Saccharomyces cerevisiae. Analysis of Chs3 activity in chitosomal and plasma membrane fractions revealed that Chs3 in the plasma membrane fraction is about sixfold more active than in the chitosome.     

Development of an Immunofluorometric, High-Capacity, Cell-Based Assay for the Measurement of Human Type I and Type II Interferons

We have developed a cell-based 96-well microtiter plate, high throughput assay for measuring both type I and type II interferon (IFN) activity on human cells. This assay makes use of a previously described IFN-specific reporter stably expressed in human HT 1080 cells. The induction of the reporter by IFN is determined by measuring the IFN-dependent expression of CD2 on the cell surface. The cytokine-induced expression of CD2 occurs within 48 h and is measured using a time-resolved fluorometric immunoassay. The limit of detection for type I IFN is.0.4 IU/ml. Interassay and intraassay coefficients of variation were 1.1% and 1.3% for the medium control (31 IU IFNb1b/ml), respectively. The limit of detection for type II IFN is.8 IU/ml, and the assay coefficients of variation are similar to those determined for type I IFNs. The level of sensitivity for this assay is comparable to other assays commonly used to measure IFN activity on cells. The current assay has an advantage over antiviral and antiproliferative assays, in that there is no requirement for the use of pathogenic virus or for determining viable cell numbers. The current assay is ideally suited for increasing sample screening and high-capacity automation, making it an excellent tool for drug discovery.     

A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases

The use of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) labeled casein in autoquenching assays of proteolytic activity has been recently described, and we have adapted this assay to measurement of calpain activity. BODIPY-FL coupled to casein at a ratio of 8 mol of BODIPY-FL/mol of casein or higher produces a BODIPY-FL-casein substrate that can be used in an autoquenching assay of calpain proteolytic activity. This assay has a number of advantages for measuring calpain activity. (1) The procedure does not require precipitation and removal of undegraded protein, so it is much faster than other procedures that require a precipitation step, and it can be used directly in kinetic assays of proteolytic activity. (2) The BODIPY-FL-casein assay is easily adapted to a microtiter plate format, so it can be used to screen large numbers of samples. (3) Casein is an inexpensive and readily available protein substrate that more closely mimics the natural substrates of endoproteinases, such as the calpains, than synthetic peptide substrates do. Casein has K(m) values for micro- and m-calpain that are similar to those of other substrates such as fodrin or MAP2 that may be "natural" substrates for the calpains, and there is no reason to believe that calpain hydrolysis of casein is inherently different from hydrolysis of fodrin or MAP2, which are much less accessible as substrates for protease assays. (4) The BODIPY-FL-casein assay is capable of detecting 10 ng ( approximately 5 nM) of calpain and is nearly as sensitive as the most sensitive calpain assay reported thus far. (5) The BODIPY-FL-casein assay is as reproducible as the FITC-casein assay, whose reproducibility is comparable to or better than the reproducibility of other methods used to assay calpain activity. The BODIPY-FL-casein assay is a general assay for proteolytic activity and can be used with any protease that cleaves casein.     

A double-reporter splicing assay for determining splicing efficiency in mammalian cells

Changes in alternative splicing patterns can result from both inherited and acquired defects, and they are increasingly recognized as causes of human diseases. Hence, improvements in the understanding of alternative splicing regulation may provide opportunities for restoring productive patterns of splicing. The identification of factors (such as proteins, nucleic acids or small molecules) that modulate the splicing pattern would be facilitated by systems with which many samples can be screened. The absence of reliable systems prompted us to develop an assay system based on dual enzymatic activities. Two distinct signals derived from spliced and unspliced RNA are measured, providing the basis for a robust, rapid and convenient assay for investigating splicing. This protocol describes how to use this system; the time required for lysing the cells and recording enzymatic activity is about 2 hours.     

Measurement of serum bone-specific alkaline phosphatase activity in cynomolgus macaques

Serum levels of bone-specific alkaline phosphatase activity (B-ALP) in cynomolgus monkeys were evaluated as an index of elevated bone turnover following ovariectomy. The enzyme immunoassay 96-well microtiter plate B-ALP assay, developed by Metra Biosystems (Mountain View, CA) for human use, was employed and compared with a standard automated assay measuring total serum levels of alkaline phosphatase activity (T-ALP). The B-ALP assay was first validated for use in these monkeys. Ovariectomy led to increased bone turnover as indicated by approximately 2-fold higher activity in both assays and this elevation was inhibited by daily estradiol administration. Although both assays provided generally similar results, several monkeys were observed to have greatly elevated values of T-ALP but not B-ALP. This discrepancy is believed to result from high levels of the liver isoform of alkaline phosphatase in monkeys with hepatic dysfunction, which are not detected by the B-ALP assay.     

Monomeric and Multimeric Blockers of Selectins: Comparison of <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> Activity

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO(3)Le(a)-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLe(x) is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO(3)Le(a)-PAA-sTyr.     

Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase

An assay method is described for measurement of absolute concentrations of the molybdenum cofactor, based on complementation of the defective nitrate reductase ('apo nitrate reductase') in extracts of the nit-1 mutant of Neurospora crassa. A number of alternative methods are described for preparing, anaerobically, molybdenum-cofactor-containing solutions from sulphite oxidase, xanthine oxidase and desulpho xanthine oxidase. For assay, these were mixed with an excess of extract of the nit-1 mutant, incubated for 24 h at 3.5 degrees C then assayed for NADPH:nitrate reductase activity. In all cases, the specific activity of the molybdenum cofactor, expressed as mumol of NO2-formed/min per ng-atom of Mo added from the denatured molybdoenzyme , was 25 +/- 4, a value that agrees with the known catalytic activity of the nitrate reductase of wild-type Neurospora crassa. This indicates that, under our conditions, there was quantitative transfer of the molybdenum cofactor from denatured molybdoenzyme to yield fully active nitrate reductase. Comparable cofactor assay methods of previous workers, apparently indicating transfer efficiencies of at best a few per cent, have never excluded satisfactorily the possibility that cofactor activity arose, not from stoichiometric constituents of the molybdoenzymes , but from contaminants. The following factors were investigated separately in developing the assay:the efficiency of extraction of the cofactor from the original enzyme, the efficiency of the complementation reaction between cofactor and apo nitrate reductase, and the assay of the resultant nitrate reductase, which must be carried out under non-inhibitory conditions. Though the cofactor is unstable in air (t1/2 about 15 min at 3.5 degrees C), it is stable when kept anaerobic in the presence of sodium dithionite, in aqueous solution or in dimethyl sulphoxide (activity lost at the rate of about 3%/24 h at 20-25 degrees C). Studies of stabilities, and investigations of the effect of added molybdate on the assay, permit conclusions to be drawn about the ligation of molybdenum to the cofactor and about steps in incorporation of the cofactor into the apoenzyme. Though the development of nitrate reductase activity is slow at 3.5 degrees C (t1/2 1.5-3 h) the complementation reaction may be carried out in high yield, aerobically. This is ascribed to rapid formation of an air-stable but catalytically inactive complex of the cofactor, as a precursor of the active nitrate reductase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of hemoglobin through its peroxidase activity on chlorpromazine

Chlorpromazine is an excellent chromogen for determining micro-quantities of hemoglobin. The oxidation of chlorpromazine by peroxidase activity of hemoglobin is coupled to a non-enzymatic reaction of second-order. Kinetic analysis of the overall system leads to a discussion about the optimal assay conditions. Spectrophotometric progress curves for the accumulation of the chlorpromazine cation radical during the reaction have been obtained, and further analyzed by non-linear regression. The use of a linear calibration curve of the enzymatic reaction rate against hemoglobin concentration is proposed for its determination.     

A rapid and sensitive quantitative kinase activity assay using a convenient 96-well format

Activation of protein kinases in response to growth factor and extracellular matrix stimulation has been implicated in regulating a number of cell functions including differentiation, gene expression, migration, and proliferation. An improved quantitative assay for measuring protein kinase activity is crucial to the detailed study of this important category of signaling proteins and their role in regulating cell behavior. We describe a modified in vitro kinase activity assay that is both sensitive and quantitative. It offers several advantages when compared to the traditional immunoprecipitation/kinase assay: (i) high sensitivity that reduces the required amount of cell lysate by an order of magnitude, (ii) an immunoseparation technique utilizing antibody immobilization onto the surface of microtiter wells that replaces the cumbersome immunoprecipitation method, (iii) a 96-well plate configuration that eases handling of multiple samples and increases throughput of the assay, and (iv) the use of 96-well filter plates that greatly reduces radioactive liquid waste generation. While we implement this technique in a case study for measuring the activity of extracellular signal-regulated kinase 2 (ERK2), this assay can be extended to studying other protein kinases by using an appropriate antibody and in vitro substrate for the kinase of interest.     

Fluorimetric detection of octopamine in high-performance liquid chromatography and its application to the assay of dopamine β-monooxygenase in human serum

A high-performance liquid chromatographic procedure is described for the determination of octopamine. The method, which is based on the separation on a microparticulate bonded strong cation-exchange resin and measurement of the native fluorescence, has been applied to give a sensitive assay of dopamine β-monooxygenase (EC 1.14.17.1) activity in human serum with tyramine as the substrate. The procedure, which has been designed for use with an-automatic sampler, has a detection limit of about 50 pmoles of octopamine, and the analysis time is approximately 10 min per sample.     

Flow cytometric detection of micronuclei and cell cycle alterations in fish-derived cells after exposure to three model genotoxic agents: mitomycin C, vincristine sulfate and benzo(a)pyrene

The measurement of cytogenetic alterations in vitro is considered an initial step in the risk assessment procedures for genotoxic agents. The concern about genotoxic pollutants in natural fish population makes the use of fish-derived cells an useful tool for these purposes. The technological improvements in well-established cytogenetic endpoints, such as micronuclei (MN) estimations by means of flow cytometry, have been proposed in the later years using mammalian cells. In this work, we test the capability of flow cytometry to evaluate MN induction and cell cycle alterations in an established fish cell line (RTG-2) using three agent-inductor models at different concentrations and exposure periods. For mitomycin C, an inverse relationship between length of exposure period and concentrations was observed. A dose-response relationship was observed after exposing RTG-2 cells to vincristine sulfate and benzo(a)pyrene. As this study shows, RTG-2 cells respond to clastogenic and aneugenic effects of the tested chemicals through the induction of MN at similar doses to mammalian cells and without the addition of exogenous metabolic activity. The possibility to check cell cycle alterations, in the same sample, gives the opportunity to evaluate early signals of cytotoxicity. The use of flow cytometry improves the assay by means of its speed and objectivity, which makes the assay very useful for genotoxicity assessment of aquatic chemicals.     

Detection of glycosylase, endonuclease and methyltransferase enzyme activities using immobilized oligonucleotides

A novel rapid assay for detection of DNA glycosylase, restriction endonuclease, and DNA methyltransferase enzyme activities is presented. The assay is based on enzyme-dependent label release (in case of glycosylase and endonuclease), or non-release (in case of methyltransferase) into solution from end-labeled DNA immobilized on solid support (CPG or Tenta Gel S-NH2). The assay has been validated for monitoring activity of repair enzyme uracil-DNA glycosylase, restriction endonucleases SsoII, MvaI and EcoRII and (cytosine-5)-DNA methyltransferase SsoII. Two types of labels have been tested and found compatible with the assay: radioactive (32P) and fluorescent (rhodamine B and fluorescein). The enzyme activity is estimated as a ratio of the label released into solution to the total amount of the label. Use of fluorescent labeling facilitates detection while use of solid phase-immobilized substrates facilitates product separation, improved assay sensitivity, and increases throughput of assay. Proposed technique provides an estimate of enzyme activity but not its specific activity. Thus, the assay will most valuable in the applications where rapid estimation of enzyme activity is necessary.