Calibration of a flow cytometric assay of glucose-6-phosphate dehydrogenase activity.

The reduction of tetrazolium salts to colored formazans is a reaction which has been exploited both in histo- and cytochemistry. Tetrazolium salts forming fluorescent formazans prove suitable for measuring defined cellular dehydrogenase activities in automated processes. This study considers an important aspect of formazan measurement in flow cytometry, namely, calibration. Calibration is performed by correlating the number (and fluorescence intensity) of formazan-bearing cells measured by flow cytometry with simultaneously performed biochemical analyses of the same material. The method is demonstrated by an example of glucose-6-phosphate dehydrogenase. Using the data of a typical experiment, the enzyme activity is expressed in femtomol of hydrogen transferred per cell during incubation time. Furthermore, through spatially resolved double excitation of formazan and nuclear DAPI fluorescence, an independent analysis of cell cycle and cellular enzymatic activity is established.     

Rapid method for the determination of choline kinase activity. Choline kinase in marine invertebrates

A quick sensitive test is suggested to determine choline kinase (EC 2.7.1.32). The method includes incubation of a substrate with enzyme in microvolume and separation of the formed phosphorylcholine from the initial choline by high-performance thin-layer silica gel chromatography. The procedure of determination is simple, reproducible and takes no more than 30 min. The method reveals high activity of choline kinase in some marine invertebrates. Certain features of the enzyme from the intestine of strongylocentrotus intermedius are described.     

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 μL of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 μL or less are now possible using a dedicated microvolume fluorospectrometer.Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ μL to 15,000 ng/ μL with minimal consumption of sample.     

Mg2＋对阿霉素引起心肌线粒体F1F0变化的保护

抗肿瘤药物阿霉素(ADM)对心肌线粒体F₁F₀-复合体呈现抑制而对F₁-ATPase无抑制,这表明ADM可能是通过膜脂起作用的,适当浓度Mg²⁺能降低ADM对复合体的抑制.经 ³¹P-NMR和标记荧光探针NBD-PE,DPH,MC-540以及内源荧光等的测定,结果表明ADM可能首先通过诱导F₁F₀膜脂形成非双层脂结构,继而影响了膜脂的堆积程度和流动性,进而引起F₁F₀-复合体酶蛋白构象的改变,最终导致酶活力的降低.Mg^2＋则可能由于与ADM竞争与心磷脂的结合,而对ADM引起F₁F₀的变化产生保护作用.     

A sensitive fluorometric assay for plasminogen, plasmin, and streptokinase

A rapid, convenient, and highly sensitive fluorometric assay for plasmin (P), plasminogen (Pg), and streptokinase (SK) as the activator complex (SK·P) is described. These assays are based on the measurement of the fluorescence of β-naphthol (βN) released from α-N-methyl α-N-tosyl-l-lysine β-naphthol ester (MTLNE) by the P present or generated during the assay. The rate of βN release may be followed by direct recording or determined subsequently, following termination of the enzyme reaction at a fixed digestion time, by the addition of p-nitrophenyl-p′-guanidinobenzoate. The latter method may be readily automated. The K_m and V values for the hydrolysis of MTLNE by P or SK·P are equivalent. The Pg activator activity of P was shown to be very small (less than 0.2% of that of SK·P).     

Inactivation of mitochondrial succinate dehydrogenase by adriamycin activated by horseradish peroxidase and hydrogen peroxide

Although human cancers are widely treated with anthracycline drugs, these drugs have limited use because they are cardiotoxic. To clarify the cardiotoxic action of the anthracycline drug adriamycin (ADM), the inhibitory effect on succinate dehydrogenase (SDH) by ADM and other anthracyclines was examined by using pig heart submitochondrial particles. ADM rapidly inactivated mitochondrial SDH during its interaction with horseradish peroxidase (HRP) in the presence of H(2)O(2) (HRP-H(2)O(2)). Butylated hydroxytoluene, iron-chelators, superoxide dismutase, mannitol and dimethylsulfoxide did not block the inactivation of SDH, indicating that lipid-derived radicals, iron-oxygen complexes, superoxide and hydroxyl radicals do not participate in SDH inactivation. Reduced glutathione was extremely efficient in blocking the enzyme inactivation, suggesting that the SH group in enzyme is very sensible to ADM activated by HRP-H(2)O(2). Under anaerobic conditions, ADM with HRP-H(2)O(2) caused inactivation of SDH, indicating that oxidized ADM directly attack the enzyme, which loses its activity. Other mitochondrial enzymes, including NADH dehydrogenase, NADH oxidase and cytochrome c oxidase, were little sensitive to ADM with HRP-H(2)O(2). SDH was also sensitive to other anthracycline drugs except for aclarubicin. Mitochondrial creatine kinase (CK), which is attached to the outer face of the inner membrane of muscle mitochondria, was more sensitive to anthracyclines than SDH. SDH and CK were inactivated with loss of red color of anthracycline, indicating that oxidative activation of the B ring of anthracycline has a crucial role in inactivation of enzymes. Presumably, oxidative semiquinone or quinone produced from anthracyclines participates in the enzyme inactivation.     

Relationship between contents of adrenomedullin and distributions of neutral endopeptidase in blood and tissues of rats in septic shock

Adrenomedullin (ADM), a multifunction peptide with important roles in regulating cardiovascular homeostasis, has the vasodilatory properties and is of particular interest in the pathophysiology of sepsis. ADM levels in plasma and tissues are regulated by the proteolysis of neutral endopeptidase (NEP), the major enzyme of ADM degradation. We observed the NEP activity in the plasma, the activity and distribution of NEP and its mRNA expression in the tissues of rats in septic shock to study the possible role of NEP in elevating tissue ADM concentration during sepsis. ADM level increases progressively during sepsis except in the jejunum. Rats in early phase of shock (ES) showed diverse changes in tissue NEP activity. Plasma NEP activity, tissue NEP activity and its protein and mRNA expression in the left ventricle, aorta, jejunum and lung in the late phase of shock (LS) rats were lower than those in ES and the control, but no statistical change of NEP activity in the kidney was observed. The level of ADM was inversely correlated with NEP activity in the plasma, ventricle and aorta and positively correlated with NEP activity in the jejunum. Thus, in sepsis, the local concentration and action of ADM in tissues may be differentially regulated by NEP.     

An intramolecularly quenched fluorescent tripeptide as a fluorogenic substrate of angiotensin-I-converting enzyme and of bacterial dipeptidyl carboxypeptidase.

The N-acyltripeptide 2-aminobenzoylglycyl-p-nitrophenylalanylproline was synthesized and applied as a substrate in the assay of angiotensin-I-converting enzyme from calf lung and human serum, and of the bacterial dipeptidyl carboxypeptidase from Escherichia coli. This compound belongs to a new class of substrates for proteolytic enzymes, having the general structure F--X--Q in which fluorescence of group F is quenched by intramolecular interaction with the group Q. Enzymatic cleavage of the peptide chain (X stands for one or more amino acid residues) generates the unquenched F-containing derivative and the resulting fluorescence is used for quantitative measurement of the hydrolysis rate. Cleavage of the Gly-Phe(NO2) peptide bond in the weakly fluorescent 2-amino-benzoylglycyl-p-nitrophenylalanylproline molecule results in appearance of the 71 times higher fluorescence (lambdamax = 415 nm) of 2-aminobenzoylglycine. Continuous recording of the rising fluorescence allows convenient, sensitive and specific determination of the enzymatic activity, applicable to crude enzyme preparations and human serum. The activity of the mammalian enzyme, measured by this method, is enhanced by Cl- ions and inhibited by low concentrations of EDTA and [Asn1, Val5]angiotensin II. Kinetic measurements showed Michaelis-Menten behavior, Km = 0.21 +/- 0.1 mM and 0.16 +/- 0.1 mM for the calf lung and the bacterial enzyme respectively.     

Determination of angiotensin I-converting enzyme activity in cell culture using fluorescence resonance energy transfer peptides

An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. The advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity. The kinetic parameter K(m) for the hydrolysis of the three substrates by ACE in this system was also determined and the values are comparable to those obtained using the purified enzyme in solution. The specificity of the activity was demonstrated by the complete inhibition of the hydrolysis by the ACE inhibitor lisinopril. Therefore, the results presented in this work show for the first time that determination of ACE activity directly on the surface of intact CHO cells is feasible and that the method is reliable and sensitive. In conclusion, we describe a methodology that may represent a new tool for the assessment of ACE activity which will open the possibility to study protein interactions in cells in culture.     

Two-photon fluorescence cross-correlation spectroscopy as a potential tool for high-throughput screening of DNA repair activity

Several lines of evidence indicate that differences in DNA repair capacity are an important source of variability in cancer risk. However, traditional assays for measurement of DNA repair activity in human samples are laborious and time-consuming. DNA glycosylases are the first step in base excision repair of a variety of modified DNA bases. Here, we describe the development of a new sensitive DNA glycosylase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. FCCS was applied to the measurement of uracil DNA glycosylase activity of human cell extracts and validated by comparison with standard gel electrophoresis assay. Our results indicate that FCCS can be adapted to efficient assays for DNA glycosylase activity in protein extracts from human cells. This method has a potential for the development of automated screening of large number of samples.     

Inhibition of the adrenomedullin/nitric oxide signaling pathway in early diabetic retinopathy

The nitric oxide (NO) signaling pathway is integrally involved in visual processing and changes in the NO pathway are measurable in eyes of diabetic patients. The small peptide adrenomedullin (ADM) can activate a signaling pathway to increase the enzyme activity of neuronal nitric oxide synthase (nNOS). ADM levels are elevated in eyes of diabetic patients and therefore, ADM may play a role in the pathology of diabetic retinopathy. The goal of this research was to test the effects of inhibiting the ADM/NO signaling pathway in early diabetic retinopathy. Inhibition of this pathway decreased NO production in high-glucose retinal cultures. Treating diabetic mice with the PKC β inhibitor ruboxistaurin for 5 weeks lowered ADM mRNA levels and ADM-like immunoreactivity and preserved retinal function as assessed by electroretinography. The results of this study indicate that inhibiting the ADM/NO signaling pathway prevents neuronal pathology and functional losses in early diabetic retinopathy.     

Screening for enzyme activity in turbid suspensions with scattered light

New screening techniques for improved enzyme variants in turbid media are urgently required in many industries such as the detergent and food industry. Here, a new method is presented to measure enzyme activity in different types of substrate suspensions. This method allows a semiquantitative determination of protease activity using native protein substrates. Unlike conventional techniques for measurement of enzyme activity, the BioLector technology enables online monitoring of scattered light intensity and fluorescence signals during the continuous shaking of samples in microtiter plates. The BioLector technique is hereby used to monitor the hydrolysis of an insoluble protein substrate by measuring the decrease of scattered light. The kinetic parameters for the enzyme reaction (V(max,app) and K(m,app)) are determined from the scattered light curves. Moreover, the influence of pH on the protease activity is investigated. The optimal pH value for protease activity was determined to be between pH 8 to 11 and the activities of five subtilisin serine proteases with variations in the amino acid sequence were compared. The presented method enables proteases from genetically modified strains to be easily characterized and compared. Moreover, this method can be applied to other enzyme systems that catalyze various reactions such as cellulose decomposition.     

A particle concentration fluorescence immunoassay for prostaglandin D synthase in the rat central nervous system

A solid phase, particle concentration fluorescence immunoassay (PCFIA) was developed for the measurement of prostaglandin (PG) D synthase in the 100,000g supernatant of various regions of the rat central nervous system. In this assay, the enzyme (in the range of 1-25 micrograms protein of brain supernatant or 1-100 ng of the purified enzyme) is attached to submicrometer carboxypolystyrene beads coated with polyclonal anti-rat brain PGD synthase IgG. The total particle-bound enzyme is assayed with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-PGD synthase IgG after incubation for 1 h. The optimum assay condition was obtained when carboxyl particles coated with ca. 500 micrograms/ml of polyclonal IgG at pH 5.0 and 5 micrograms/ml of FITC-IgG were used. No significant fluorescence was observed when FITC conjugates or carboxyl particles were prepared using IgG from nonimmunized rabbits. Heat treatment of the brain supernatant decreased the specific binding of the enzyme in parallel with the loss of enzyme activity, indicating that the denatured enzyme is not recognized by this assay method. The PGD synthase immunoreactivity was widely distributed in the brain regions and was highest in the paraflocculus. Although slight discrepancy was observed between the concentration by PCFIA and the enzyme activity measured by using [14C]PGH2 in some brain regions, there is a considerable correlation (0.727) between the values by both methods in the same brain regions. The PCFIA now developed showed higher sensitivity (around 10 times), greater reliability, and larger number of samples measurable at once than the radio-TLC assay using [14C]PGH2. This method could provide valuable information concerning the regulatory mechanisms of PGD synthase.     

Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity

Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.     

An automated continuous assay of membrane-bound and solube ATPases and related enzymes.

A simple, rapid, and precise method is described for the continuous automated determination of the activity of membrane-bound enzymes which deliberate inorganic phosphate, e.g., ATPases and phosphatases. The characteristics of this method, which is based on the determination of liberated phosphate in the presence of nucleotides, are: (A) the enzyme reaction can be followed continuously during a certain period, thus providing a higher precision, as compared to other methods in which the enzyme reaction is measured by few distinct determinations; (B) the enzyme protein and other (membrane) proteins of the enzyme preparation have not to be removed during the continuous determination of enzyme activity because they remain solubilized after denaturation; and (C) low or moderate concentration of nonionic detergents do not disturb the reading of the absorbancy.     

A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay

Angiotensin I-converting enzyme (ACE) is involved in various physiological and physiopathological conditions; therefore, the measurement of its catalytic activity may provide essential clinical information. This protocol describes a sensitive and rapid procedure for determination of ACE activity using fluorescence resonance energy transfer (FRET) substrates containing o-aminobenzoic acid (Abz) as the fluorescent group and 2,4-dinitrophenyl (Dnp) as the quencher acceptor. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that can be detected continuously, allowing quantitative measurement of the enzyme activity. The FRET substrates provide a useful tool for kinetic studies and for ACE determination in biological fluids and crude tissue extracts. An important benefit of this method is the use of substrates selective for the two active sites of the enzyme, namely Abz-SDK(Dnp)P-OH for N-domain, Abz-LFK(Dnp)-OH for C-domain and Abz-FRK(Dnp)P-OH for somatic ACE. This methodology can be adapted for determinations using a 96-well fluorescence plate reader.     

Phagocytosis, intracellular pH, and cell volume in the multifunctional analysis of granulocytes by flow cytometry

Phagocytosis of Escherichia coli K12 strain bacteria was used to measure by flow cytometry the functional activities of human granulocytes in whole blood or buffy coat preparations. In a first measurement, the increase in electric cell volume and acridine orange (AO) green and red fluorescence were used to quantify the degree of phagocytosis. In a second measurement, the intracellular pH and esterase activity of each cell were determined with 1,4-diacetoxy-2,3-dicyanobenzene to obtain information on the metabolic activities during phagocytosis and degradation of bacteria. The DNA of dead cells was simultaneously counterstained with propidium iodide in both assays. The volume, the AO green and red fluorescence, the internal pH, and esterase activity were automatically averaged for all granulocytes or lymphocytes of a measurement. The calculated mean values were transferred into the self-learning database of the DIAGNOS1-program system. The functional granulocyte parameters of normal healthy individuals can be used as reference values for the automated diagnosis of abnormal granulocytes in various infectious disease states. The assays require 1 ml of heparinized whole blood and the results are available within 1 hour.     

A critical role for adrenomedullin-calcitonin receptor-like receptor in regulating rheumatoid fibroblast-like synoviocyte apoptosis

Rheumatoid arthritis (RA) is characterized by fibroblast-like synoviocyte (FLS) hyperplasia, which is partly ascribable to decreased apoptosis. In this study, we show that adrenomedullin (ADM), an antiapoptotic peptide, is constitutively secreted in larger amounts by FLS from joints with RA (RA-FLS) than with osteoarthritis (OA-FLS). ADM secretion was regulated by TNF-alpha. Peptidylglycine alpha-amidating monooxygenase, the ADM-processing enzyme, was expressed at the mRNA level by both RA-FLS and OA-FLS. Constituents of the ADM heterodimeric receptor calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)-2 were up-regulated at the mRNA and protein levels in cultured RA-FLS compared with OA-FLS. ADM induced rapid intracellular cAMP production in FLS and reduced caspase-3 activity, DNA fragmentation, and chromatin condensation in RA-FLS exposed to apoptotic conditions, indicating that CRLR/RAMP-2 was fully functional. ADM-induced cAMP production was less marked in OA-FLS than in RA-FLS, suggesting differences in receptor regulation and expression. ADM dose-dependently inhibited RA-FLS apoptosis, and this effect was reversed by the 22-52 ADM antagonist peptide. ADM inhibited RA-FLS apoptosis triggered by extrinsic and intrinsic pathways. Our data suggest that ADM may prevent or reduce RA-FLS apoptosis, via up-regulation of its functional receptor CRLR/RAMP-2. Regulation of ADM secretion and/or CRLR/RAMP-2 activation may constitute new treatment strategies for RA.     

A fluorimetric method for measuring the activity of soil enzymes

Summary A fluorimetric method is described for the measurement of the activity of a range of soil enzymes. The method is based on the measurement of 4-methylumbelliferone (MUB), a fluorescent product liberated on hydrolysis of the enzyme substrate. The main advantage of the method over colorimetric techniques is that separation of MUB from the soil is unnecessary and the method is therefore suitable for routine, automated analyses. The method was used to measure the activity of β-cellobiase, β-galactosaminidase, β-glucosidase and β-xylosidase over a wide range of substrate concentration and in a range of soils. Kinetic parameters are reported for these enzymes. The method was also shown to be suitable for the assay of arylsulphatase and acid and alkaline phosphatase in soil. The technique should be applicable to a wide range of soil hydrolases, using the same assay methods.