A single-cell assay of beta-galactosidase in recombinant Escherichia coli using flow cytometry

A flow cytometric method was developed for the assay of beta-galactosidase in single Escherichia coli cells. A new fluorogenic substrate for beta-galactosidase, C(12)FDG, contains a lipophilic group that allows the substrate to penetrate through cell membranes under normal conditions. When the substrate is hydrolyzed by intracellular beta-galactosidase, a green fluorescent product is formed and retained inside the cell. Consequently, the stained beta-galactosidase-positive cells exhibit fluorescence, which is detected by flow cytometry. This new assay was used to analyze the segregational instability caused by a reduction in specific growth rate of the plasmid-bearing cells in the T7 expression system. Induction results in a substantial accumulation of intracellular beta-galactosidase along with a rapid increase in the fraction of plasmid-free cells. Once the cells lose the plasmid, they no longer produce beta-galactosidase, which is reduced by at least half every generation; thus, after staining, the fluorescent, plasmid-bearing cells can be distinguished from the nonfluorescent, plasmid-free cells using flow cytometry. This article describes the feasibility of the flow cytometric assay for single E. coli cells and reports the optimal assay conditions. A direct relationship between beta-galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of induction.     

A single-cell assay of beta-galactosidase activity in Saccharomyces cerevisiae

A novel assay of single-cell exogenous beta-galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin-beta-D-galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell. The cells are permeabilized with Triton X-100, and the assay is performed at 0 degrees C. These conditions were chosen to minimize intercellular fluorescence communication. Free resorufin in the extracellular space is bound by bovine serum albumin to prevent its uptake by cells. Two regimes of fluorescence accumulation are observed, one limited by the rate of diffusion of substrate into the cell, and one limited by the rate of enzymatic cleavage of the substrate. A quantitative correlation between fluorescence and beta-galactosidase activity is obtained under optimized assay conditions.     

Kinetic assay of fluorescein mono-beta-D-galactoside hydrolysis by beta-galactosidase: a front-face measurement for strongly absorbing fluorogenic substrates

A novel enzymatic assay method was developed for fluorogenic substrates that have significant intrinsic absorbance and fluorescence under the assay conditions. Fluorescein mono-beta-D-galactoside (FMG) was chosen as the substrate for the fluorescence enzymatic assay because of the high fluorescence of its hydrolytic product (fluorescein) and suitability of being hydrolyzed by beta-galactosidase. The fluorescence-concentration relationships for fluorescein and for FMG in both the right-angle detection mode of a fluorometer and the front-face detection mode of a fluorescence plate reader were exactly established and used to determine the kinetics of the enzyme assay. The results show that only front-face detection in the fluorescence plate reader can overcome the fluorescence concentration quenching that inevitably results from high absorbance by the intrinsically absorbing substrate in the conventional fluorometer, which utilizes right-angle detection. Only with front-face detection was the fluorescent assay of FMG hydrolysis under conditions of high optical density possible. The enzymatic measurements on the fluorescence plate reader were particularly efficient for determination of the enzyme kinetics because of the high rate of data collection. In this assay system, Michaelis-Menten constant Km and enzymatic catalysis rate k2 of FMG were determined as 117.6 microM and 22.7 mumol-(min.mg)-1, respectively. The results and methods described in this paper can be generalized for any assay using a fluorogenic substrate whether or not it has a high background absorbance.     

Construction of a flocculent Saccharomyces cerevisiae strain secreting high levels of Aspergillus niger β-galactosidase

A flocculent Saccharomyces cerevisiae strain secreting Aspergillus niger beta-galactosidase activity was constructed by transforming S. cerevisiae NCYC869-A3 strain with plasmid pVK1.1 harboring the A. niger beta-galactosidase gene, lacA, under the control of the ADH1 promoter and terminator. Compared to other recombinant S. cerevisiae strains, this recombinant yeast has higher levels of extracellular beta-galactosidase activity. In shake-flask cultures, the beta-galactosidase activity detected in the supernatant was 20 times higher than that obtained with previously constructed strains (Domingues et al. 2000a). In bioreactor culture, with cheese-whey permeate as substrate, a yield of 878.0 nkat/gsubstrate was obtained. The recombinant strain is an attractive alternative to other fungal beta-galactosidase production systems as the enzyme is produced in a rather pure form. Moreover, the use of flocculating yeast cells allows for enzyme production with high productivity in continuous fermentation systems with facilitated downstream processing.     

A new resorufin-based α-glucosidase assay for high-throughput screening

Mutations in α-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, α-glucosidase is a therapeutic target for type II diabetes, and α-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the α-glucosidase enzyme assay, resorufin α-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-α-d-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK_a of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the α-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.     

Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations

A new fluorescent stain has been developed for detecting cloned beta-galactosidase activity in individual cells of Saccharomyces cerevisiae by flow cytometry. The staining reaction is based on enzymatic cleavage of alpha-naphthol-beta-D-galactopyranoside by intracellular beta-galactosidase and trapping of the liberated naphthol by hexazoniumpararosaniline yielding a fluorescent, insoluble end product. This stain, in connection with an appropriate host strain, has been applied for detecting plasmids encoding inducible beta-galactosidase in unstable recombinant cell populations carrying plasmids with different origins of replication. The method enables rapid determination of the fraction of plasmid-containing cells as well as quantitation of intracellular beta-galactosidase content by kinetic enzyme assay. Inducibility of the marker enzyme is important for maintaining correlation between enzyme and gene content.     

Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems.

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase. Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.     

Fluorogenic substrate turnover in single living cells

Synopsis Intracellular diffusion properties and enzyme activities in single living cells can be analysed by means of fluorogenic substrates that diffuse into the cells where they are converted into a fluorescent product by an enzymic reaction. The reaction-kinetic analysis of this process as a system of consecutive reactions provides information on the diffusion of the substrate into the cells, on intracellular enzyme activities and on the efflux of the fluorescent product. Separation of diffusion and enzyme-mediated processes is obtained by inducing specific changes of the cellular membrane using gramicidin D. A model for the functional interpretation of the experimental findings is proposed. Application of this method as a viability test for freshly prepared and frozen platelets is discussed.Paper given at the Royal Microscopical Society's European Histochemistry Meeting at Nottingham in September 1975.     

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the experimentally injured rabbit cornea and tear fluid using the sensitive substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC)

Comparative histochemical and biochemical studies on acid beta-galactosidase activity in the rabbit eye after various experimental injuries were performed using the same sensitive fluorogenic substrate beta-galactoside-4-trifluoromethylumbelliferyl (HFC). The aim of the study was to examine whether the severity of corneal damage corresponds with the level of the enzyme activity in the tear fluid. As until recently the substrate beta-galactoside-4-HFC had not been used for the histochemical detection of acid beta-galactosidase in the cornea, results obtained with this substrate in a fluorescent method were compared in parallel cryostat sections with results obtained using the substrate 5-bromo-4-chloro-3-indoxyl beta-galactoside in the indigogenic method (previously shown to be very sensitive for the detection of acid beta-galactosidase activity in the cornea). Both methods revealed similar localization and changes in enzyme activity; using beta-galactoside-4-HFC an acceptable cellular localization was achieved. For the measurement of acid beta-galactosidase activity in the tear fluid a semiquantitative biochemical method was elaborated using filter paper punches with the substrate (beta-galactoside-4-HFC) soaked with tears and incubated at 37 degrees C. The time of the first appearance of a greenish-yellow fluorescence (enzyme positivity) was recorded by UV lamp and compared with the appearance of fluorescence in calibrated punches containing known acid beta-galactosidase activities. The results show that beta-galactoside-4-HFC is useful for the biochemical assessment of acid beta-galactosidase activity in the tear fluid. Comparing histochemical and biochemical results, it can be concluded that increased enzymatic activity in tears parallels the severity of corneal damage. Further studies are necessary to evaluate whether the detection of acid beta-galactosidase activity in tears might be useful for diagnostic purposes in humans.     

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.     

Quantitative immunofluorescence in single Saccharomyces cerevisiae cells

We have developed a staining procedure that allows the simultaneous determination of intracellular amounts of DNA and an antigen in Saccharomyces cerevisiae with a single laser flow cytometer. The antigen, beta-galactosidase from a cloned lacZ gene, is inducible and is detected with an indirect immunofluorescent stain. Cell preparation procedures, specifically cell fixation and cell wall removal, have significant effects on measured levels of immunofluorescence and have been optimized to prevent cell loss and maximize immunofluorescence. Average immunofluorescent levels of cell populations expressing different levels of beta-galactosidase show excellent correlation with measurements of average beta-galactosidase activity per cell based on cleavage of o-nitrophenyl-beta-D-galactopyranoside. Experiments with yeast populations containing various numbers of copies of the cloned gene indicate that the relationship between immunofluorescence and antigen content also holds at the single-cell level. Correlated measurements of DNA and beta-galactosidase content on a single-cell level permit the investigation of cellular enzyme content as a function of cell cycle position under various conditions. The procedure can be easily modified to detect other antigens by changing the primary antibody used.     

Improved FACS-Gal: flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs

The previously reported FACS-Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603-2607, 1988) measures E. coli lacZ-encoded beta-galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, using a fluorogenic substrate, which is hydrolyzed and retained intracellularly. In this system, lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence-activated sorting of cells based on their lacZ expression level. This report details the following improvements of the original assay: 1) use of phenylethyl-beta-D-thiogalactoside, a competitive inhibitor, to inhibit beta-galactosidase activity; 2) reduction of false positives by two-color measurements; and 3) inhibition of interfering mammalian beta-galactosidases by the weak base chloroquine. We found an exponential relationship between fluorescence generated by beta-galactosidase in this assay and the intracellular concentration of beta-galactosidase molecules. Finally, we report conditions for optimal loading of the substrate (FDG) and retention of the product, fluorescein. Under these conditions, we found uniform loading of FDG in all cells of a clone in individual experiments. Together, these improvements make FACS-Gal an extremely powerful tool for investigation of gene expression in eukaryotic cells.     

A quantitative cytochemical assay of beta-galactosidase in single cultured human skin fibroblasts

A quantitative cytochemical method for the measurement of beta-galactosidase activity in cultured human skin fibroblasts has been developed using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside as the indigogenic substrate. The method relies upon the oxidation of the primary reaction product by ferro/ferricyanide during which an insoluble indigo dye is generated as the final reaction product. The reaction was linear with time up to 60 min using the final cytochemical standard procedure. The enzyme showed maximum activity at pH 4.0 to 4.1. The concentration optima of indigogenic substrate and potassium ferro/ferricyanide were 3.67 mM and 3.13 mM respectively. The presence of sodium chloride activated beta-galactosidase up to 100 mM, but was inhibitory above that concentration. The enzyme was inhibited by N-ethylmaleimide, N-acetyl-D-galactosamine and heparin. The enzyme molecules were shown to diffuse out of the cells using media without a suitable inert colloid stabilizer. However, diffusion was completely prevented by using polyvinyl alcohol (PVA) grade G18/140. Air-drying of cells was essential to make the cell membrane permeabel to the substrate and, thereby, to avoid a pronounced lag phase. However, in a biochemical analysis, air-drying itself caused a decrease in enzyme activity to 43% of the control. Even after air-drying lysosomal latency could still be demonstrated by using PVA grade G04/140. Control persons, one carrier of and two patients with beta-galactosidase deficiency were easily identified as belonging to three separate groups by using the cytochemical assay. It is proposed that the quantitative cytochemical approach may also be applied to cultured human amniotic fluid cells or chorion biopsies giving a rapid prenatal diagnosis of beta-galactosidase deficiency due to the small number of cells needed in the analysis.     

A gel diffusion assay for visualization and quantification of chitinase activity

Higher plants, bacteria, fungi, insects, and crustaceans all produce chitinases. Chitinase genes in many organisms are currently under investigation. Chitinase activity is usually assayed with radiolabeled or fluorogenic substrates. We developed a simple, inexpensive, nonradioactive gel-diffusion assay for chitinase that can be used to screen large numbers of samples. In this assay, chitinase diffuses from a small circular well cut in an agarose or agar gel containing the substrate glycol chitin, a soluble, modified form of chitin. Chitinase catalyzes the cleavage of glycol chitin as it diffuses through the gel, leaving a dark, unstained circular zone around the well, because the fluorescent dye calcofluor binds only to undigested chitin. Sample activities can be determined from linear regression of logstandard enzyme concentration versus the zone diameter of internal standards on each Petri dish used for a diffusion assay.     

Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase.

A fluorescent method for monitoring the activity of the secreted Candida carboxyl (aspartic) proteinase (EC 3.4.23.6) was developed using a fluorogenic substrate based on resonance energy transfer. The fluorescent assay was used to monitor proteinase production, purification, and inhibition. The Km for the fluorogenic substrate, 4-(4-dimethylaminophenylazo)benzoyl-gamma-aminobutyryl-Ile-His-Pro - Phe-His-Leu-Val-Ile-His-Thr- [5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid, was found to be 4.3 microM at the optimum pH of 4.5. Reaction products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis or by 252Cf plasma desorption mass spectrometry. Cleavage of the fluorogenic substrate was between the histidine-threonine residues, releasing the fluorescent product, threonine-[5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid. Proteolytic activity was expressed as nanomoles of fluorescent product released at 22 degrees C/60 min, pH 4.5, and the release of 0.9 nmol product was equivalent to one hemoglobin proteolytic unit (O.D.A700 increase of 0.100) produced at 37 degrees C/60 min, pH 3.5. The aspartic proteinase inhibitor pepstatin had an IC50 of 27 nM when tested in a dose-response study with the purified enzyme. The apparent Ki for pepstatis was 2.9 nM. Several synthetic inhibitors of the enzymes were identified with IC50's in the nanomolar range. The most potent compound, A70450, was characterized as a fast, tight-binding inhibitor having an IC50 of 1.3 nM and apparent Ki of 0.17 nM.     

N-DEVD-N'-morpholinecarbonyl-rhodamine 110: novel caspase-3 fluorogenic substrates for cell-based apoptosis assay

A novel caspase-3 substrate N-Ac-DEVD-N'-MC-R110, which is a fluorogenic substrate cleavable in a single step, has been prepared. It has a significantly higher enzyme turnover rate and sensitivity for detecting caspase-3 activity both in solution and living cells than existing fluorogenic substrates.     

Development of a sensitive chemiluminescent neuraminidase assay for the determination of influenza virus susceptibility to zanamivir

Determination of the sensitivity of influenza viruses to neuraminidase (NA) inhibitors is presently based on assays of NA function because, unlike available cell culture methods, the results of such assays are predictive of susceptibility in vivo. At present the most widely used substrate in assays of NA function is the fluorogenic reagent 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (MUN). A rapid assay with improved sensitivity is required because a proportion of clinical isolates has insufficient NA to be detectable in the current fluorogenic assay, and because some mutations associated with resistance to NA inhibitors reduce the activity of the enzyme. A chemiluminescence-based assay of NA activity has been developed that uses a 1,2-dioxetane derivative of sialic acid (NA-STAR) as the substrate. When compared with the fluorogenic assay, use of the NA-STAR substrate results in a 67-fold reduction in the limit of detection of the NA assay, from 200 pM (11 fmol) NA to 3 pM (0.16 fmol) NA. A panel of isolates from phase 2 clinical studies of zanamivir, which were undetectable in the fluorogenic assay, was tested for activity using the NA-STAR substrate. Of these 12 isolates with undetectable NA activity, 10 (83%) were found to have detectable NA activity using the NA-STAR substrate. A comparison of sensitivity to zanamivir of a panel of influenza A and B viruses using the two NA assay methods has been performed. IC(50) values for zanamivir using the NA-STAR were in the range 1.0-7.5 nM and those for the fluorogenic assay in the range 1. 0-5.7 nM (n = 6). The NA-STAR assay is a highly sensitive, rapid assay of influenza virus NA activity that is applicable to monitoring the susceptibility of influenza virus clinical isolates to NA inhibitors.     

Scanning the prime-site substrate specificity of proteolytic enzymes: a novel assay based on ligand-enhanced lanthanide ion fluorescence

A novel method for assaying the substrate specificity of proteolytic enzymes has been developed utilizing ligand-enhanced lanthanide ion fluorescence. This approach was used to develop peptide libraries to probe substrate specificity in the prime sites of proteolytic enzymes. A positional scanning synthetic combinatorial library of fluorogenic peptides was synthesized and used to determine the extended prime site specificity of bovine -chymotrypsin. The enzyme showed a preference for Lys and Arg in the P1′ position, rather broad specificity in the P2′ position, and a slight Arg specificity in the P3′ position. The specificity profile of bovine -chymotrypsin agrees well with previously reported data, and the substrate library reported herein should provide valuable information about the prime site substrate specificities of other proteolytic enzymes as well. Furthermore, the continuous fluorogenic assay described may prove useful in analyzing the activity of other hydrolytic enzymes.     

Peptide substrate for caspase-3 with 2-aminoacridone as reporting group

Synthesis and properties of a new fluorescent/fluorogenic substrate Ac-DEVD-AMAC for caspase-3 are reported. The substrate is obtained by conventional Fmoc-based solid phase peptide synthesis and its properties are investigated with regard to fluorescence, sensitivity, applicability and kinetic constants. A non-traditional approach to assay the proteases activity using 2-aminoacridone labeled peptides is proposed. This approach utilizes the decrease of fluorescence intensity of a sample as a measure for the enzyme activity.     

Pyrene-methyl lauryl ester, a new fluorescent substrate for lipases: use for diagnosis of acid lipase deficiency in Wolman's and cholesteryl ester storage diseases

Fluorescent pyrene-methyl lauryl ester (PMLes) was synthesized and used for the determination of cellular lipase activities in lymphoblasts and fibroblasts from normal subjects and from patients affected with Wolman's or cholesteryl ester storage diseases (both exhibiting a deficiency of the lysosomal acid lipase). The hydrolysis of PMLes by acid lipase could be followed directly in a spectrofluorometer; this was possible because of the very high fluorescence emission of pyrene-methanol at 378 nm (monomeric form) in aqueous medium, whereas the substrate has practically no monomeric emission at 378 nm but emits only at 475 nm (excimeric form) in the experimental conditions used: this property permitted us to use PMLes as a fluorogenic substrate. In an alternative procedure, the enzymatic reaction could be determined after partition of the reaction mixture in a biphasic system of heptane and aqueous ethanol; the residual undegraded substrate partitioned into the upper heptane phase and the fluorescence of the product (i.e. pyrene-methanol) was read in the lower aqueous-ethanolic phase, at 378 nm. PMLes was hydrolyzed in extracts of normal lymphoblasts and fibroblasts by at least two lipases, one acidic lipase (pH 4.0) and a second more neutral enzyme (pH 6.5). The acidic lipase activity was practically absent in lymphoblasts and fibroblasts from Wolman's or cholesteryl ester storage diseases. This demonstrates that the fluorescent PMLes is hydrolyzed by the lysosomal acid lipase and can be used as a very sensitive fluorogenic substrate which permits direct recording of product formation and is suitable for the enzymatic diagnosis of either of these diseases.