Microfluorometric kinetic analysis of cathepsin B activity in single human thyroid follicular epithelial cells using image analysis and continuous monitoring

Summary The activity of a cysteine proteinase, cathepsin B (EC 3.4.22.1), was determined in unfixed single human thyroid follicular epithelial cells at room temperature using an image analysis system. The formation of the reaction product was monitored every minute by measuring the increasing fluorescence intensity of emitted light from a Schiff-base product formed by the substrateN-CBZ-ala-arg-arg-4-methoxy-2-naphthylamide and the coupling agent 5-nitrosalicylaldehyde. Non-specific fluorescent signals were eliminated by adjusting the video signal to zero using leupeptin, a specific inhibitor of cathepsins B, H and L. The enzyme activity was expressed as fluorescence intensity versus time. After a mean lag period of 5 min (range 4–8 min,n=4), the enzyme activity increased linearly lasting on average 5 min (range 3–8 min), followed by a marked decrease in reaction rate for 3 min (range 1–4 min), and another linear increase for 11 min (range 8–14 min). The second linear part of the curve was not as steep as the first one. The reaction velocity recorded in individual granules resulted either in a biphasic curve or straight line, suggesting the presence of two distinct organelle compartments with differences in membrane permeability. It is concluded that human thyroid follicular epithelial cells in culture exhibit cathepsin B activity which can be monitored continuously by videomicrofluorometry without interference from non-specific fluoresence. Part of this study has been presented in a preliminary form at the 9th International Congress of Histochemistry and Cytochemistry 1992 (Kayser & H?yer, 1992).     

Flow microfluorometric identification of liver cells with elevated gamma-glutamyltranspeptidase activity after carcinogen exposure.

We have developed a fluorometric cytochemical assay for gamma-glutamyltranspeptidase (gamma-GT) using the substrate gamma-glutamyl-4-methoxy-2-naphthylamide in which the released methoxynaphthylamine was coupled with 5-nitrosalicylaldehyde to form a yellow fluorescent crystalline product within the cells. Single cell suspensions were obtained by collagenase perfusion of livers from rats that had either received a two-thirds partial hepatectomy followed 24 hr later by a single injection of diethylnitrosamine (DEN) or received a partial hepatectomy alone. Cultured HTC cells were used as a source of gamma-GT+ cells. Fluorescence (gamma-GT activity) was low in most of the cells from both DEN-exposed and control rats, but high in HTC cells. The livers of both DEN-exposed and control rats had a subpopulation of cells that were gamma-GT+; this population could be quantitated and sorted by flow cytometry. Five weeks post injection the number of GT+ cells from the rats exposed to DEN was more than 20 times that from the control rats. Increased gamma-GT activity may be a useful cytochemical marker for preneoplastic liver cells.     

Localization of cathepsin B activity in fibroblasts and chondrocytes by continuous monitoring of the formation of a final fluorescent reaction product using 5-nitrosalicylaldehyde

Summary The histochemical fluorescence method using 5-nitrosalicylaldehyde for the demonstration of cathepsin B activity has been used. Precipitation of the fluorescent final reaction product was analysed continuously during incubation for cathepsin B activity. Unfixed cultured human fibroblasts as well as cryostat sections of mouse metacarpal bone explants were used. Continuous monitoring of the formation of the fluorescent reaction product showed that after a certain lag phase, depending on the enzyme activity in the tissue, discrete granules appeared which became increasingly fluorescent with incubation time. Subsequently, recrystallization and redistribution of the final reaction product started to occur. It is concluded that the coupling reaction with 5-nitrosalicylaldehyde is sufficiently fast for a proper localization of proteinase activity and can be used for kinetic analysis of enzyme activity. The method provides indications of relative amounts of cathepsin B activity in different cell types within a tissue section. It appeared from the study on metacarpal bone explants that fibroblasts in perichondrium and periosteum contained a relatively high cathepsin B activity whereas chondrocytes showed a low but distinct activity. This observation suggests that cysteine proteinases are not only involved in collagen degradation by fibroblasts but that they also play a role in the intracellular digestion of collagen by chondrocytes.     

Fluorescence histochemical detection of hydrolases in tissue sections and culture cells

Fluorescence and dye histochemical methods are compared for the investigation of hydrolases in sections and culture cells. At present, only some of the synthetic substrates with fluorescent leaving groups may be used for the fluorescence localization of these enzymes in sections. This limitation is due to a reduced fluorescence intensity and/or diffusion of the fluorescent tags. Satisfactory results are obtained for alkaline phosphatase, non-specific esterases and proteases with naphthol AS and 4-methoxy-2-naphthylamine coupled to nitrosalicylaldehyde. If, however, cultured monolayer cells are investigated, all synthetic substrates with fluorescent tags are suitable, including those that have so far only been used for biochemical hydrolase measurements. The fluorescent leaving groups are naphthol AS and its derivates, 4-methoxy-2-naphthylamine, aminomethylcoumarin, aminomethyltrifluoromethylcoumarin, methylumbelliferon, fluorescein and, with some limitations, also 1- and 2-naphthol. These fluorescence methods are more sensitive than the corresponding dye procedures. In addition, the fluorescence techniques allow the use of more synthetic substrates and therefore more information become available than with dye histochemistry about the enzymic properties of culture cells.     

Identification of neuropeptide-degrading enzymes in the pancreas.

Neutral endopeptidase (NEP) and aminopeptidase M (APM) were identified in the pancreas by enzymatic assays and Western blotting. The NEP activity, assessed by the phosphoramidon- and DL-thiorphan-inhibitable degradation of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine, was 28.8 pmol/h/micrograms of pancreatic membrane protein and 124 pmol/h/10(6) pancreatic acinar cells. The APM enzymatic activity, assessed by the actinonin- and amastatin-inhibitable degradation of Ala-4-methoxy-2-naphthylamine, was 633 pmol/h/micrograms pancreatic membrane protein and 17.4 nmol/h/10(6) pancreatic acinar cells. Proteins corresponding to NEP (95 kDa) and APM (140 kDa) were identified in membranes by Western blotting. Both NEP and APM on acinar cells may degrade neuropeptides and regulate their effects on exocrine secretion.     

The use of hexazonium-p-rosanilin in the histochemical demonstration of peptidases.

The suitability of hexazonium-p-rosanilin (HP) in the histochemical demonstration of peptidases was investigated. The detection was carried out in cold mictrotome sections adherent to slides or semipermeable membranes. Alanyl-1-naphthylamide, alanyl-2-naphthylamide, leucyl-2-naphthylamide, leucyl-4-methoxy-2-naphthylamide (all substrates in concentration of 0.4 mg/1 ml of citrate phosphate buffer pH 6.5), gamma-L-glutamyl-1-naphthylamide, gamma-L-glutamyl-2-naphthylamide (both substances in concentration of 0.24 mg/1 ml of acetate buffer pH 6.5) were used as the substrates. Results were compared with those obtained with Fast Blue B and Fast Garnet GBC. In comparison with Fast Blue B and Fast Garnet GBC HP is a faster coupler, furnishes azodyes which are stable, amorphous (even without lipid extractions from sections), more substantive and in the case of 1-naphthylamine almost insoluble in ordinary lipid solvents used for the dehydration and clearing of sections before mounting. The molecular extinction coefficient of azodyes furnished by HP is 1.5X higher for 1-naphthylamine than for 2-naphthylamine. It is higher than that of Fast Garnet GBC, however, lower than that of Fast Blue B. The inhibitory influence of individual diazonium salts on enzyme activity (activities) splitting leucyl-2-naphthylamide amounts to 36% (Fast Garnet GBC), 37% (Fast Blue B), 52% (HP, 0.03 ml/1 ml) and 63% (HP, 0.09 ml/1 ml) at pH 6.5. For gamma-glutamyl-transpeptidase the corresponding values are 50%, 59%, 62% and 67%. The higher inhibitory influence of HP is compensated by the possibility of its using in the technic of semipermeable membranes. HP improves greatly the localization of peptidases in cold microtome sections from which lipids were not extracted. The best results are furnished by 1-naphthylamine dervatives. In the case of 4-methoxy-2-naphthylamine derivatives the localization is very sharp, however, the azodye is less distinct than that of 2-naphthylamine. The localization as obtained with HP in combination with substrates derived of simple naphthylamines is similar or even better than with 4-methoxy-2-naphthylamine derivatives applied with Fast Blue B. Typical examples are shown.     

Colorimetric and fluorimetric microplate assays for legumain and a staining reaction for detection of the enzyme after electrophoresis.

The cysteine endopeptidase legumain was recently discovered in mammalian cells, predominantly localized in the lysosomal system where it is believed to contribute to antigen processing for MHC class II. Here we describe rapid assay procedures for the enzyme in 96-well microplates with two substrates, a novel compound, succinyl-Ala-Ala-Asn-4-methoxy-2-naphthylamide, and benzyloxycarbonyl-Ala-Ala-Asn-4-methyl-7-coumarylamide. Both substrates are suitable for fluorimetric assays, but the naphthylamide also allows colorimetric detection of legumain activity, since the released 4-methoxy-2-naphthylamine gives a red product when coupled with the Fast Garnet color reagent. We show that the naphthylamide substrate can be used to visualize active legumain after electrophoresis in polyacrylamide gel. Both substrates provide assays that are reproducible and sufficiently sensitive to allow the assay of legumain in crude samples such as tissue homogenates, although the coumarylamide is the more sensitive. The specificity of both assay methods for legumain was verified by the lack of inhibition by E-64 and total inhibition by egg white cystatin.     

gamma-Glutamyl transpeptidase demonstrated in tissue sections embedded in glycol methacrylate resin

Summary A method is described for the histochemical demonstration of -glutamyl transpeptidase in tissue sections embedded in glycol methacrylate at low temperature. Enzyme activity was preserved by a short (3 h) fixation of tissue in 4% paraformaldehyde at 4° C prior to embedding at 4° C. Tissue embedded in glycol methacrylate combined good morphology with accurate enzyme localization. Blocks of the embedded tissue could be stored at room temperature for at least 3 months without loss of enzyme activity. The resin is non-fluorescent, allowing the use of the fluorescent coupling agent 5-nitrosalicylaldehyde to visualize the reaction product.     

Fluorescence demonstration of cathepsin B activity in fractionated alveolar macrophages.

Histochemical localization of cathepsin B in alveolar macrophages (AM) that separated into four different density fractions (I, II, III and IV) by discontinuous Percoll gradient centrifugation was demonstrated in fluorescence microscope using CBZ-Arg-Arg-4-methoxy-2- naphthylamide as a substrate and 5-nitrosalicylaldehyde as a coupling reagent. The least dense AM (fraction I) was found numerous bright yellow fluorescing particles with high intensity in small granules distributed throughout the cytoplasm when compared to the most dense cells (fraction IV). The different localization of cathepsin B activity in the fractionated cells suggested differentiation of lysosomal system and existence of maturational (or aging) sequence in rat AM.     

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.     

Stereospecific synthesis of l-2-amino-3-(7-methoxy-4-coumaryl)propionic acid, an alternative to tryptophan in quenched fluorescent substrates for peptidases

A high-yielding synthesis of the highly fluorescent amino acid l-2-amino-3-(7-methoxy-4-coumaryl)propionic acid (l-Amp) has been developed via (2R)-bornane-10,2-sultam glycinate. l-Amp facilitates the synthesis of sensitive fluorogenic proteinase substrates with N-terminal solubilising or reactive groups.     

Improved zymographic detection of bovine acrosin.

Acrosomal materials extracted from bovine spermatozoa contain a trypsin-like proteinase termed acrosin (1–3) (EC 3.4.21.10). The presence of multiple molecular forms of this spermatozoal enzyme has been demonstrated both by gel filtration (2) and by polyacrylamide gel electrophoresis (1,3).The enzymic reactions of proteinases can be detected in electrophoretic patterns by incorporating proteinaceous substrates in the electrophoretic media (4) or by using specific amino acid derivatives of β-naphthylamine (1,5,6). Andary and Dabich (6) recently reported that the former method was improved by diffusing the proteinaceous substrate into the gel during a 1-hr incubation period following electrophoresis. The zymogram then required an additional incubation of the gel in buffer solution for 2–12 hr before the transparent zones of proteinase activity were detectable. Incubation periods of less than 1 hr are normally required for the zymographic staining methods that use synthetic arginine derivatives of β-napthylamine to detect acrosin activity. These systems do, however, suffer from a lack of sensitivity and fading of the diazonium-coupled product (1). An improved method for rapid detection of acrosin activity in gels would, therefore, be useful. The present communication describes an improved version of the staining system for detecting acrosin activity using a synthetic arginine derivative of β-naphthylamine. The application of this staining system for the detection of the multiple forms of bovine acrosin is presented. In addition, the stability of the zymograms resulting from three different coupling dyes was investigated using a miniature polyacrylamide gel electrophoretic system.     

The evolution of proteinase substrates with special reference to dipeptidylpeptidase IV

Summary The design and development of specific substrates for proteolytic enzymes is reviewed. Particular attention is given to substrates containing the leaving groups 4-methoxy-2-naphthylamide (MNA) and 7-amino-4-trifluoromethylcoumarin (AFC). The MNA substrates are used for histochemical and cytochemical purposes, and they yield a coloured final reaction product when azo-coupled with a diazonium salt, an osmiophilic product for electron microscopy when coupled with hexazotized Pararosaniline, or a fluorescent final reaction product when coupled with 5-nitrosalicylaldehyde. AFC substrates are considerably more sensitive, and they yield the fluorescent product AFC after enzymatic cleavage of the substrate. AFC is not sufficiently water-insoluble to allow (intra)cellular localization, but AFC substrates are successfully used for incubations in microwells (Immu-Probe technique) and for the demonstration of banding patterns after gel electrophoresis (enzyme-directed overlay membrane technique). The methods are discussed with the example of the elucidation of the role of dipeptidylpeptidase IV in autoimmune diseases.As presented by R.E.S. for the Pearse Prize Lecture at the Annual Histochemistry Meeting of the Royal Microscopical Society on 8 January 1991 in London, UK. The oral presentation appeared in written form inProc. Roy. Microsc. Soc. 26, 135–43 (1991).     

Cytochemical localization and biochemical evaluation of a lysosomal serine protease in lung: dipeptidyl peptidase II in the normal rat

Dipeptidyl peptidase II (DPP II) in normal rat lung was evaluated by the enzymes' ability to hydrolyze Lys-Ala or Lys-Pro derivatives of 4-methoxy-2-naphthylamine (MNA). For visualization of this activity, the liberated MNA was coupled with fast blue B for light microscopy (LM) or hexazotized pararosaniline with osmication for electron microscopy (EM). Granular to diffuse reaction product was noted in many lung cells in frozen sections for LM, including alveolar and tissue macrophages, fibroblasts, chondrocytes, bronchial and bronchiolar epithelial cells and mast cells. Reaction product at the EM level was seen in the lysosomal structures of the above cells, although lysosomal heterogeneity with regard to reactivity was noted. Cellular content of reaction product by EM correlated with LM staining intensity. Additional structures, not obviously reactive by LM, such as the lamellar bodies of type II cells and lysosomes in other cell types, were seen to contain reaction product ultrastructurally. A modified biochemical assay for the quantitation of DPP II in tissue homogenates was used to determine the activity of the enzyme in rat lung. Enzyme activity in polyacrylamide isoelectric focusing gels indicate that Lys-Ala-MNA was the more specific substrate but, by virtue of its rapid hydrolysis, Lys-Pro-MNA was more sensitive.     

Application of an organic solvent extraction to the determination of catechol-O-methyltransferase activity by high-performance liquid chromatography in human mononuclear cells

We report here a method for measuring mononuclear cell catechol-O-methyltransferase (COMT) activity which is ideally adapted to clinical studies. The method measures the O-methylation of dopamine to 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine. Whole mononuclear cell sonicate is incubated with saturating concentrations of dopamine, S-adenosyl-l-methionine and magnesium chloride in sodium—potassium phosphate buffer at pH 7.3. An organic solvent extraction using ethyl acetate is then used for product separation, followed by high-performance liquid chromatography with electrochemical detection for product separation and quantification. This method allows both O-methylated products, 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine, to be isolated and quantified separately. The apparent Michaelis constants for dopamine and S-adenosyl-l-methionine using this method are similar to values reported previously (0.51 and 14 μM, respectively). The optimal concentration of magnesium chloride is eight to ten times higher than previously reported. No endogenous inhibitors were apparent using this assay. The within-day coefficient of variation using this method is 7% when measuring 3-methoxytyramine and 5% when measuring 4-methoxy-3-hydroxyphenethylamine. The between-day coefficient of variation is 11%. Mononuclear cell COMT activity can be detected using protein concentrations as low as 0.75 mg/ml, corresponding to 2–3 ml of whole blood. The small amount of blood required per sample allows multiple sample analysis from a single patient, including infants.     

Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation.

Processing of the hepatitis C virus polyprotein is mediated by host cell signalases and at least two virally encoded proteinases. Of these, the serine-type proteinase encompassing the amino-terminal one-third of NS3 is responsible for cleavage at the four sites carboxy terminal of NS3. The activity of this proteinase is modulated by NS4A, a 54-amino-acid polyprotein cleavage product essential for processing at the NS3/4A, NS4A/4B, and NS4B/5A sites and enhancing cleavage efficiency between NS5A and NS5B. Using the vaccinia virus-T7 hybrid system to express hepatitis C virus polypeptides in BHK-21 cells, we studied the role of NS4A in proteinase activation. We found that the NS3 proteinase and NS4A form a stable complex when expressed as a single polyprotein or as separate molecules. Results from deletion mapping show that the minimal NS4A domain required for proteinase activation is located in the center of NS4A between amino acids 1675 and 1686 of the polyprotein. Amino acid substitutions within this domain destabilizing the NS3-NS4A complex also impair trans cleavage at the NS4A-dependent sites. Similarly, deletion of amino-terminal NS3 sequences impairs complex formation as well as cleavage at the NS4B/5A site but not at the NS4A-independent NS5A/5B site. These results suggest that a stable NS3-NS4A interaction is important for cleavage at the NS4A-dependent sites and that amino-terminal NS3 sequences and the central NS4A domain are directly involved in complex formation.     

Purification and characterization of a high molecular weight proteinase (macropain) from human erythrocytes

An alkaline proteinase, previously identified in rat liver and heart, has been purified from the soluble fraction of human erythrocytes. The proteinase has an apparent molecular weight of 600 000 and is composed of eight subunits with molecular weights ranging from 32 000 to 21 000. The proteinase degrades both protein and synthetic peptide substrates with a broad pH optimum of 7.5-11.0. Among the synthetic peptides tested, tripeptides with arginine at the P1 position (e.g. Z-Val-Leu-Arg-4-methoxy-2-napthylamine and Boc-Leu-Gly-Arg-4-methylcoumarin-7-amide) are particularly good substrates. The proteinase appears to be sulfhydryl-dependent and is inhibited completely by mersalyl acid and by hemin; inhibitors of serine and metallo-type proteinases have no effect on proteinase activity. Interestingly, a variety of other proteinase inhibitors such as leupeptin, chymostatin and N-ethylmaleimide failed to completely inhibit protein-hydrolyzing activities of the enzyme. These results indicate that these activities may be accounted for by at least two different catalytic sites. Proteinase activity is stable in the presence of 1 M urea, 0.5% Triton X-100 or 0.03% SDS and is not affected by ATP. Based on the high molecular weight and sulfhydryl-dependence, we have named this proteinase macropain.     

Antiprotozoal and cytotoxic naphthalene derivatives from Diospyros assimilis

Chemical investigation of the roots of Diospyros assimilis had led to the isolation and characterization of six naphthalene derivatives, two 2-naphthaldehyes, namely 4-hydroxy-3,5-dimethoxy-2-naphthaldehyde 1, 4-hydroxy-5-methoxy-2-naphthaldehye 2, its related isomer 5-hydroxy-4-methoxy-2-naphthaldehyde 3 and three commonly occurring naphthoquinones, diospyrin 4, 8'-hydroxyisodiospyrin 5 and the simple monomer, plumbagin 6. Their chemical structures were established by detailed NMR investigations including 1H and 13C NMR, HSQC, HMBC and NOESY experiments. In addition, the naphthalene derivatives 1-5 were evaluated for their in vitro antiprotozoal activity against protozoan parasites belonging to the genera Trypanosoma, Leishmania and Plasmodium. Among the tested compounds, naphthaldehyde 1 showed moderate inhibition of the growth of the parasites, T. brucei, T. cruzi, L. donovani with IC50 values of 19.82, 12.28 and 38.78 microM and displayed cytotoxicity towards rat skeletal myoblasts (L-6 cells) with IC50 of 174.94 microM, while 2 and 3 were found to be comparatively less active to 1. The dimeric quinones 4 and 5 exhibited good activity against T. brucei and L. donovani with IC50 of 1.12 and 8.82 microM and 12.94 and 16.66 microM respectively.     

Synthesis, antimalarial, antileishmanial, antimicrobial, cytotoxicity, and methemoglobin (MetHB) formation activities of new 8-quinolinamines

We report the synthesis, in vitro antiprotozoal (against Plasmodium and Leishmania), antimicrobial, cytotoxicity (Vero and MetHb-producing properties), and in vivo antimalarial activities of two series of 8-quinolinamines. N1-{4-[2-(tert-Butyl)-6-methoxy-8-quinolylamino]pentyl}-(2S/2R)-2-aminosubstitutedamides (21-33) and N1-[4-(4-ethyl-6-methoxy-5-pentyloxy-8-quinolylamino)pentyl]-(2S/2R)-2-aminosubstitutedamides (51-63) were synthesized in six steps from 6-methoxy-8-nitroquinoline and 4-methoxy-2-nitro-5-pentyloxyaniline, respectively. Several analogs displayed promising antimalarial activity in vitro against Plasmodium falciparum D6 (chloroquine-sensitive) and W2 (chloroquine-resistant) clones with high selectivity indices versus mammalian cells. The most promising analogs (21-24) also displayed potent antimalarial activity in vivo in a Plasmodium berghei-infected mouse model. Most interestingly, many analogs exhibited promising in vitro antileishmanial activity against Leishmania donovani promastigotes, and antimicrobial activities against a panel of pathogenic bacteria and fungi. Several analogs, notably 21-24, 26-32, and 60, showed less MetHb formation compared to primaquine indicating the potential of these compounds in 8-quinolinamine-based antimalarial drug development.