Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.     

New fluorogenic substrates for N-arginine dibasic convertase

N-Arginine dibasic (NRD) convertase is a recently described peptidase capable of selectively cleaving peptides between paired basic residues. The characterization of this unique peptidase has been hindered by the fact that no facile assay procedure has been available. Here we report the development of a rapid and sensitive assay for NRD convertase, based on the utilization of two new internally quenched fluorogenic peptides: Abz-GGFLRRVGQ-EDDnp and Abz-GGFLRRIQ-EDDnp. These peptides contain the fluorescent 2-aminobenzoyl moiety that is quenched in the intact peptide by a 2, 4-dinitrophenyl moiety. Cleavage by NRD convertase at the Arg-Arg sequence results in an increase of fluorescence. NRD convertase cleaves these peptides efficiently and with high specificity as observed by both HPLC and fluorescence spectroscopy. The rate of hydrolysis of the fluorogenic substrates is proportional to enzyme concentration, and obeys Michaelis-Menten kinetics. The kinetic parameters for the fluorescent peptides (Km values of approximately 1.0 microM, and Vmax values of approximately 1 microM/(min. mg) are similar to those obtained with peptide hormones as substrates.     

Solubilization and properties of GDP-fucose: xyloglucan 1,2-alpha-L-fucosyltransferase from pea epicotyl membranes

GDP-fucose:xyloglucan 1,2-alpha-L-fucosyltransferase from pea (Pisum sativum) epicotyl microsomal membranes was readily solubilized by extraction with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps). When using GDP-[14C]fucose as fucosyl donor and tamarind xyloglucan (XG) as acceptor, maximum activation was observed at 0.3% (w/v) Chaps and the highest yield of solubilized activity at 0.4%. The reaction product was hydrolyzed by Trichoderma cellulase to yield labeled oligosaccharides that peaked on gel permeation chromatography at the same elution volume as pea XG nona- and decasaccharide subunits. The apparent Km for fucosyl transfer to tamarind XG by the membrane-bound or solubilized enzyme was about 80 microM GDP-fucose. This was 10 times the apparent Km for fucosyl transfer to endogenous pea nascent XG. Optimum activity was between pH 6 and 7, and the isoelectric point was close to pH 4.8. The solubilized enzyme showed no requirement for, or stimulation by, added cations or phospholipids, and was stable for several months at -70 degrees C. Solubilization and gel permeation chromatography on columns of Sepharose CL-6B enriched the specific activity of the enzyme by about 20-fold relative to microsomes. Activity fractionated on columns of CL-6B with an apparent molecular weight of 150 kDa. The solubilized fucosyltransferase was electrophoresed on nondenaturing polyacrylamide slab gels containing 0.02% (w/v) tamarind XG, and its activity located by incubation in GDP-[14C]fucose, washing, and autoradiographing the gel. A single band of labeled reaction product appeared with an apparent molecular weight of 150 kDa.     

Enzymic transfer of α-L-arabinopyranosyl residues to exogenous 1,4-linked β-D-galacto-oligosaccharides using solubilized mung bean (Vigna radiata) hypocotyl microsomes and UDP-β-L-arabinopyranose

A single alpha-L-arabinopyranosyl (alpha-L-Arap) residue was shown, by a combination of chemical and spectroscopic methods, to be transferred to O-4 of the nonreducing terminal galactosyl (Gal) residue of 2-aminobenzamide (2AB)-labeled galacto-oligosaccharides when these oligosaccharides were reacted with UDP-ss-L-arabinopyranose (UDP-ss-L-Arap) in the presence of a Triton X-100-soluble extract of microsomal membranes isolated from mung bean (Vigna radiata, L. Wilezek) hypocotyls. Maximum-(1-->4)-arabinopyranosyltransferase activity was obtained at pH 6.0-6.5 and 20 degrees C in the presence of 25 mM Mn2+. The enzyme had an apparent K m of 45 microM for the 2AB-labeled galactoheptasaccharide and 330 microM for UDP-ss-L-Arap. A series of 2AB-labeled galacto-oligosaccharides with a degree of polymerization (DP) between 6 and 10 that contained a single alpha-L-Arap residue linked to the former nonreducing terminal Gal residue were generated when the 2AB-labeled galactohexasaccharide (Gal6-2AB) was reacted with UDP- ss-L-Ara p in the presence of UDP-beta-D-Galp and the solubilized microsomal fraction. The mono-arabinosylated galacto-oligosaccharides are not acceptor substrates for the galactosyltransferase activities known to be present in mung bean microsomes. These results show that mung bean hypocotyl microsomes contain an enzyme that catalyzes the transfer of Arap to the nonreducing Gal residue of galacto-oligosaccharides and suggest that the presence of a alpha-L-Arap residue on the former terminal Gal residue prevents galactosylation of galacto-oligosaccharides.     

Characterization of dolichyl diphosphate phosphatase from rat liver

Dolichyl diphosphate phosphatase (DolPPase) has been characterized in rat liver. Subcellular distribution studies indicate that the enzyme is localized in the endoplasmic reticulum. The in vitro enzymatic activity is stimulated by EDTA, due to release of inhibition by trivalent cations found in the assay tubes. All di- and trivalent cations tested were inhibitory, with the trivalent ions Al3+ and Fe3+ showing greater than 70% inhibition at a concentration of 10 microM. The assay requires the presence of a detergent for optimal activity, with Triton X-100 giving maximum activity at 0.1%. The substrate specificity of DolPPase toward polyprenyl diphosphates has been determined and indicates that there is little preference of the enzyme for substrates of different chain length, and either stereochemical orientation or degree of saturation of the alpha-isoprene unit. Km values of 11-14 microM were obtained for all substrates tested. Preliminary studies on the transmembrane topology of the DolPPase using latency assays, indicate that the active site of the enzyme may reside on the cytoplasmic face of the endoplasmic reticulum.     

Simple assay for sialyltransferase activity with a new fluorogenic substrate

A new fluorogenic acceptor for sialyltransferase, 2-[(2-pyridyl)amino]ethyl O-beta-D-galactopyranosyl-(1----4)-beta-D-glucopyranoside, was prepared from lactose as a starting material. Sialyltransferase activity was assayed by incubation of the enzyme with the acceptor and CMP-N-acetylneuraminic acid, separation of the fluorogenic sialylated product from the enzymatic reaction mixture by HPLC, and measurement of the product. Compared to assays so far reported that use radioactive substrates, this assay is simple and rapid. This method was used to assay sialyltransferase activity in human serum.     

Preparation of UDP-galacturonic acid using UDP-sugar pyrophosphorylase

UDP-galacturonic acid, the activated form of galacturonic acid (GalUA), is synthesized both de novo and by salvage pathways. The UDP-GalUA pyrophosphorylase gene involved in the salvage pathway has not been identified. Here we show that UDP-sugar pyrophosphorylase from Pisum sativum with a broad specificity has UDP-GalUA pyrophosphorylase activity. The enzyme catalyzed the formation of UDP-GalUA and pyrophosphate from GalUA 1-phosphate and UTP with an equilibrium constant value of 0.24. The recombinant UDP-sugar pyrophosphorylase had optimal pH of 6.0, and the apparent K(m) values for GalUA 1-phosphate, UTP, UDP-GalUA, and pyrophosphate were 2.27, 1.15, 0.70, and 1.26 mM, respectively. In the presence of inorganic pyrophosphatase, the recombinant enzyme produced UDP-GalUA in an 84% yield (based on the GalUA 1-phosphate substrate) on a preparative scale. Thus, this UDP-sugar pyrophosphorylase is useful for the highly efficient production of UDP-GalUA for studies on pectin biosynthesis.     

New fluorogenic substrates for renin

A simple and sensitive fluorometric assay was developed to test renin activity within several hours. Two new fluorogenic peptides, Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (octapeptide-MCA) and a succinyl derivative of the octapeptide-MCA were synthesized and used as a renin substrate. Renin cleaved the substrates at the Leu-Leu bond, releasing Leu-Val-Tyr-MCA. Three amino acids of this product were then successively split off by the auxiliary enzyme, leucine aminopeptidase, to liberate free 7-amino-4-methylcoumarin (AMC). The generation of the fluorescent 7-amino-4-methylcoumarin was proportional to renin concentrations up to 100 mGoldblatt U/tube. The optimal pH of renin reaction for both substrates was 6.5 to 7.0. As low as 5 mGoldblatt U of renin could be detected by this method. This method was applied to the assay of renin during its purification.     

Development of inductively coupled plasma-mass spectrometry-based protease assays

Rapid, sensitive, and quantitative assays for proteases are important for drug development and in the diagnosis of disease. Here an assay for protease activity that uses inductively coupled plasma-mass spectrometry (ICP-MS) detection is described. Peptidic α-chymotrypsin substrates were synthesized containing a lanthanide ion chelate at the N terminus to provide a distinct elemental tag. A biotin label was appended to the C terminus of the peptide, allowing separation of uncleaved peptide from the enzymatic digestion. The enzyme activity was determined by quantifying the lanthanide ion signal of the peptide cleavage products by ICP-MS. Biotinylated substrates synthesized include Lu-DTPA-Asp-Leu-Leu-Val-Tyr∼Asp-Lys(biotin) and Lu-DTPA-βAla-βAla-βAla-βAla-Gly-Ser-Ala-Tyr∼Gly-Lys-Arg-Lys(biotin)-amide. Parallel assays with a commercially available fluorogenic substrate (Suc-AAPF-AMC) for α-chymotrypsin were performed for comparison. Using the ICP-MS assay, enzyme concentrations as low as 2 pM could be readily detected, superior to the detection limit of an assay using the α-chymotrypsin fluorogenic substrate (Suc-AAPF-AMC). Furthermore, we demonstrated the use of this approach to detect chymotrypsin activity in HeLa cell lysates.     

Actin polymerization promotes the reversal of streaming in the apex of pollen tubes

Actin polymerization is important in the control of pollen tube growth. Thus, treatment of pollen tubes with low concentrations of latrunculin B (Lat-B), which inhibits actin polymerization, permits streaming but reversibly blocks oscillatory growth. In the current study, we employ Jasplakinolide (Jas), a sponge cyclodepsipeptide that stabilizes actin microfilaments and promotes polymerization. Uniquely, Jas (2 microM) blocks streaming in the shank of the tube, but induces the formation of a toroidal-shaped domain in the swollen apex, of which longitudinal optical sections exhibit circles of motion. The polarity of this rotary motion is identical to that of reverse fountain motility in control pollen tubes, with the forward direction occurring at the edge of the cell and the rearward direction in the cell interior. Support for the idea that actin polymerization in the apical domain contributes to the formation of this rotary motility activity derives from the appearance therein of aggregates and flared cables of F-actin, using immunofluorescence, and by the reduction in G-actin as indicated with fluorescent DNAse. In addition, Jas reduces the tip-focused Ca2+ gradient. However, the alkaline band appears in the swollen apex and is spatially localized with the reverse fountain streaming activity. Taken together, our results support the idea that actin polymerization promotes reversal of streaming in the apex of the lily pollen tube.     

Post-proline cleaving enzyme. Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man.

Synthesis and application of the first fluorogenic substrate, N-carbobenzoxyglycylprolyl-4-methylcoumarinyl amide (Z-Gly-Pro-MeCouNH) for the determination of the post-proline cleaving enzyme (EC 3.4.21.-) were reported. Maximal activity of the enzyme purified from lamb kidney for the new substrate was observed at pH 7.0. This substrate showed a higher affinity (Km = 0.02 mM) for the enzyme than the proline containing substrates studied previously and allowed the detection of 10-50 ng post-proline cleaving enzyme activity per ml sample after a 1 min incubation period. Distribution of post-proline cleaving enzyme and other proline specific peptidases in rat tissues was studied using Z-Gly-Pro-MeCouNH and other proline-containing substrates. High post-proline cleaving enzyme activity was observed in testis, liver and skeletal muscle. Inhibition experiments indicated that post-proline cleaving enzyme activity was completely inactivated by 0.1 mM diisopropylphosphofluoridate and Z-Gly-Pro-chloromethylketone, as had been found in the case of the enzyme isolated from lamb kidney. Activity in human body fluids was also tested for levels of post-proline cleaving enzyme activity using Z-Gly-Pro-MeCouNH and semen was found to show the highest cleaving activity.     

The Metabolism of Dopamine by Both Forms of Monoamine Oxidase in the Rat Brain and Its Inhibition by Cimoxatone

In the rat brain, dopamine is metabolised by both A and B forms of monoamine oxidase (MAO), although the A form of the enzyme is the major component. The Km of MAO-A toward dopamine (120 microM) is lower than the Km of MAO-B toward this substrate (340 microM). The activity of MAO-A was lower in old rats than in young rats, and the same degree of decrease was found for 5-hydroxytryptamine as for dopamine as substrates for this enzyme form. The activity of MAO-B was higher in the old rats, the degree of increase being the same for dopamine as for beta-phenethylamine as substrates for this enzyme form. The Ki values of the inhibition of MAO-A by cimoxatone and MD770222 (the principal plasma metabolite of cimoxatone) were independent of the substrate used to assay for activity, but were lower than the Ki values for the inhibition of MAO-B by these compounds.     

Solubilization of human prostatic 5 alpha-reductase

A sensitive assay for 5 alpha-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5 alpha-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5 alpha-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 microM testosterone was found to stabilise 5 alpha-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5 alpha-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5 alpha-reductase was studied. All detergents caused a concentration-dependent solubilization of 5 alpha-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5 alpha-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX.     

Human Platelet Phenol Sulfotransferase: Partial Purification and Detection of Two Forms of the Enzyme

To begin to study the usefulness of platelet phenol sulfotransferase (PST) as a possible measure of the enzyme activity in other organs such as the brain, we purified human platelet PST 36-120-fold. Activity toward 3-methoxy-4-hydroxyphenylglycol (MHPG), dopamine, 5-hydroxytryptamine (5-HT), and phenol eluted in the same Sephadex G-100 and Affi-Gel Blue column fractions. Specific activities of the enzyme with MHPG, dopamine, 5-HT, and phenol as substrates were 1198, 1068, 401, and 408 units/mg protein, respectively. Optimal assay conditions were established for each substrate. Apparent Km values were 598 microM, 21 microM, 19 microM, and 500 microM for MHPG, dopamine, phenol, and 5-HT, respectively. Apparent Km values for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) with the same four substrates ranged from 0.11 to 0.25 microM. The pH optima were 6.3 for phenol, 6.8 for dopamine, and 7.0 for MHPG and 5-HT. An additional pH optimum at 8.6 was present for 5-HT. A thermolabile form of the enzyme measured with dopamine and 5-HT, as well as a thermostable form measured with phenol, were present. Dichloronitrophenol (10(-5) M) noncompetitively inhibited the thermostable enzyme activity by 96% but decreased the thermolabile activity by only 36%. These studies provide the basis for a more accurate comparison of human platelet PST with the enzyme in the human brain and in other tissues.     

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.     

Solubilization of a mannose-polymerizing enzyme from Phaseolus aureus

A soluble enzyme preparation, which catalyses the polymerization of mannose, was obtained by Triton X-100 extraction of a particulate fraction derived from Phaseolus aureus hypocotyls. The product that resulted when GDP-alpha-d-mannose was used as a substrate was a beta-(1-->4)-linked mannan, about three-quarters of which was alkali-insoluble. The mannose-polymerizing enzyme activity was at least as great in the soluble preparation as in the particulate preparation, and the specific activity of the solubilized enzyme was greater by a factor of at least 3.5. Kinetic studies of the soluble enzyme indicate that the apparent K(m) is 55-62mum, and a disproportionate increase in rate is observed at high concentrations. GDP-alpha-d-glucose is a strong competitive inhibitor of the mannose-polymerizing reaction, with an apparent K(i) of 6.2mum. The soluble enzyme is relatively unstable, losing about two-thirds of its original activity in 5h at 0 degrees C or in 24h at -20 degrees C. A solvent (acetone, butanol, diethyl ether)-extracted particulate preparation, which also exhibits the same enzyme activity, is more stable, retaining full activity for at least 5 days at -20 degrees C. There was no polymerizing-enzyme activity in the soluble enzyme preparation when UDP-d-glucose, UDP-d-galactose, UDP-d-xylose, UDP-l-arabinose or UDP-d-glucuronic acid were used as substrates. However, the soluble enzyme preparation would catalyse the polymerization of glucose, with GDP-d-glucose as substrate.     

Application of rapid enzyme assay techniques for monitoring of microbial water quality

Rapid enzyme assay techniques based on direct measurement of beta-d-galactosidase (GALase) or beta-d-glucuronidase (GLUase) activity without selective cultivation are used for rapid estimation of the level of coliform bacteria and Escherichia coli in water samples. Reported detection limits using fluorogenic substrates correspond to culturable target bacteria concentrations that can be appropriate within present guidelines for recreational waters. The rapidity, that is detection within one hour, compromises the specificity of the assay; enzyme activity contributions from other than target bacteria need to be considered, particularly at low levels of target bacteria. Enzyme activities are more persistent than the culturability of target bacteria to environmental and disinfection stress, thus water samples may express enzyme activities of both culturable and viable non-culturable cells.     

Activation of dynein 1 adenosine triphosphatase by organic solvents and by Triton X-100

The presence of low concentrations of methanol or isopropyl alcohol (2-5%, v/v) in the assay medium stabilizes the latency of dynein 1 from sea urchin sperm flagella, with about a 50% decrease in ATPase level compared to that in the absence of solvent. Somewhat higher concentrations (10-20%, v/v) of these solvents in the assay give a 5-10-fold activation of ATPase activity. Dioxane, formamide, and dimethylformamide, on the other hand, always activate the ATPase activity, with a 5-10-fold increase observed at about 15% (v/v). The activation of latent ATPase activity by solvents is reversible for short exposures, especially in the presence of ATP and at low temperature, but the activation becomes irreversible upon more prolonged exposure. The rate constant for irreversible activation by 16% methanol at 21 degrees C is 0.08 min-1, compared to rates of 0.44 and 0.02 min-1 for activation by 0.05% Triton X-100 at 21 and 0 degree C, respectively. The slowness of this reversible activation induced by methanol and by Triton X-100 suggests that it is the result of large-scale conformational changes in the structure of the dynein. However, the activation by methanol occurs without the dissociation of the alpha and beta subunits of dynein that is observed with Triton X-100. The presence of 1 mM MgATP, or of 100 microM MgATP and 10 microM vanadate substantially protects latent dynein from activation by 0.05% Triton X-100.     

Guanylate cyclase from bovine rod outer segments: solubilization, partial purification, and regulation by inorganic pyrophosphate

In spite of its pivotal role in visual transduction, very little is known about guanylate cyclase of retinal photoreceptor cells. The enzyme has not yet been purified principally because of the difficulty in solubilizing it. We report here a simple method for solubilization of 67% of the cyclase activity from the retinal rod disk membranes (RDM). With Nonidet P-40 as detergent, the solubilization of cyclase is favored by a high concentration of KCl and exclusion of manganese. The solubilized and the residual insoluble enzymes are both highly unstable but could be partially stabilized by dithiothreitol. They were both insensitive to calcium, calmodulin, and atrial natriuretic factor. They also responded similarly to varying the manganese concentration in the assay. For the activity in both fractions, the Km for GTP was about 230 microM, Line-weaver-Burk plots showed that substrate binding was cooperative, and Hill plots suggested that there are two substrate binding sites. Cumulatively, these observations showed that while the entire activity could not be solubilized, the solubilized and the residual insoluble activities probably belonged to the same enzyme. Partial purification resolved the solubilized enzyme into two activities refered to as enzymes 1 and 2. Both had substrate saturation kinetics similar to the solubilized enzyme and were inhibited competitively by inorganic pyrophosphate, one of the products of the cyclase reaction. The Ki for PPi for enzyme 1 was 70-100 microM and 150-200 microM for enzyme 2. cGMP at concentrations up to 800 microM had no influence on the activity of either enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)