Human and mouse homo-oligomeric meprin A metalloendopeptidase: substrate and inhibitor specificities

Meprin metalloproteinases have been implicated in the susceptibility to and progression of diabetic nephropathy and inflammatory bowel diseases. Our studies with experimental models of these diseases in mice are congruent with the conclusion that meprins modulate the inflammatory responses and tissue damage. To determine whether the mouse and human enzymes differ, recombinant forms of meprin A from the two species were compared with respect to structure, substrates and inhibitors. Human homo-oligomeric meprin A formed oligomers ranging from 950,000 to 1,500,000 Da vs. 900,000 Da for mouse meprin A. Human and mouse meprin A exhibited similar activity against azocasein, fibronectin, collagen IV, and peptides such as parathyroid hormone, ghrelin, and gastrin-releasing peptide. The human enzyme had lower activity against gelatin, bradykinin, alpha-melanocyte-stimulating hormone and neurotensin, and higher activity against secretin and orcokinin. Human meprin A showed a preference for acidic residues in the P1' position of the substrate, unlike mouse meprin A. Several metalloproteinase inhibitors had IC(50) values in the nanomolar range, but potency ranged from similar values to a difference of several orders of magnitude for meprins from the two species. This work provides valuable data to improve predictability for human systems based on meprin functions in mouse models.     

A chromogenic substrate for a beta-xylosidase-coupled assay of alpha-glucuronidase

4-Nitrophenyl 2-(4-O-methyl-alpha-d-glucopyranuronosyl)-beta-d-xylopyranoside obtained on deesterification of 4-nitrophenyl 2-O-(methyl 4-O-methyl-alpha-d-glucopyranosyluronate)-beta-d-xylopyranoside (Hirsch et al., Carbohydr. Res. 310, 145-149, 1998) was found to be an excellent substrate for the measurement of hemicellulolytic alpha-glucuronidase activity. A new precise alpha-glucuronidase assay was developed by coupling the alpha-glucuronidase-catalyzed formation of 4-nitrophenyl beta-d-xylopyranoside with its efficient hydrolysis by beta-xylosidase. A recombinant strain of Saccharomyces cerevisiae, harboring and expressing the beta-xylosidase gene xlnD of Aspergillus niger under control of the alcohol dehydrogenase II promoter on a multicopy plasmid, was used as a source of beta-xylosidase. The activity values of beta-xylosidase in the assay required to achieve a steady-state rate of 4-nitrophenol formation shortly after starting the alpha-glucuronidase reaction were obtained both experimentally and by calculation using the kinetics of coupled enzyme reactions.     

A new chromogenic substrate for assay and detection of alpha-amylase

A new soluble chromogenic substrate for alpha-amylase was prepared by coupling partially hydrolyzed starch with a dye, Ostazin brilliant red H-3B. The substrate is precipitable from buffered solutions with ethanol and is equally suitable for assay of alpha-amylase, detection of separated alpha-amylase isoenzymes in gels, and selection of microbial producers of the enzyme.     

New chromogenic substrates for the assay of alpha-amylase and (1 leads to 4)-beta-D-glucanase

New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1 leads to 4)-beta-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1 leads to 4)-beta-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.     

A novel 2D-based approach to the discovery of candidate substrates for the metalloendopeptidase meprin

In the past, protease-substrate finding proved to be rather haphazard and was executed by in vitro cleavage assays using singly selected targets. In the present study, we report the first protease proteomic approach applied to meprin, an astacin-like metalloendopeptidase, to determine physiological substrates in a cell-based system of Madin-Darby canine kidney epithelial cells. A simple 2D IEF/SDS/PAGE-based image analysis procedure was designed to find candidate substrates in conditioned media of Madin-Darby canine kidney cells expressing meprin in zymogen or in active form. The method enabled the discovery of hitherto unknown meprin substrates with shortened (non-trypsin-generated) N- and C-terminally truncated cleavage products in peptide fragments upon LC-MS/MS analysis. Of 22 (17 nonredundant) candidate substrates identified, the proteolytic processing of vinculin, lysyl oxidase, collagen type V and annexin A1 was analysed by means of immunoblotting validation experiments. The classification of substrates into functional groups may propose new functions for meprins in the regulation of cell homeostasis and the extracellular environment, and in innate immunity, respectively.     

Application of a direct spectrophotometric assay employing a chromogenic substrate for tryptophanase to the determination of pyridoxal and pyridoxamine 5′-phosphates

Improved procedures for the isolation of apotryptophanase and its use in estimation of the vitamin B-6 coenzymes are presented. An excess of the apoenzyme is allowed to react with limiting amounts of pyridoxal-P. Estimation of the holotryptophanase thus formed by use of the chromogenic substrate. S-o-nitrophenyl-l-cysteine, provides a sensitive (1–400 pmol) and conveniently direct spectrophotometric assay for pyridoxal-P. For the specific estimation of pyridoxamine 5′-phosphate, samples are first reduced with NaBH₄ to convert pyridoxal-P to pyridoxine-P (inactive). By nonenzymatic transamination with glyoxylate, pyridoxamine-P is then converted quantitatively to pyridoxal-P and estimated with apotryptophanase. The method gives excellent recoveries of the added coenzymes and indicates that in many tissue extracts pyridoxamine-P surpasses pyridoxal-P in concentration.     

A simple enzymatic synthesis of 4-nitrophenyl beta-1,4-D-xylobioside, a chromogenic substrate for assay and differentiation of endoxylanases

A simple procedure has been elaborated for preparation of 4-nitrophenyl beta-d-xylopyranosyl-1,4-beta-d-xylopyranoside (NPX(2)), a chromogenic substrate of some endo-beta-1,4-xylanases. The procedure is based on a self-transfer reaction from 4-nitrophenyl beta-d-xylopyranoside catalyzed by an Aureobasidium pullulans and Aspergillus niger beta-xylosidases. Both enzymes catalyzed only the formation of 4-nitrophenyl glycosides of beta-1,4-xylobiose with a small admixture of 4-nitrophenyl glycoside of beta-1,3-xylobiose. The highest yields of the NPX(2) (19.4%) was obtained at pH 5.5. The removal of the beta-1,3-isomer from NPX(2) is not necessary for quantification of endo-beta-1,4-xylanase activity since it is not attacked by endo-beta-1,4-xylanases. In contrast to GH family 5 xylanase from Erwinia chrysanthemi, which did not attack NPX(2), all family 10 and 11 xylanases cleaved the chromogenic substrate exclusively between xylobiose and the aromatic aglycone. Significant differences in the K(m) values of GH10 and GH11 xylanases suggested that activities of these enzymes could be selectively quantified in the mixtures using various concentrations of NPX(2). Moreover, NPX(2) could serve as an ideal substrate to follow the interaction of endo-beta-1,4-xylanases with various xylanase inhibitors.     

N-acetyl-(L-Ala) 3 -p-nitroanilide as a new chromogenic substrate for elastase

The introduction of a useful new chromogenic substrate for the determination of elastase (EC 3.4.4.7) activity is described. N-acetyl-L-Ala-L-Ala-L-Ala-$\text{p}$-nitroanilide (AcAla₃NA) is a new specific elastase substrate whose hydrolysis can be followed spectrophotometrically at 410 nm in a wide pH range. Its rate of hydrolysis by α-chymotrypsin (EC 3.4.4.5) and trypsin (EC 3.4.4.4.) is 0.02% and 0.001% respectively compared to its rate of hydrolysis by elastase. As little as 0.1 μg elastase/ml can be satisfactorily determined. At pH 8, K_m = 0.88 mM and k_cat = 11.9 sec^?1.     

A continuous spectrophotometric assay for the Bacillus cereus phospholipase C using a thiophosphate substrate analog: evaluation of assay conditions and chromogenic agents

A thiophosphate analog of dioctanoylphosphatidylcholine has been used as the substrate in a continuous spectrophotometric assay for the Bacillus cereus phospholipase C. The reaction has been monitored at 412 nm using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and at 324 nm using 4,4'-dithiopuridine (DTP) as the respective thiol-reactive chromogenic agents. An optimum pH 6.0 was determined for the phospholipase C-catalyzed reaction which was independent of the chromogen utilized. Although the reaction rates observed when DTP was used were increased over those seen with DTNB, the rates were insensitive to changes in the concentration of the chromogen normally used for the assay. The initial velocities were shown to be linearly dependent upon the amount of enzyme added over at least a 20-fold enzyme concentration range. The dependency of the initial velocity on the concentration of substrate showed a discontinuity at [S] = 40 microM when either DTP or DTNB was used. This was consistent with a value of 56 microM estimated for the substrate critical micelle concentration by an independent measurement. While the substrate data measured using DTP could not be fit to existing equations based on Michaelis-Menten kinetics, the data obtained using DTNB as the chromogen conformed with the model proposed by Wells for enzymes acting upon micelle-forming substrates (M. A. Wells (1974, Biochemistry 13, 2248-2257). This allowed for the estimation of monomer and micelle Michaelis-Menten parameters for the B. cereus phospholipase C-catalyzed reaction with a thiophosphate analog substrate.     

Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-D-N-acetylneuraminate) substrate.

This report describes the preparation of a sodium (4-methylumbelliferyl-α-d-N-acetylneuraminate) substrate and its use in a sensitive fluorometric assay of neuraminidase (EC 3.2.1.18) from Vibrio cholerae, cultured fibroblasts, and human leucocytes. V. cholerae neuraminidase showed maximum activity at pH 4.6 and an apparent K_m of 1.5 mm and was activated by CaCl₂ and inhibited by ethylenediaminetetraacetate, NaCl, and N-acetylneuraminic acid. The inhibition by N-acetylneuraminic acid was competitive (K_i = 6.1 mm). Cultured fibroblast and leucocyte neuraminidases showed maximum activity between pH 4.2 and 4.4 and apparent K_m values of 0.13 and 0.22 mm, respectively. Neuraminidase activity was considerably reduced in cultured fibroblasts of patients with mucolipidosis types I, II, and III.     

Synthesis of 2-n-(hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine-hydroxide, a chromogenic substrate for assaying sphingomyelinase activity.

2-N-(Hexadecanoyl)-amino-4-nitrophenyl phosphorylcholine-hydroxide a compound resembling sphingomyelin is synthesized. It is cleaved by sphingomyelinase to the chromogenic N-acylaminonitrophenyl moiety. Phospholipase C preparations do not hydrolyze this compound. The starting material is 2-amino-4-nitrophenol which when acylated with palmitoyl chloride yields the hexadecananilide. Reaction with beta-bromoethylphosphoryldichloride gives the phosphate which is quaternized with trimethylamine to give the title compound.     

Biodegradation kinetics of a mixture containing a primary substrate (phenol) and an inhibitory co-metabolite (4-chlorophenol)

Batch experiments on the simultaneous utilization of phenol (primary substrate) and 4-chlorophenol (cometabolic secondary substrate) demonstrated two critical substrate interactions. First, the cometabolic degradation of 4-chlorophenol was proportional to the rate of phenol oxidation, which provided the electrons for the initial monooxygenase reaction. Second, 4-chlorophenol inhibited the oxidation of the primary substrate, phenol. Modeling analyses of the degradation of phenol alone and of phenol and 4-chlorophenol together showed that the proportionality between phenol and 4-chlorophenol degradation rates averaged 0.1 mg 4-CP/mg phenol, which corresponds to 0.5% of the electrons generated by phenol oxidation being used as a cosubstrate for the monooxygenase reaction of 4-chlorophenol. In addition, modeling analyses suggest that 4-chlorophenol was a noncompetitive inhibitor of phenol oxidation for high phenol concentrations, but a competitive inhibitor for low phenol concentrations.Abbreviations GC gas chromatography - FID flame-ionization detector - DO dissolved oxygen - 4-CP 4-chlorophenol - Ph phenol - RLS relative least squares criterion - NAD nicotinamide adenine dinucleotide - NADP nicotinamide adenine dinucleotide phosphate     

3-Methoxy-4-(2-nitrovinyl)phenyl glycosides as potential chromogenic substrates for the assay of glycosidases

Selective glycosidation of 2,4-dihydroxybenzaldehyde with either 2,3,4, 6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide, 2-acetamido-3,4,6-tri-O-acetyl-alpha-D-glucopyranosyl chloride, or 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide afforded the corresponding 4-O-glycosyl derivatives. Subsequent O-methylation, O-deacetylation, and condensation with nitromethane afforded the appropriate beta-glycoside of 3-methoxy-4-(2-nitrovinyl)phenol. The phenol is highly coloured at alkaline pH so that these glycosides may be suitable as chromogenic substrates for the assay of glycosidases.     

Structure and expression of the human MEP1A gene encoding the alpha subunit of metalloendopeptidase meprin A

The human genome contains several sequences that are similar to the MEP1A gene encoding the alpha subunit of metalloendopeptidase meprin A. We now report the first genomic structure for the human MEP1A gene that maps to chromosome 6p21. The gene spans approximately 45 kb and consists of 14 exons and 13 introns. Overall, about 6.7% (3 kb) of the MEP1A gene corresponds to the exon sequences. Tissue specificity of the MEP1A gene expression was examined by dot blot analysis of poly(A) RNA from 50 different human tissues. The MEP1A mRNA was detected for the first time in kidney and appendix in addition to colon and small intestine previously known to express the gene. The elucidated gene structure and tissue-specific expression of the MEP1A gene set the stage for investigating regulation and function of the gene and related sequences in the human genome.     

A spectrophotometric assay for allicin,alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates

Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.     

Preparation of non-reducing-end substituted p-nitrophenyl alpha-maltopentaoside (FG5P) as a substrate for a coupled enzymatic assay for alpha-amylases

p-Nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D - glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside, FG5P, was prepared, taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase on a mixture of O-6-deoxy-6-[(2-pyridyl)-amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha- D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-D-glucose and p-nitrophenyl alpha-glucoside. The maltopentaose derivative is resistant to alpha-glucosidase and is suitable as a substrate for the alpha-amylase assay coupled with alpha-glucosidase in which the activity of alpha-amylase is determined by measuring the amount of p-nitrophenol liberated by alpha-glucosidase from p-nitrophenyl alpha-glucoside and p-nitrophenyl alpha-maltoside produced by the action of alpha-amylase. This alpha-amylase assay method was applied for determination of alpha-amylases in human serum.     

A chromogenic substrate for phosphatidylinositol-specific phospholipase C: 4-nitrophenyl myo-inositol-1-phosphate.

A chromogenic water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized starting from myo-inositol employing isopropylidene and 4-methoxytetrahydropyranyl protecting groups. In this analogue of phosphatidylinositol, 4-nitrophenol replaces the diacylglycerol moiety, resulting in synthetic, racemic 4-nitrophenyl myo-inositol-1-phosphate. Using this synthetic substrate a rapid, convenient and sensitive spectrophotometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus was developed. Initial rates of the cleavage of the nitrophenol substrate were linear with time and the amount of enzyme used. At pH 7.0, specific activities for the B. cereus enzyme were 77 and 150 mumol substrate cleaved min-1 (mg protein)-1 at substrate concentrations of 1 and 2 mM, respectively. Under these conditions, less than 50 ng quantities of enzyme were easily detected. The chromogenic substrate was stable during long term storage (6 months) as a solid at -20 degrees C.     

Automation of chromogenic substrate Limulus amebocyte lysate assay method for endotoxin by robotic system.

The chromogenic substrate Limulus amebocyte lysate (LAL) assay method for the detection of endotoxin was automated by a Zymate robotic system. The software developed enables the robot to automatically dilute a stock reference endotoxin standard (20,000 endotoxin units per ml) for the construction of a five-point standard curve, make sample dilutions to the proper testing concentration, and perform chromogenic substrate LAL assays in duplicate. The linearity of the standard curve and the endotoxin concentration in each sample are calculated and results are printed automatically. In 48 min the automated system assays three samples and a reference standard in duplicate along with a water blank. Sensitivity of the assay is a function of incubation time. The assay is linear (r greater than 0.99) in the region of 0 to 1.0 endotoxin units per ml or 0 to 0.2 endotoxin units per ml with incubation times of 10 or 16 min, respectively. The method can be made very sensitive, detecting as low as 0.003 endotoxin units per ml with 30 min of incubation. The precision of the assay method, determined by assaying an endotoxin reference solution eight times, is ca. 6%. The LAL reagent designed for gel-clot assay was modified for the chromogenic substrate assay. We describe the optimum conditions for the performance of the chromogenic substrate LAL assay and stability of the LAL reagent.