Highly increased levels of active stromelysin in rheumatoid synovial fluid determined by a selective fluorogenic assay.
AuthorsBeekman B, van El B, Drijfhout JW, Ronday HK, TeKoppele JM
JournalFEBS Lett
PubMed ID9428733
'Stromelysin-1 (MMP-3) is an important member of the matrix metalloproteinase family. In joint-degrading diseases like arthritis, elevated levels of MMP-3 protein are detected in synovial fluid using immunological methods. However, these methods do not discriminate between active and inactive enzyme. In the present study, a specific stromelysin activity assay was ... More
A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis.
AuthorsMaggiora LL, Smith CW, Zhang ZY
JournalJ Med Chem
PubMed ID1433187
'A general scheme for obtaining a fluorescent donor/acceptor peptide substrate via solid-phase synthesis methodology is presented. The key feature of this method is the design of a glutamic acid derivative that has been modified on the carboxyl side chain with a 5-[(2''-aminoethyl)-amino]naphthelenesulfonic acid (EDANS) to create a fluorescent donor moiety ... More
Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH(2)-Gly(3) substrates.
AuthorsTon-That H, Mazmanian SK, Faull KF, Schneewind O
JournalJ Biol Chem
PubMed ID10734144
'Staphylococcus aureus sortase anchors surface proteins to the cell wall envelope by cleaving polypeptides at the LPXTG motif. Surface proteins are linked to the peptidoglycan by an amide bond between the C-terminal carboxyl and the amino group of the pentaglycine cross-bridge. We find that purified recombinant sortase hydrolyzed peptides bearing ... More
A bifunctionalized fluorogenic tetrasaccharide as a substrate to study cellulases.
AuthorsArmand S, Drouillard S, Schülein M, Henrissat B, Driguez H
JournalJ Biol Chem
PubMed ID9006908
'Cellulases are usually classified as endoglucanases and cellobiohydrolases, but the heterogeneity of cellulose, in terms of particle size and crystallinity, has always represented a problem for the biochemical characterization of the enzymes. The synthesis of a bifunctionalized tetrasaccharide substrate suitable for measuring cellulase activity by resonance energy transfer is described. ... More
Substrate recognition through a PDZ domain in tail-specific protease.
'Tail-specific protease (Tsp) is a periplasmic enzyme that selectively degrades proteins bearing a nonpolar C-terminus. Its substrate specificity suggests that Tsp may contain a substrate recognition domain, which selectively binds to the nonpolar C-termini of substrate proteins, separate from its catalytic site. In this work, we show that substrate recognition ... More
Identification and characterization of falcilysin, a metallopeptidase involved in hemoglobin catabolism within the malaria parasite Plasmodium falciparum.
AuthorsEggleson KK, Duffin KL, Goldberg DE
JournalJ Biol Chem
PubMed ID10542284
'The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in ... More
A continuous fluorescence-based assay of human cytomegalovirus protease using a peptide substrate.
AuthorsHolskin BP, Bukhtiyarova M, Dunn BM, Baur P, de Chastonay J, Pennington MW
JournalAnal Biochem
PubMed ID7668375
'The 28-kDa protease from human cytomegalovirus (hCMV) has been successfully cloned, expressed, and purified to homogeneity. An internally quenched fluorescent substrate (4-4''-dimethylaminophenazo)benzoyl-Arg-Gly-Val-Val-Asn-Ala-Ser-Ser -Arg-Leu-Ala-5-[(2''-aminoethyl)-amino]-naphthalene-1-sulfonic acid; DABCYL-CMV-EDANS) based on the maturational cleavage site (M-site) junction was synthesized in an effort to develop a fluorescence-based assay. This substrate is cleaved specifically between the ... More
Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction.
AuthorsLiu Y, Kati W, Chen CM, Tripathi R, Molla A, Kohlbrenner W
JournalAnal Biochem
PubMed ID10036138
'A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important ... More
Synthesis and characterization of two fluorescent sulfhydryl reagents.
AuthorsHudson EN, Weber G
JournalBiochemistry
PubMed ID4745664
Singlet energy transfer studies of the arrangement of proteins in the 30 S Escherichia coli ribosome.
AuthorsHuang KH, Fairclough RH, Cantor CR
JournalJ Mol Biol
PubMed ID241859
New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain.
AuthorsGarcía-Echeverría C, Rich DH
JournalFEBS Lett
PubMed ID1551413
A series of new substrates for determining the catalytic activity of cysteine proteinases is described. The rate of hydrolysis by papain was monitored by a fluorescence continuous assay based on internal resonance energy transfer using 5-[(2-aminoethyl)amino]naphtalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) as fluorescent donor and quenching acceptor, respectively, in ... More
Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS).
AuthorsGershkovich AA, Kholodovych VV
JournalJ Biochem Biophys Methods
PubMed ID9029259
A prospective class of intramolecular fluorescence energy transfer substrates (IFETS) is described. In contrast to the known chromogenic and fluorogenic substrates that are used widely in the scientific and medical research, IFETS allow to control the enzymatic cleavage at any point of the peptide chain and thus permit simultaneous studies ... More
Molecular beacons: probes that fluoresce upon hybridization.
AuthorsTyagi S, Kramer FR
JournalNat Biotechnol
PubMed ID9630890
We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fluorogenic conformational change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains ... More
Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media.
AuthorsBeekman B, Drijfhout JW, Bloemhoff W, Ronday HK, Tak PP, te Koppele JM
JournalFEBS Lett
PubMed ID8706864
Matrix metalloproteinases (MMPs) are involved in physiological tissue remodeling and pathological conditions like tumour metastasis and joint destruction. Until now, no convenient and sensitive MMP-activity assay in crude media like synovial fluid has been available. Therefore, the highly soluble fluorogenic substrate TNO211 (Dabcyl-Gaba-Pro-Gln-Gly-Leu-Glu(EDANS)-Ala-Lys-NH2), containing the MMP cleavable Gly-Leu bond and ... More
Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer.
AuthorsPennington MW, Thornberry NA
JournalPept Res
PubMed ID8012123
Interleukin 1 beta converting enzyme (ICE) is responsible for processing an inactive 31-kDa precursor to the active, mature 17-kDa Il-1 beta with cleavage occurring between the Asp116-Ala117 amide bond. We have prepared a peptide substrate that contains the protease cleavage site situated between two fluorophores located at the termini of ... More
Engineering the S1' subsite of trypsin: design of a protease which cleaves between dibasic residues.
AuthorsKurth T, Grahn S, Thormann M, Ullmann D, Hofmann HJ, Jakubke HD, Hedstrom L
JournalBiochemistry
PubMed ID9708978
The serine protease trypsin was converted into a site-specific protease which hydrolyzes peptides between dibasic residues. Trypsin exhibits a high S1 specificity for Arg and Lys residues. However, the S1' specificity of trypsin is very broad, with only a slight preference for hydrophobic residues in P1'. We replaced Lys60 with ... More
A fluorescence-quenched chitopentaose for the study of endo-chitinases and chitobiosidases.
AuthorsCottaz S, Brasme B, Driguez H
JournalEur J Biochem
PubMed ID10951219
A new fluorogenic substrate displaying intramolecular fluorescence energy transfer (FRET) has been synthetized from NI,NII,NIII, NIV-tetra-acetyl-chitopentaose. Two molecules, a fluorophore (5-(2-aminoethyl) amino-1-naphtalene-sulfonic acid; EDANS) and a quenching group (dimethylaminophenylazophenyl; DAB) were chemically introduced on to the chitopentaose, one at each end. Among eight enzymes tested, only endo-chitinase and chitobiosidase activities ... More
The herpesvirus protease: mechanistic studies and discovery of inhibitors of the human cytomegalovirus protease.
AuthorsFlynn DL, Becker DP, Dilworth VM, Highkin MK, Hippenmeyer PJ, Houseman KA, Levine LM, Li M, Moormann AE, Rankin A, Toth MV, Villamil CI, Wittwer AJ, Holwerda BC
JournalDrug Des Discov
PubMed ID9332827
The herpesvirus protease is a recently identified enzyme which is essential for viral replication. It is found in all herpesviruses and offers a new molecular target for therapeutic intervention. Its genomic structure has recently been described and consists of a large open reading frame which encodes a fusion protein containing ... More
A new, sensitive fluorogenic substrate for papain based on the sequence of the cystatin inhibitory site.
AuthorsGauthier F, Moreau T, Lalmanach G, Brillard-Bourdet M, Ferrer-Di Martino M, Juliano L
JournalArch Biochem Biophys
PubMed ID8215429
We have designed and tested a new papain substrate with intramolecularly quenched fluorescence. It is based on a highly conserved sequence in all members of the cystatin superfamily that participates in the inhibition of cysteine proteinases. This substrate, O-aminobenzoyl (Abz)-QVVAGA-ethylenediamine-2-4-dinitrophenyl (EDDnp) is very sensitive to papain with a second-order rate ... More
Purification and characterization of the yeast glycosylphosphatidylinositol-anchored, monobasic-specific aspartyl protease yapsin 2 (Mkc7p).
AuthorsKomano H, Rockwell N, Wang GT, Krafft GA, Fuller RS
JournalJ Biol Chem
PubMed ID10446224
The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase. Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain. Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular ... More
The use of poly(ethylene glycol)-block-poly(lactic acid) derived copolymers for the rapid creation of biomimetic surfaces.
AuthorsTessmar J, Mikos A, Göpferich A
JournalBiomaterials
PubMed ID12922157
For many tissue engineering applications biomimetic or bioactive polymers would allow for a more precise control of cell behavior in growing tissues than has so far been possible. For this application recently developed amine reactive diblock copolymers (N-succinimidyl tartrate monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) [ST-NH-PEGxPLAy]) were investigated concerning their reactivity in ... More
Internally consistent libraries of fluorogenic substrates demonstrate that Kex2 protease specificity is generated by multiple mechanisms.
AuthorsRockwell NC, Wang GT, Krafft GA, Fuller RS
JournalBiochemistry
PubMed ID9048578
Kex2 protease from the yeast Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases. To clarify understanding of the interactions responsible for substrate recognition in this family of enzymes, we have carried out a systematic examination of Kex2 substrate specificity using internally consistent sets of substrates ... More
Fluorescence characterization of structural transitions at the strong actin binding motif in skeletal myosin affinity labeled at cysteine 540 with novel spectroscopic cysteaminyl mixed disulfides.
AuthorsBertrand R, Derancourt J, Kassab R
JournalBiochemistry
PubMed ID11087419
We have synthesized the luminescent and fluorescent lanthanide chelate S-(2-nitro-5-thiobenzoic acid)cysteaminyldiethylenetriaminepentaacetate-5-[(2-aminoethyl)am ino ]naphthalene-1-sulfonic acid as well as the fluorescent analogue S-(2-nitro-5-thiobenzoic acid)cysteaminyl-5-carboxyfluorescein using the procedure we recently described [Bertrand, R., Capony, J.-P., Derancourt, J., and Kassab, R. (1999) Biochemistry 38, 11914-11925]. Both mixed disulfides react with the skeletal myosin motor ... More
Alpha-ketoacids are potent slow binding inhibitors of the hepatitis C virus NS3 protease.
AuthorsNarjes F, Brunetti M, Colarusso S, Gerlach B, Koch U, Biasiol G, Fattori D, De Francesco R, Matassa VG, Steinkühler C
JournalBiochemistry
PubMed ID10677236
The replication of the hepatitis C virus (HCV), an important human pathogen, crucially depends on the proteolytic maturation of a large viral polyprotein precursor. The viral nonstructural protein 3 (NS3) harbors a serine protease domain that plays a pivotal role in this process, being responsible for four out of the ... More
Direct measurement of acylenzyme hydrolysis demonstrates rate-limiting deacylation in cleavage of physiological sequences by the processing protease Kex2.
AuthorsRockwell NC, Fuller RS
JournalBiochemistry
PubMed ID11297433
Saccharomyces cerevisiae Kex2 protease is the prototype for the family of eukaryotic proprotein convertases that includes furin, PC1/3, and PC2. These enzymes belong to the subtilase superfamily of serine proteases and are distinguished from degradative subtilisins by structural features and by their much more stringent substrate specificity. Pre-steady-state studies have ... More
On the sequential determinants of calpain cleavage.
AuthorsTompa P, Buzder-Lantos P, Tantos A, Farkas A, Szilágyi A, Bánóczi Z, Hudecz F, Friedrich P
JournalJ Biol Chem
PubMed ID14988399
The structural clues of substrate recognition by calpain are incompletely understood. In this study, 106 cleavage sites in substrate proteins compiled from the literature have been analyzed to dissect the signal for calpain cleavage and also to enable the design of an ideal calpain substrate and interfere with calpain action ... More