Application of Bimane-Peptide Substrates to Spectrofluorometric Assays of Metalloendopeptidases
| Autor: | Shin-ichi Ishii, Kenta Kajiwara, Yuichi Kanaoka, Takashi Kumazaki, Eisuke Sato |
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| Rok vydání: | 1991 |
| Předmět: |
Molecular Sequence Data
Fluorescence spectrometry Biochemistry Streptomyces Substrate Specificity Bridged Bicyclo Compounds chemistry.chemical_compound Bimane Thermolysin Amino Acid Sequence Molecular Biology Pancreatic Elastase biology Chemistry Streptomyces griseus Metalloendopeptidases Substrate (chemistry) General Medicine Bridged Bicyclo Compounds Heterocyclic biology.organism_classification Kinetics Spectrometry Fluorescence Metalloendopeptidase activity Pseudomonas aeruginosa Metalloendopeptidase Oligopeptides |
| Zdroj: | The Journal of Biochemistry. 110:345-349 |
| ISSN: | 1756-2651 0021-924X |
| DOI: | 10.1093/oxfordjournals.jbchem.a123583 |
| Popis: | A spectrofluorometric method for sensitive determination of metalloendopeptidase activity has been developed by using a bimane-peptide containing a tryptophan residue, i.e. 1,7-dioxo-2,5,6-trimethyl-1H,7H-pyrazolo[1,2-alpha]pyrazol-3-yl-methyl- thiomethylcarbonyl-phenylalanyl-tryptophanyl-leucine (Bim-SCH2CO-Phe-Trp-Leu-OH). Such an "intramolecularly quenched" substrate was originally designed for a sensitive assay of angiotensin I converting enzyme (ACE) [Sato, E. et al. (1989) Chem. Pharm. Bull. 37, 145-147]. All the typical metalloendopeptidases tested, such as thermolysin, Pseudomonas aeruginosa (Ps.) elastase, Streptomyces griseus metalloendopeptidases I and II (SGMPI and SGMPII), and alkinonase A, a metalloendopeptidase from Streptomyces violaceorectus, cleaved this substrate strictly at a Phe-Trp bond, leading to a marked increase in fluorescence. Kinetic parameters of the enzymatic hydrolyses of five kinds of analogous bimane substrates were compared to examine how the nature of neighboring amino acid residues on either side of the cleavable bond affects the catalytic efficiency of each of the metalloendopeptidases. Bim-SCH2CO-Phe-Trp-Leu-OH was most efficiently hydrolyzed by all of these enzymes. The use of this substrate made it possible to determine minute amounts of metalloendopeptidases, especially those originating from Streptomycetes (for example, as little as 10 fmol of SGMPII). |
| Databáze: | OpenAIRE |
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