Application of bimane-peptide substrates to spectrofluorometric assays of metalloendopeptidases.
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).
Kajiwara
K
K
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University.
Kumazaki
T
T
Sato
E
E
Kanaoka
Y
Y
Ishii
S
S
D000595:Amino Acid Sequence; D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D007700:Kinetics; D008666:Metalloendopeptidases / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010196:Pancreatic Elastase / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013305:Streptomyces griseus / Q000201:enzymology; D013379:Substrate Specificity
D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D009842:Oligopeptides; C021149:bimanes; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases