Biotinylated and cysteine-modified peptides as useful reagents for studying the inhibition of cathepsin G.
An assay for studying the proteolytic activity of endopeptidases using a biotinylated and cysteine-modified peptide has been developed. This assay is rapid, sensitive, and reproducible. Although used here specifically for the enzyme which cleaves at the amino terminus (N-terminus) of beta-amyloid peptide (BAP); this type of radiolabeled substrate is readily applied to the analysis and detection of other endoprotease activities. This method relies on a peptide substrate which contains: (a) the amino acids flanking the enzymatic cleavage site, (b) an added cysteine at the carboxy-terminus to allow for incorporation of radiolabel via an addition reaction with tritiated N-[ethyl-1,2-3H]maleimide (3H-NEM), and (c) a biotin at the N-terminus to allow for binding to avidin-coated scintillation proximity assay (SPA) beads. It has been suggested that the enzyme involved in the N-terminal cleavage of amyloid precursor peptide to generate BAP is a chymotrypsin-like serine protease such as cathepsin G. To study this enzymatic activity and to screen for its inhibitors, we have synthesized the peptide biotin-SEVKMDAEFdC which contains the amino acids flanking the N-terminal cleavage site of BAP. Tritiated NEM is covalently bound to the cysteine at the carboxy-terminal end and the labeled peptide is purified by reverse-phase high-performance liquid chromatography. Following digestion of 3H-NEM-labeled peptide by cathepsin G, the biotinylated side of the cleaved peptide is bound to the SPA bead, while the tritiated end of the cleaved peptide remains in solution. Enzymatic hydrolysis is measured as the loss of 3H-induced scintillation signal. This method has allowed us to rapidly determine kinetic constants and develop a high throughput screen to study inhibition of cathepsin G cleavage in a native peptide context.
Brown
A M
AM
Department of CNS Biological Research, Lederle Laboratories, American Cyanamid Company, Pearl River, New York 10965.
George
S M
SM
Blume
A J
AJ
Dushin
R G
RG
Jacobsen
J S
JS
Sonnenberg-Reines
J
J
D000595:Amino Acid Sequence; D001710:Biotin / Q000378:metabolism*; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D003545:Cysteine / Q000494:pharmacology*; D005033:Ethylmaleimide; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012697:Serine Endopeptidases; D014316:Tritium; D000514:alpha 1-Antichymotrypsin
D010455:Peptides; D000514:alpha 1-Antichymotrypsin; D014316:Tritium; D001710:Biotin; D002403:Cathepsins; D012697:Serine Endopeptidases; D056649:Cathepsin G; D003545:Cysteine; D005033:Ethylmaleimide