A novel [125I]iodinated carboxypeptidase A substrate detects a metallopeptidase activity distinct from carboxypeptidase A in brain.
We have designed two radioactive substrates, hippuryl-L-[3H]phenylalanine and 3-(p-hydroxy, m-[125I]phenyl)propionic acid ([125I]Bolton reagent) derivative of L-arginyl-L-phenylalanine, i.e. [125I]BRF, for a highly sensitive assay of carboxypeptidase A (CPA) activity. After cleavage of the C-terminal phenylalanine residue by CPA, the radioactive product of the reaction was conveniently separated by polystyrene bead chromatography. Using [125I]BRF, typical CPA activity inhibited by 1 microM 2-benzyl-3-mercaptopropanoic acid was detected in extracts from rat pancreas or intestine. In brain and some other tissues, however, [125I]BRF-hydrolyzing activity was only inhibited by this compound in 1000-fold higher concentration, suggesting the participation of a metallopeptidase distinct from CPA.
Normant
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Unité de Neurobiologie et de Pharmacologie de l'INSERM, Paris, France.
Schwartz
J C
JC
Gros
C
C
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002268:Carboxypeptidases / Q000378:metabolism*; D043422:Carboxypeptidases A; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008297:Male; D008666:Metalloendopeptidases / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D011422:Propionates / Q000138:chemical synthesis / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence; D013388:Succinimides
D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D011422:Propionates; D013388:Succinimides; D010649:Phenylalanine; C104271:3-(4-hydroxy-3-iodophenyl)propionic acid; C022581:Bolton-Hunter reagent; C049123:N-(hippuryl)phenylalanine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D008666:Metalloendopeptidases