Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides.
Endopeptidase 24.15, a metalloendopeptidase (EC 3.4.24.15) with an Mr of about 70,000, was purified to homogeneity from rat testes. The enzyme cleaves preferentially bonds on the carboxyl side of hydrophobic amino acids. Secondary enzyme-substrate interactions at sites removed from the scissile bond are indicated by the finding that a hydrophobic or bulky residue in the P3' position greatly contributes to substrate binding and catalytic efficiency. The isolated enzyme is inhibited by metal chelators and by thiols. Loss of enzymic activity after dialysis against EDTA can be restored by low concentrations of Zn2+ and Co2+ ions. The rate of reaction of the Co2+ enzyme with a synthetic substrate was higher than that of the Zn2+ enzyme. These results are consistent with the classification of the enzyme as a metalloendopeptidase. N-Carboxymethyl peptides that fulfil the binding requirements of the substrate recognition site of the enzyme act as potent competitive inhibitors. Biologically active peptides such as luteinizing hormone-releasing hormone, bradykinin and neurotensin are cleaved at sites consistent with the specificity of the enzyme deduced from studies with synthetic peptides. Dynorphin A (1-8)-peptide, beta-neoendorphin, metorphamide, and Metenkephalin-Arg6-Gly7-Leu8 are rapidly converted to the corresponding enkephalins. The testis enzyme is catalytically and immunologically closely related to the previously identified brain enzyme.
Orlowski
M
M
Department of Pharmacology, Mount Sinai School of Medicine City University of New York, NY 10029.
Reznik
S
S
Ayala
J
J
Pierotti
A R
AR
D000818:Animals; D004745:Enkephalins; D008297:Male; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*
D004745:Enkephalins; D010455:Peptides; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase