Isolation and characterization of a dibasic selective metalloendopeptidase from rat testes that cleaves at the amino terminus of arginine residues.
A metalloendopeptidase that selectively cleaves doublets of basic amino acids on the amino-terminal side of arginine residues was purified to homogeneity from rat testes and analyzed further. Two catalytically active forms with apparent relative molecular masses of 110,000 and 140,000 Da, respectively, were present in the purified preparation of the enzyme. Antibodies raised against the purified testis endopeptidase revealed by immunoblot both the 110- and 140-kDa forms in both rat testis and brain cortex extracts. The isolated enzyme was inhibited by metal chelators and divalent cations. Its activity, lost after preincubation with EDTA, was restored by low concentrations of Zn2+ and Mn2+, thus demonstrating the metallopeptidase nature of the enzyme. This endopeptidase also exhibited a high sensitivity to amastatin (100% inhibition at 20 microM), an aminopeptidase inhibitor. A substrate specificity study using physiologically important or synthetic peptides containing a processing dibasic site indicated that cleavage occurred selectively at the amino-terminal side of an arginine residue, independent of the nature of the basic doublet. The enzyme produced such a cleavage at the Arg-Lys doublet of somatostatin 28 (Km = 43 microM), at the Arg-Arg doublet of dynorphin A (Km = 6.45 microM) and atrial natriuretic factor (Km = 6.25 microM), and at the Lys-Arg doublet of preproneurotensin-(154-170) (Km = 17.3 microM). Moreover, cleavage efficiency was found to be higher for the larger substrates. The distinctive properties of this endopeptidase imply that this protein is a member of a novel class of proteolytic enzymes that may be involved in the endoproteolytic maturation of hormonal precursors.
Chesneau
V
V
Laboratoire de Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, Université Pierre et Marie Curie, Paris, France.
Pierotti
A R
AR
Barré
N
N
Créminon
C
C
Tougard
C
C
Cohen
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D002413:Cations, Divalent / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D011480:Protease Inhibitors; D008345:Manganese; D001120:Arginine; D004492:Edetic Acid; D008666:Metalloendopeptidases; D015032:Zinc