PURIFICATION AND PROPERTIES OF A PROLYL ENDOPEPTIDASE FROM RABBIT BRAIN

Authors

  • Marian Orlowski,

    1. Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, Fifth Avenue and 100th Street, New York, NY 10029, U.S.A.
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  • Elizabeth Wilk,

    1. Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, Fifth Avenue and 100th Street, New York, NY 10029, U.S.A.
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  • Stevens Pearce,

    1. Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, Fifth Avenue and 100th Street, New York, NY 10029, U.S.A.
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  • Sherwin Wilk

    1. Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, Fifth Avenue and 100th Street, New York, NY 10029, U.S.A.
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Abstract

Abstract— An enzyme with the specificity of a prolyl endopeptidase was purified about 880-fold from rabbit brain. The enzyme hydrolyzes peptidylprolyl-peptide and peptidylprolyl-amino acid bonds. Several biologically active peptides such as angiotensin, bradykinin, neurotensin. substance P and thyrotropin releasing hormone are degraded by hydrolysis of the bond between the carboxyl group of proline and the adjacent amino acid or ammonia respectively. The enzyme is activated by dithiothreitol and inhibited by heavy metals and thiol blocking agents. The serine protease inhibitor phenylmethanesulfonylfluoride has no effect on activity; however, inhibition was obtained with diisopropylfluorophosphate. Prolyl endopeptidase has a molecular weight of about 66,000 and a pH optimum of about 8.3. A new chromogenic substrate, N-benzyloxycarbonylglycyl-L-prolylsulfamethoxazole, was used for determination of enzyme activity. The substrate is hydrolyzed to N-benzyloxycarbonylglycyl-L-proline and free sulfamethoxazole which can be conveniently determined by a colorimetric procedure.

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