Endopeptidase 24.15: A neuropeptide metabolizing enzyme. Isolation, localization, possible function.
Item
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Title
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Endopeptidase 24.15: A neuropeptide metabolizing enzyme. Isolation, localization, possible function.
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Identifier
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AAI9108167
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identifier
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9108167
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Creator
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Reznik, Sandra Eve.
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Contributor
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Adviser: Marian Orlowski
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Date
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1990
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Endopeptidase 24.15, metalloendopeptidase (EC 3.4.24.15)with an M{dollar}\sb{lcub}\rm r{rcub}{dollar} of about 70,000 that rapidly converts dynorphin(1-8), {dollar}\alpha{dollar}-neo-endorphin, {dollar}\beta{dollar}-neo-endorphin, Met-enkephalin-Arg-Gly-Leu, and metorphamide into the repsective enkephalins, was purified to homogeneity from rat testes. 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 Zn{dollar}\sp{lcub}2+{rcub}{dollar} and Co{dollar}\sp{lcub}2+{rcub}{dollar} ions. These results are consistent with the classification of the enzyme as a metalloendopeptidase. The testis enzyme is catalytically and immunologically closely related to the previously identified brain enzyme.;The enzyme was purified from rat brain by immunoaffinity chromatography. Both forms of the brain enzyme, soluble and membrane-bound, were obtained; the soluble form was purified to homogeneity. As previous efforts to isolate the brain enzyme had failed to remove an inactive component from the preparation, this is the first time the brain enzyme has been so highly purified. The brain enzyme was inhibited by EDTA and EGTA in accordance with its classification as a metalloenzyme. The brain and testis enzymes appeared to be closely related catalytically and immunologically. The testis enzyme migrated somewhat more slowly in SDS PAGE than the brain enzyme, suggesting a higher molecular weight than the brain enzyme. The membrane-bound brain enzyme was found to be catalytically and immunologically similar to the soluble brain enzyme, but of less mobility in SDS PAGE. The slower migration suggests that the membrane-bound enzyme may contain an additional hydrophobic sequence for anchoring in the plasma membrane.;Endopeptidase 24.15 is most highly concentrated in testis, brain, anterior pituitary, and spinal cord. The high relative activity in testis prompted us to examine the localization of the enzyme within the testis immunocytochemically. Two cell populations containing the enzyme were identified immunocytochemically: Leydig cells and spermatazoa. Preincubation of the immune serum with the enzyme resulted in dramatically diminished or abolished staining. The enzyme may act on the peptide precursors of Met- and Leu-enkephalin to release these enkephalins in the testis and/or modulate the paracrine or autocrine function of LHRH in the testis.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
