Abstract
The determination of enzyme activity or inhibition in intact living cells is a problem in the development of inhibitors for intracellular proteases. The production of fluorescent protoporphyrin IX (PpIX) from the nonfluorescent (N)-Gly/Pro-5-aminolevulinic acid (ALA) substrates was used to evaluate the prolyl/glycyl-specific dipeptidylpeptidase IV (DPPIV)-like and prolyloligopeptidase (POP)-like activities of human cells. The results demonstrated that whereas POP-like activity could be attributed to the actual POP, the DPPIV-like activity could be related to actual DPPIV only in one colon cell line. In the other breast and colon cell lines, DPPIV-like activity was intracellular and displayed by other prolyl-specific aminopeptidases. Our experiments also demonstrated the involvement of glycyl-specific proteases in the processing of ALA precursors. These observations have important consequences for the development and evaluation of selective inhibitors for these enzymes.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Deaminase / metabolism
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Aminolevulinic Acid / analogs & derivatives
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Aminolevulinic Acid / metabolism
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Breast Neoplasms / enzymology
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Breast Neoplasms / pathology
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Cell Line, Tumor
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Colonic Neoplasms / enzymology
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Colonic Neoplasms / pathology
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Cysteine Endopeptidases / analysis*
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Dipeptidyl Peptidase 4 / metabolism
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Drug Design
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Female
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Fluorescent Dyes*
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Glycoproteins / metabolism
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Humans
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Kinetics
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Male
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Methods
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Prolyl Oligopeptidases
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Protease Inhibitors / chemical synthesis
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Protease Inhibitors / pharmacology
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Protoporphyrins / analysis
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Protoporphyrins / biosynthesis*
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Serine Endopeptidases / analysis*
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Substrate Specificity
Substances
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Fluorescent Dyes
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Glycoproteins
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Protease Inhibitors
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Protoporphyrins
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Aminolevulinic Acid
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protoporphyrin IX
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DPP4 protein, human
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Dipeptidyl Peptidase 4
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Serine Endopeptidases
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PREPL protein, human
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Prolyl Oligopeptidases
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Cysteine Endopeptidases
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glycyl endopeptidase
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Adenosine Deaminase