Probing the cathepsin D using a BODIPY FL-pepstatin A: Applications in fluorescence polarization and microscopy

Chii Shiarng Chen, Wan Nan U Chen, Mingjie Zhou, Seksiri Arttamangkul, Richard P. Haugland

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

Redistribution of cathepsin D, a major lysosomal aspartic endopeptidase, has been related to various pathological progressions during tumor formation and oxidation stress. We have synthesized a fluorescent probe for cathepsin D, where the pepstatin A was covalently conjugated with the BODIPY (Boron dipyrromethene difluoride) fluorophore. In vitro, BODIPY FL-pepstatin A inhibits cathepsin D activity with an IC50 of 10 nM. The nature of its binding to cathepsin D was further characterized using a fluorescence polarization measurement. Results showed that BODIPY FL-pepstatin A selectively binds to cathepsin D at pH 4.5. In fixed cells, BODIPY FL- pepstatin A stained lysosomes, where it co-localized with cathepsin D. This staining was depleted when cells were co-incubated with unlabeled pepstatin A in acidic buffer. In live cells, BODIPY FL-pepstatin A is internalized and transported to lysosomes. The staining in the lysosomes can be competed with unlabeled pepstatin A. These properties, along with the good photostability of the BODIPY FL fluorophore, make this probe a novel tool for the study of the secretion and trafficking of cathepsin D. (C) 2000 Elsevier Science B.V.

Original languageEnglish (US)
Pages (from-to)137-151
Number of pages15
JournalJournal of Biochemical and Biophysical Methods
Volume42
Issue number3
DOIs
StatePublished - Mar 16 2000
Externally publishedYes

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Polarization Microscopy
Cathepsin D
Fluorescence Polarization
Boron
Fluorescence Microscopy
Microscopic examination
Fluorescence
Polarization
Lysosomes
Fluorophores
Aspartic Acid Endopeptidases
Staining and Labeling
boron difluoride
dipyrromethene
pepstatin
Fluorescent Dyes
Inhibitory Concentration 50
Tumors
Buffers
Oxidation

Keywords

  • Aspartyl proteases
  • Enzyme inhibitors
  • Image processing
  • Lysosomes

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

Cite this

Probing the cathepsin D using a BODIPY FL-pepstatin A : Applications in fluorescence polarization and microscopy. / Chen, Chii Shiarng; Chen, Wan Nan U; Zhou, Mingjie; Arttamangkul, Seksiri; Haugland, Richard P.

In: Journal of Biochemical and Biophysical Methods, Vol. 42, No. 3, 16.03.2000, p. 137-151.

Research output: Contribution to journalArticle

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