Verapamil inhibitioa of enzymatic product efflux leads to improved detection of β-galactosidase activity in lacZ-transfected cells

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The β-galactosidase activity encoded by the lacZ gene of Escherichia coli is widely used to monitor successful expression of transfected genes. Fluorogenic substrates allow detection of enzyme activity in viable cells, which, subsequently, can be selected for further study on the basis of fluorescence emission. We analyzed three fluorogenic substrates (FDG, C12FDG, and CMFDG), all of which incorporate fluorescein as their fluorophore, regarding intensity of fluorescent signal and selectivity toward the transfected β-galactosidase activity vs. lysosomal enzyme activity. Among these substrates, 5-chloromethylfluoresecein di-β-D-galactopyranoside (CMFDG) showed the strongest selectivity toward the lacZ-encoded enzyme activity. An attempt to improve this selectivity by alkalinization of the lysosomal pH with chloroquine, such that the endogenous enzyme would be exposed to a suboptimal pH, led to significant cell death. In contrast, inhibition of dye efflux with verapamil enhanced the selectivity of CMFDG toward the lacZ-encoded enzyme activity by approximately threefold. Incubation with probenecid, on the other hand, showed little effect.

Original languageEnglish (US)
Pages (from-to)36-41
Number of pages6
JournalCytometry
Volume28
Issue number1
DOIs
StatePublished - May 1 1997
Externally publishedYes

Fingerprint

Galactosidases
Verapamil
Galactose
Enzymes
Fluorescent Dyes
Probenecid
Lac Operon
Chloroquine
Fluorescein
Cell Death
Coloring Agents
Fluorescence
Escherichia coli
Gene Expression

Keywords

  • β-galactosidase
  • CMFDG
  • FDG
  • Fluorescence
  • Fluorochromasia
  • LacZ
  • Lysosomal enzyme activity
  • Verapamil

ASJC Scopus subject areas

  • Biophysics
  • Cell Biology
  • Endocrinology
  • Hematology
  • Pathology and Forensic Medicine

Cite this

Verapamil inhibitioa of enzymatic product efflux leads to improved detection of β-galactosidase activity in lacZ-transfected cells. / Poot, Martin; Arttamangkul, Seksiri.

In: Cytometry, Vol. 28, No. 1, 01.05.1997, p. 36-41.

Research output: Contribution to journalArticle

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