English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

A Catalytic Antibody Produces Fluorescent Tracers of Gap Junction Communication in Living Cells

MPS-Authors
/persons/resource/persons58764

List,  Benjamin
Dept. of Chemistry, The Scipps Research Institute;
Research Department List, Max-Planck-Institut für Kohlenforschung, Max Planck Society;

Locator
There are no locators available
Fulltext (public)
There are no public fulltexts available
Supplementary Material (public)
There is no public supplementary material available
Citation

Subauste, M. C., List, B., Guan, X., Hahn, K. M., Lerner, R. A., & Gilula, N. B. (2001). A Catalytic Antibody Produces Fluorescent Tracers of Gap Junction Communication in Living Cells. The Journal of Biological Chemistry, 276(52), 49164-49168. doi:10.1074/jbc.M105700200.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0014-A4D3-B
Abstract
The antibody 38C2 efficiently catalyzed a retro-Michael reaction to convert a novel, cell-permeable fluorogenic substrate into fluorescein within living cells. In vitro, the antibody converted the substrate to fluorescein with ak cat of 1.7 × 10−5s−1 and a catalytic proficiency (k cat /k uncat Km ) of 1.4 × 1010 M −1(Km = 7 μm). For hybridoma cells expressing antibody or Chinese Hamster Ovarian (CHO) cells injected with antibody, incubation of the substrate in the extracellular medium resulted in bright intracellular fluorescence distinguishable from autofluorescence or noncatalyzed conversion of substrate. CHO cells loaded with antibody were 12 times brighter than control cells, and more than 85% of injected cells became fluorescent. The fluorescein produced by the antibody traveled into neighboring cells through gap junctions, as demonstrated by blocking dye transfer using the gap junction inhibitor oleamide. The presence of functional gap junctions in CHO cells was confirmed through oleamide inhibition of lucifer yellow transfer. These studies demonstrate the utility of the intracellular antibody reaction, which could generate tracer dyes in specific cells within complex multicellular environments simply by bathing the system in substrate.