HORSERADISH PEROXIDASE-GLUTARALDEHYDE (HRP-GLA) REACTION ON THE PLASMA MEMBRANE OF FROG UROTHELIAL CELLS

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In the presence of glutaraldehyde (GLA), horseradish peroxidase (HRP) was found to bind to the basolateral cell membrane, but not to the apical membrane, of dissociated frog urothelial cells. To clarify the mechanism of the binding, the effect of various modifications to the reaction was studied. The binding of HRP, i. e. "the HRP-GLA staining or reaction", revealed the following characteristics: 1) The staining was observed only in the cells dissociated with cation chelation or enzyme treatment, and not in the cells scraped mechanically. 2) GLA should coexist in the same solution with HRP. Paraformaldehyde (FA) or dimethyl suberimidate (DMS) could not replace GLA. 3) Prefixation with GLA, FA or DMS could prevent the staining. 4) Diazotization of amino groups by prolonged sodium nitrite incubation abolished the reaction totally. 5) Pretreatment of cell with poly-L-lysine caused the apical membrane to show positive staining. These results indicate that the IIRP-GLA reaction stains the amino groups,which are exposed only after some dissociating treatments, and can be used as a cell membrane marker at an electron microscopic level.
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