FLUORESCENT CYTOCHEMISTRY OF ACID-PHOSPHATASE AND DEMONSTRATION OF FLUID-PHASE ENDOCYTOSIS USING AN AZO-DYE METHOD

The aim of this work was the development of a fluorescent microscopy t echnique to visualize acid phosphatase activity in living and pre-fixe d cells. We have shown that a coupling azo dye method, using naphthol AS-MX phosphate (NP) as substrate and fast red TR (FR) as a diazonium salt coupling agent, gives rise to a fluorescent azo dye reaction prod uct which permits a highly sensitive demonstration of lysosomal acid p hosphatase in both living and pre-fixed monolayer cell cultures. The g ranular staining is prevented by inhibition of acid phosphatase activi ty using fluoride and/or orthovanadate in both living and pre-fixed pr eparations. Lysosomal staining in living cells is also abolished by in hibition of fluid-phase endocytosis using low temperatures or inhibiti on of oxidative phosphorylation. It was shown that whilst NP entered l iving cells by passive diffusion, occurrence of FR in lysosomes result ed from fluid-phase endocytosis. Spectroscopic analysis of the emissio n and absorption features of FR, NP, naphthol AS-MX (N), and the N-FR azo dye reaction product in solution corroborated our microscopic resu lts. The differing uptake mechanisms, and the occurrence of lysosomall y localized azo dye, were also in keeping with the predictions of quan titative structure-activity relationship models of this system.