A novel fluorogenic substrate for detecting alkaline phosphatase activity in situ.
We describe here the in situ detection of alkaline phosphatase (APase) activity with a new fluorogenic substrate, 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (CPPCQ). CPPCQ is very soluble and colorless. APase converts it into a rapidly precipitating product, whose strong fluorescence marks the sites of APase activity. The detected APase was either a probing enzyme anchored to epidermal growth factor (EGF) receptors of fixed human epidermoid carcinoma cell line (A431) by biotinylated EGF and streptavidin-APase conjugates or an endogenous marker existing in a fixed canine kidney cell line (MDCK). With CPPCQ staining, the EGF receptors and the endogenous APase were both visualized by fluorescence microscopy as contrasting, photostable, and well-resolved fluorescent stains. The EGF receptor staining was specific since it could be blocked by excessive unlabeled EGF. In contrast, fluorescein-labeled EGF failed to specifically stain the EGF receptors under the same fluorescent microscope. The endogenous APase staining with CPPCQ was sensitive to heating, levamisole and L-homoarginine, showing an APase tissue specificity of the liver/bone/kidney type. Therefore, CPPCQ appears to be a novel substrate dye for sensitive fluorescence APase histochemistry.
Huang
Z
Z
You
W
W
Haugland
R P
RP
Paragas
V B
VB
Olson
N A
NA
Haugland
R P
RP
D000469:Alkaline Phosphatase / Q000032:analysis*; D000818:Animals; D002460:Cell Line; D004285:Dogs; D066246:ErbB Receptors / Q000032:analysis*; D005456:Fluorescent Dyes; D006651:Histocytochemistry / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence; D009943:Organophosphorus Compounds; D011799:Quinazolines; D052999:Quinazolinones; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D009943:Organophosphorus Compounds; D011799:Quinazolines; D052999:Quinazolinones; C079177:2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone; D066246:ErbB Receptors; D000469:Alkaline Phosphatase