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A sensitive staining method for alkali-labile phosphoproteins has been developed. As little as 0.2 nmol bound P/mm2 can be detected. The procedure is based on alkaline hydrolysis, phosphate capture, and formation of an insoluble rhodamine B-phosphomolybdate complex. A further modification for the qualitative detection of alkaline phosphatase activity on polyacrylamide gels is proposed. During incubation, the released Pi is precipitated as lead phosphate and subsequently stained with rhodamine B.

1. The non-specific hen's egg yolk alkaline phosphatase is a metalloprotein (Zn2+?) composed of two identical inactive subunits. 2. A second metal site preferably binds Mg2+ (15-fold activation). Me(II)(H2O)H+, a charged arginine, and tyrosine in the active site are involved in positioning and binding of the substrate and metal ion. 3. Substrate inhibition differs with pH. This may be related to the presence of two active sites in the enzyme, one in each subunit. 4. Uncompetitive inhibition with l-phenylalanine and analogues suggests a phosphorylated intermediate. 5. Inhibition is weakly competitive with Pi, strong non-competitive with PPi as compared to Mg2+- free PPi, and partially competitive with arsenate. 6. The purified enzyme is stabilized and activated by amines and proteins.

1. Hen's egg yolk alkaline phosphatase can transfer the donor substrate phosphoryl to a hydroxyl containing acceptor. 2. This acceptor contains preferably an alpha-amino or alpha-imino group. 3. The optimal pH of the transfer reaction is at 9.45 with Tris and at 9.60 with diethanolamine as acceptor. 4. Transfer products were isolated and characterized. 5. The ratio of products is independent of the donor substrate. ROH and R'OP production increase linearly with the acceptor concentration, while Pi release and Km remain constant. 6. This points to a mechanism involving two phosphorylated intermediates. 7. The acceptor bypasses the isomerization step of the phosphoryl enzyme, which is necessary before hydrolysis can take place.