Label extenders discovery

The whole question of the specificity was reopened with the discovery that E. coli phosphatase, contrary to an earlier statement (114), hydrolyzed a variety of polyphosphates including metaphosphate of average chain length 8 (97). It was subsequently reported that partially purified phosphatases from several mammalian tissues had appreciable PPi-ase activity at pH 8.5 (115). This was confirmed (116) and extended to include ATPase and fluorophosphatase activities (117). Proof that the same enzyme is responsible for the monoesterase and PPi-ase activities was afforded by heat inactivation studies, cross inhibition experiments, and inhibition of PPi-ase activity by L-phenylalanine, a specific inhibitor of intestinal phosphatase. It was also found that calf intestinal phosphatase couid be phosphorylated by 32P-PP and the number of sites so labeled agreed with the number of active sites determined with a monoester substrate using a stopped-flow technique (118). It would seem that the main reason for the confusion with regard to the PPi-ase activity results from the inclusion of Mg2+ in the assay. This stimulates the monoesterase activity but almost completely inhibits PPi-ase activity (117). 

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