Experimental determination of the phosphorylation state of phenylalanine hydroxylase

A. K. Green, R. G.H. Cotton, I. Jennings, M. J. Fisher

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Abstract

A monoclonal antibody (PH 7), which recognizes the phosphorylated form of phenylalanine hydroxylase from human liver, has been used for the analysis of the enzyme in crude cell extracts from rat. In immunoblot analyses of rat liver cell extracts, the extent of binding of PH 7 closely correlates with the phosphorylation state of phenylalanine hydroxylase, as judged by [32P]P(i) incorporation. These observations have made possible the rapid non-radioactive quantification of hormonal effects on phenylalanine hydroxylase phosphorylation state. In particular, the glucagon-dependent phosphorylation of phenylalanine hydroxylase in liver cells was investigated. Epidermal growth factor was shown to modulate this process. In addition, this technique was used to demonstrate, for the first time, that dibutyryl cyclic AMP, unlike the Ca2+ ionophore A23187, stimulates the phosphorylation of phenylalanine hydroxylase in isolated kidney tubules from rat.

Original languageEnglish
Pages (from-to)563-568
Number of pages6
JournalBiochemical Journal
Volume265
Issue number2
DOIs
Publication statusPublished - 1 Jan 1990
Externally publishedYes

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