[33] Preparation of protein phosphatase-resistant substrates using adenosine 5′-O-(γ-Thio)triphosphate

Adenosine 5'-O-(γ-thio)-triphosphate (ATPγS) can replace ATP as an effective substrate of many kinases, particularly protein kinases. On the other hand, ATPγS is hydrolyzed very slowly by phosphatases and by most ATPases in comparison with ATP. Similarly, proteins thiophosphorylated by ATPγS are also resistant to protein phosphatases. Thus, thiophosphorylation of phosphorylase b by phosphorylase kinase leads to its conversion to the activated a form that is resistant to dethiophosphorylation and inactivation by phosphorylase phosphatase activity. The metabolic stability of thiophosphorylated proteins has also been demonstrated for myosin light chain in smooth muscle contraction, and for the receptor of epidermal growth factor in the epidermal carcinoma cell line A431. Therefore, thiophosphorylation of protein by ATPγS can serve as a powerful tool for investigating the regulatory mechanism of enzymes and metabolic processes that are controlled by a cycle of protein phosphorylation and dephosphorylation.