Enzymatic -Phosphate Incorporation into Proteins

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A lot of biologic membrane systems and cellular organelles contain kinases, which transfer phosphate groups to proteins, especially to serine, threonine, and tyrosine residues. Self-phosphorylation of enzymes leading to acylphosphates or phosphoamides can be observed, too. With respect to their chemical stability, these phos-phoproteins are classified into acid-stable (alkali labile), hydroxyl-amine-sensitive, and acid labile. 

Weller M (1979) Protein phosphorylation the nature, function, and metabolism of proteins with covalently bound phosphorus. Pion, London 

The catalytic subunit of the cycloAMP-dependent protein kinase (cAMP PrK) transfers the terminal (y) phosphate residue of ATP to serine residues of numerous proteins. The assay described in this 

Pipet the assay according to Table 6.7, cool the tubes in an ice bath, then add the protein solution or membrane suspension which 

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ATP Synthetase. Membrane-boimd proteins that utilize the proton gradient across the membrane to form ATP from ADP and phosphate are known as ATP synthetases. The synthetase complex, composed of a hydrophobic part (FO) and a water-soluble domain (FI, catalsdic center) from Rhodospirillum rubrum, was incorporated into hposomes of a polymerizable sulfolipid (235). Lipid-free synthetase has a very low enzymatic activity. If the sulfolipid liposome is partially photopolymerized and the protein complex is incorporated into the liposome, the observed enzymatic activity is comparable to that of the natural lipid membranes. 

The in situ renaturation procedure can also be adapted to measure kinase activity as incorporation of phosphate into exogenously added peptide substrate (Shackelford and Zivin, 1993). This allows detection of CaM kinase II that may be enzymatically active but has the sites of autophosphorylation altered by mutagenesis, proteolysis, or posttranslational modification. By the judicious choice of peptide substrate and required activators, the method can be used to selectively detect different protein kinases. 

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