Enzyme assays by fluorescence polarization in the presence of polyarginine: study of kinase, phosphatase, and protease reactions.
We have previously reported that the kinase catalyzed conversion of fluorescently labeled phosphate acceptor peptides to the corresponding phosphopeptides can be conveniently followed by measuring the fluorescence polarization signal in the presence of polyarginine. In the present work, we demonstrate that the method can be used for other enzymes besides kinases, such as phosphatases and proteases. By adjustment of the ionic strength of the buffer it is possible to use this method in cases where both the substrate and the enzymatic product are highly negatively charged. All of these enzymatic transformations can be followed in real time, by performing the reactions in the presence of polyarginine and continuously measuring the fluorescence polarization signal. Polyarginine was found to have no effect on the rate of enzymatic conversion of the protease studied (cathepsin G), but its presence decreased the observed rate of phosphorylation by protein kinase A, presumably by decreasing the concentration of free ATP in the reaction solution. Leukocyte antigen related phosphatase catalyzed dephosphorylation reactions were faster in the presence of polyarginine. For all three enzymes, the reaction rates in the presence of polyarginine were found to be sensitive to the presence of known enzyme inhibitors, but the IC(50) values of the kinase inhibitors H-89 and PKI were higher in the presence than in the absence of polyarginine.
Simeonov
Anton
A
Caliper Technologies Corp., 605 Fairchild Drive, Mountain View, CA 94043, USA.
Bi
Xiahui
X
Nikiforov
Theo T
TT
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D056649:Cathepsin G; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009994:Osmolar Concentration; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D011956:Receptors, Cell Surface; D012697:Serine Endopeptidases; D017186:Titrimetry
D005456:Fluorescent Dyes; D010455:Peptides; D011956:Receptors, Cell Surface; C015462:polyarginine; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D017027:Protein Tyrosine Phosphatases; D054630:Receptor-Like Protein Tyrosine Phosphatases, Class 4; D002403:Cathepsins; D012697:Serine Endopeptidases; D056649:Cathepsin G