Method for Detecting Transferase Enzymatic Activity

Abstract

Methods and kits for detecting transferase activity in a sample by measuring ATP using a composition comprising an ATP-dependent bioluminescence-generating enzyme, a luminogenic molecule and one or more transferase quenching agents.

1 - 26 . (canceled) 
     
     
         27 . A kit for measuring transferase enzymatic activity comprising:
 (a) a reconstitution buffer solution comprising one or more transferase quenching agents, wherein the transferase quenching agent selectively stops transferase activity without substantially affecting bioluminescent-generating enzyme activity;   (b) a composition comprising a luminogenic molecule and a bioluminescence-generating enzyme; and   (c) directions for using the kit.   
     
     
         28 . The kit of  claim 27  wherein the transferase enzymatic activity comprises kinase activity or ion channel/pump activity. 
     
     
         29 . The kit of  claim 28  wherein the transferase enzymatic activity comprises protein kinase activity, protein lipid-dependent kinase activity, polynucleotide kinase activity, or sugar kinase activity. 
     
     
         30 . The kit of claim  5  wherein the protein kinase comprises a Ser/Thr protein kinase, a protein tyrosine kinase, or a protein lipid-dependent kinase. 
     
     
         31 . The kit of  claim 30  wherein the Ser/Thr protein kinase comprises cAMP-dependent protein kinase (PKA), calcium and phosphlipids dependent protein kinase (PKC), cGMP-dependent protein kinase (PKG), calcium and calmodulin dependent protein kinase (CaM KII) or a dual specificity protein kinase. 
     
     
         32 . The kit of  claim 31  wherein the dual specificity protein kinase comprises mitogen activated protein kinase (MAPK) or mitogen activated protein kinase kinase (MEK). 
     
     
         33 . The kit of  claim 30  wherein the tyrosine kinase comprises Rous sarcoma related protein kinases (Src), or Src family protein tyrosine kinases. 
     
     
         34 . The kit of  claim 33  wherein the Src family protein tyrosine kinases comprise Src, Lck, Fyn, or Lyn. 
     
     
         35 . The kit of  claim 33  wherein the growth factor receptors comprise epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR) or steel growth factor receptor (c-KIT). 
     
     
         36 . The kit of  claim 30  wherein the lipid dependent protein kinases comprises Type I phosphoinositide 3-OH phosphotidylinositol kinase (PI3K). 
     
     
         37 . The kit of claim  26  wherein the transferase quenching agent comprises a detergent. 
     
     
         38 . The kit of  claim 37  wherein the detergent comprises a cationic detergent, anionic detergent, or zwitterionic detergent. 
     
     
         39 . The kit of  claim 38  wherein the cationic detergent comprises dodecyltrimethylammonium bromide, cetyltrimethylammonium bromide and benzyldimethyldodecylammonium bromide. 
     
     
         40 . The kit of  claim 38  wherein the cationic detergent is dodecyltrimethylammonium bromide. 
     
     
         41 . The kit of  claim 38  wherein the anionic detergent comprises SDS and deoxycholate. 
     
     
         42 . The kit of  claim 38  wherein the zwitterionic detergent comprises sulfobetaine 3-10. 
     
     
         43 . The kit of claim  26  wherein the transferase quenching agent comprises a metal chelating agent. 
     
     
         44 . The kit of  claim 43  wherein the transferase quenching agent comprises EDTA or EGTA. 
     
     
         45 . The kit of claim  26  wherein the transferase inhibitor comprises staurosporine, or a peptide or protein inhibitor. 
     
     
         46 . The kit of claim  26  wherein the luminogenic molecule comprises D-luciferin or a luciferin derivative and the bioluminescent enzyme comprises a luciferase. 
     
     
         47 . The kit of  claim 46  wherein the luciferase comprises a thermostable luciferase.