Methods for transferases

Abstract

Methods for detecting transferase activity of a sample are provided. Transferase activity detected includes kinase and phosphatase activity. The method includes providing a substrate having a reporter compound conjugated thereto. A quantity of the substrate is added to a solution containing the sample. The sample is incubated with the substrate under conditions where the sample is active for a time sufficient for kinase or phosphatase activity to take place. An exopeptidase is added to the solution containing the sample. Output of the reporter compound is detected.

1 . A method for detecting kinase or phosphatase activity of a sample, the method comprising: 
 (A) contacting the sample with a peptide substrate and at least one of a phosphate group donor and a phosphate group acceptor, wherein the substrate includes a reporter compound and amino acids;    (B) adding an exopeptidase, wherein phosphorylation of the substrate affects cleavage by the exopeptidase, wherein adding the exopeptidase comprises adding an exopeptidase that hydrolyzes the peptide substrate from a carboxy-terminal end of the substrate; and    (C) detecting output of the reporter compound, wherein the reporter compound exhibits a different output property when bound to at least one amino acid of the substrate when compared to when it is not bound to amino acids.    
     
     
         2 . A method of detecting kinase or phosphatase activity of a sample comprising: 
 (A) providing a substrate having a reporter compound conjugated thereto;    (B) adding a quantity of the substrate to a solution containing the sample;    (C) incubating the sample with the substrate under conditions where the sample is active for a time sufficient for kinase or phosphatase activity to take place;    (D) adding an exopeptidase to the solution containing the sample;    (E) detecting output of the reporter compound.    
     
     
         3 . A method of  claim 2 , wherein in step (A) a substrate having a fluorescent molecule conjugated thereto is provided.  
     
     
         4 . A method of  claim 2 , wherein in step (A) a substrate having a luminogenic molecule conjugated thereto is provided.  
     
     
         5 . A method of  claim 2 , further comprising terminating the kinase or phosphatase reaction.  
     
     
         6 . A method of  claim 2 , further comprising terminating the exopeptidase reaction.  
     
     
         7 . A method of  claim 2 , further comprising linking the substrate to a solid support.  
     
     
         8 . A method of  claim 7 , wherein the substrate is linked to the solid support via a first functional group on the substrate and a second functional group on the solid support.  
     
     
         9 . A method for detecting peptide transferase activity of a sample, the method comprising: 
 (A) contacting the sample with a peptide substrate and at least one of a group donor and a group acceptor, wherein the substrate includes a reporter compound and amino acids;    (B) adding an exopeptidase, wherein transfer of a group from one substrate to another affects cleavage of the substrate by the exopeptidase; and    (C) detecting output of the reporter compound.    
     
     
         10 . The method of  claim 9 , wherein the transferase activity detected is kinase activity.  
     
     
         11 . The method of  claim 9 , wherein the transferase activity detected is phosphatase activity.  
     
     
         12 . A method for detecting peptide transferase activity of a sample, the method comprising: 
 (A) contacting the sample with a peptide substrate and at least one of a group donor and a group acceptor, wherein the substrate includes a reporter compound and two peptides, each peptide being separately linked to the reporter compound; and    (B) adding an exopeptidase, wherein transfer of a group from one substrate to another affects cleavage of the substrate by the exopeptidase; and    (C) detecting output of the reporter compound.    
     
     
         13 . The method of  claim 12 , wherein the transferase activity detected is kinase activity.  
     
     
         14 . The method of  claim 12 , wherein the transferase activity detected is phosphatase activity.  
     
     
         15 . A method for detecting peptide transferase activity of a sample, the method comprising: 
 (A) contacting the sample with a peptide substrate and at least one of a group donor and a group acceptor, wherein the substrate includes a reporter compound and amino acids; and    (B) adding an exopeptidase, wherein transfer of a group from one substrate to another affects cleavage of the substrate by the exopeptidase; and    (C) liberating the reporter compound from the substrate.    
     
     
         16 . The method of  claim 15 , wherein the liberated reporter compound has increased output when compared to when the reporter compound is not liberated from the substrate.  
     
     
         17 . The method of  claim 15 , wherein the transferase activity detected is kinase activity.  
     
     
         18 . The method of  claim 15 , wherein the transferase activity detected is phosphatase activity.