Enzyme assay and use thereof
An assay and kit for determining the activity of an enzyme such as kinase, ATPase and GTPase is disclosed. The assay and kit are useful in drug screening to select modulators of such an enzyme.
1. A method for determining the activity of an ATPase or kinase that catalyzes a biochemical reaction producing ADP, comprising:
contacting a substrate with ATP in the presence of said ATPase or kinase under conditions such that at least a portion of said ATP is converted to ADP;
reacting the ADP with a phosphoryl group acceptor which is a hexose or hexose phosphate, in the presence of an ADP-dependent hexokinase, a dehydrogenase that oxidizes or dehydrogenates said hexose or hexose phosphate in the presence of, NAD(P) or an analog thereof, resazurin or a salt thereof, under conditions such that fluorescent resorufin is produced; and
determining the activity of said ATPase or kinase based on measurement of fluorescence emitted by said resorufin.
2. The method of claim 1 , wherein said hexose is D-glucose and said dehydrogenase is glucose-6-P dehydrogenase.
3. The method of claim 2 , wherein said resazurin is converted to resorufin in the presence of diaphorase.
4. The method of claim 1 , wherein the reacting step is conducted in the presence of a divalent cation.
5. The method of claim 1 , wherein the reacting step is conducted in the presence of at least 1 mM of magnesium ion.
6. A method for determining the activity of an ATPase or kinase that catalyzes a biochemical reaction producing ADP, comprising:
contacting a substrate with ATP in the presence of said ATPase or kinase under conditions such that at least a portion of said ATP is converted to ADP wherein said contacting step is conducted in the presence of one or more test compounds to be tested for their ability to inhibit the ATPase or kinase;
reacting the ADP with a phosphoryl group acceptor which is a hexose or hexose phosphate, in the presence of an ADP-dependent hexokinase, a dehydrogenase that oxidizes or dehydrogenates said hexose or hexose phosphate in the presence of NAD(P) or an analog thereof, resazurin or a salt thereof, under conditions such that fluorescent resorufin is produced; and
determining the activity of said ATPase or kinase based on measurement of fluorescence emitted by said resorufin.
7. A method for determining the activity of an ATPase or kinase that catalyzes a biochemical reaction producing ADP, comprising:
(a) contacting a substrate with ATP in the presence of said ATPase or kinase and under conditions such that at least a portion of said ATP is converted to ADP;
(b) reacting the ADP with D-glucose in the presence of an ADP-dependent glucokinase, a glucose-6-P dehydrogenase, NAD(P), resazurin, diaphorase and at least 1 mM of magnesium ion, under conditions such that fluorescent resorufin is produced;
(c) determining the activity of said ATPase or kinase based on measurement of fluorescence emitted by said resorufin;
(d) repeating the steps of (a) (b) and (c) in the presence of one or more test compounds; and
(e) comparing the measurements of fluorescence in the presence and absence of said test compounds.