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US 2004/0002129 A1Published application
Method for measuring no synthase activity
AssigneeGruenenthal GmbH
InventorsHagen-Heinrich Hennies, Bernd Sundermann
Life of this patent
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Abstract
A method for measuring the activity of NO-synthase, a corresponding method for identification of NO-synthase modulators, in particular for application in a HTS (High Throughput Screening) and use of the corresponding cation-exchange filter-plate membranes.
Claims45
A high-throughput screening method for an agent that modulates NO synthase activity, the method comprising,
- (a) substantially simultaneously incubating in a plurality of reaction vessels a reaction mixture comprising NO synthase and labeled arginine as substrate, whereby arginine is converted into citrulline by the NO synthase, wherein each reaction vessel contains a candidate agent;
- (b) substantially simultaneously separating non-converted arginine from the reaction mixture in each of the plurality of reaction vessels and capturing the non-converted arginine by means of a filter plate membrane;
- (c) measuring the amount of captured arginine from each of the plurality of reaction vessels corresponding to each individual candidate agent on the filter plate membrane as an indication of the NO synthase activity;
- and
- (d) comparing the NO synthase activity in the presence of the candidate agent with NO synthase activity measured in the absence of the candidate agent, wherein a change in the NO synthase by virtue of the presence of candidate agent indicates that the candidate agent modulates NO synthase activity.
2 more elements
A method of measuring a NO synthase activity, comprising the steps of:
- (a) incubating NO synthase with labeled arginine as substrate in a reaction mixture contained in a reaction vessel, whereby arginine is converted into citrulline by the NO synthase,
- (b) separating nonconverted arginine from the reaction mixture and capturing non-converted arginine by means of a filter plate membrane;
- and
- (c) measuring the amount of captured arginine on the filter plate membrane as an indication of the NO synthase activity.
1 more element
A screening method for an agent that modulates NO synthase activity, the method comprising,
- (a) incubating, in the presence of a candidate agent, NO synthase with labeled arginine as substrate in a reaction vessel, whereby arginine is converted into citrulline by the NO synthase;
- (b) separating non-converted arginine from the reaction mixture and capturing non-converted arginine by means of a filter plate membrane;
- (c) measuring the amount of captured arginine on the filter plate membrane as an indication of the NO synthase activity;
- and
- (d) comparing the NO synthase activity in the presence of the candidate agent with the NO synthase activity measured in the absence of the candidate agent, wherein a change in the NO synthase by virtue of the presence of candidate agent indicates that the candidate agent modulates NO synthase activity.
2 more elements
Dependencies are inferred from the claim text.