Glyoxylate assays and their use of inden tifying natural amidated compounds
First Claim
1. A method for detecting the presence of glyoxylate in a test sample comprising the steps of:
- (A) contacting said test sample with one or more agents that convert glyoxylate, directly or indirectly, to hydrogen peroxide as one reaction product; and
(B) assaying for the presence of hydrogen peroxide.
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Abstract
Methods for detecting and assaying for glyoxylate, include enzyme-based assays and/or assays for hydrogen peroxide following liberation of hydrogen peroxide from glyoxylate, are disclosed. In some embodiments, the invention is directed to methods for assaying for glyoxylate produced by the reaction of peptidylglycine alpha-amidating monooxygenase (PAM). The subject invention also concerns methods for assaying for the enzyme peptidylglycine alpha-amidating monooxygenase and/or its substrates. The detection of glyoxylate is indicative of the presence of PAM and/or its substrates. The subject invention also concerns methods for screening for peptide hormones, amidated fatty acids, any N-acyl-glycine or N-aryl-glycine conjugated molecule, and generally compounds having a glycine reside in free acid form and attached to a carbonyl group.
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Citations
44 Claims
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1. A method for detecting the presence of glyoxylate in a test sample comprising the steps of:
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(A) contacting said test sample with one or more agents that convert glyoxylate, directly or indirectly, to hydrogen peroxide as one reaction product; and
(B) assaying for the presence of hydrogen peroxide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for detecting the presence of compounds having a glycine residue, in free acid form and attached to a carbonyl group, said method comprising the steps of:
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(A) contacting a sample suspected of containing said glycine compounds with either (i) peptidylglycine alpha-amidating monooxygenase, (ii) a combination of peptidylglycine alpha-hydroxylating monooxygenase and peptidyl alpha-hydroxyglycine alpha-amidating lyase or (iii) a combination of peptidylglycine alpha-hydroxylating monooxygenase and a Lewis base;
to form an amidated compound and glyoxylate;
(B) assaying for the presence of glyoxylate. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
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32. A method of identifying amidated hormones or amidated fatty acids comprising the steps of:
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(A) incubating cells suspected of producing said hormones or fatty acids;
(B) fractionating compounds produced by said cells into a series of fractions for further analysis;
(C) contacting a fraction from step (B) with either (i) peptidylglycine alpha-amidating monooxygenase, (ii) a combination of peptidylglycine alpha-hydroxylating monooxygenase and peptidyl alpha-hydroxyglycine alpha-amidating lyase or (iii) a combination of peptidylglycine alpha-hydroxylating monooxygenase and a Lewis base;
to form an amidated compound and glyoxylate when said fraction includes a compound having a glycine residue, in free acid form and attached to a carbonyl group; and
(D) Assaying the product of step (C) for the presence of glyoxylate. - View Dependent Claims (33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44)
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Specification