Materials and Methods for Assaying for Glyoxylate

A method for detecting peptidylglycine α-amidating monooxygenase (PAM) in a sample, said method comprising detecting the presence of glyoxylate using a method selected from:

• i) contacting said sample with one or more reagents, wherein the reaction of glyoxylate in said sample with said reagents results in the production of a detectable reaction product and detecting said detectable reaction product;
• or,
• ii) a) contacting said sample with acetyl-CoA, malate synthase, and malate dehydrogenase, wherein NADH is produced if glyoxylate is present in said sample;
• and
• b) detecting said NADH produced, wherein said NADH corresponds to the presence of glyoxylate in said sample;
• or,
• iii) a) contacting said sample with lactate dehydrogenase and glycolate oxidase, whereby glycolate and hydrogen peroxide are produced if glyoxylate is present in said sample;
• and
• b) detecting said hydrogen peroxide produced in the presence of glyoxylate;
• or,
• iv) a) contacting said sample with glycolate oxidase or glyoxal oxidase, whereby oxalate and hydrogen peroxiderroduced if glyoxylate is present in said sample;
• and
• b) detecting said hydrogen peroxide produced;
• or,
• v) a) contacting said sample with glyoxylate reductase and NADPH, whereby glycolate and NADP+ is produced if glyoxylate is present in said sample;
• and
• b) detecting said NADP+ produced,
• wherein the presence of glyoxylate is indicative of the presence of PAM.

A method for screening for the presence of an α-amidated peptide or fatty acid in a sample, said method comprising:

• a) growing cells in a medium in which said cells will grow;
• b) preparing cell extract and/or obtaining spent media from the cells grown in step a);
• c) chromatographically fractionating said cell extract and/or spent media from step b);
• d) contacting the fractionated samples obtained from step c) with peptidylglycine α-amidating monooxygenase;
• e) assaying said fractionated sample for the presence of glyoxylate using a method selected from:
• i) contacting said sample with one or more reagents, wherein the reaction of glyoxylate in said sample with said reagents results in the production of a detectable reaction product;
• and detecting said detectable reaction product;
• or,
• ii) a) contacting said sample with acetyl-CoA, malate synthase, and malate dehydrogenase, wherein NADH is produced if glyoxylate is present in said sample;
• and
• b) detecting said NADH produced, Wherein said NADH corresponds to the presence of glyoxylate in said sample;
• or,
• iii) a) contacting said sample with lactate dehydrogenase and glycolate oxidase, whereby glycolate and hydrogen peroxide are produced if glyoxylate is present in said sample:
• and
• b) detecting said hydrogen peroxide produced in the presence of glyoxylate;
• or
• iv) a) contacting said sample with glycolate oxidase or glyoxal oxidase, whereby oxalate and hydrogen peroxide are produced if glyoxylate is present in said sample;
• and
• b) detecting said hydrogen peroxide produced;
• or,
• v) a) contacting said sample with glyoxylate reductase and NADPH whereby glycolate and NADP is produced if glyoxylate is present in said sample;
• and
• b) detecting said NADP produced,
• wherein fractions that test positive for glyoxylate are indicative of the presence of an α-amidated peptide or fatty acid.

A kit comprising in one or more containers:

• acetyl-CoA;
• malate synthase;
• and malate dehydrogenase;
• or
• lactate dehydrogenase;
• and glycolate oxidase;
• or
• glycolate oxidase or glyoxal oxidase;
• a peroxidase;
• and a substrate that reacts in the presence of said peroxidase to produce a fluorescent molecule, wherein said substrate is optionally Amplex Red;
• or
• glyoxylate reductase.