A method for the isolation of the amide nitrogen of glutamine from biological samples for mass spectrometry

The amide nitrogen from L-glutamine has been isolated from an artificial plasma, in a form suitable for mass spectrometry, by a macromodification of the glutaminase reaction. The prior removal of free ammonia was carried out by alkaline aeration. When this was performed at 0§C for 3 h, spontaneous hydrolysis of glutamine was 1.4 percent. Cross-contamination with nitrogen liberated from the amide group of asparagine can be avoided by preincubation with asparaginase for 2h and removal of the freed ammonia prior to reacting with glutaminase. Hydrolysis of glutamine during this step is 12 percent. Measurements of enrichment can be made on samples yielding more than 1 æmol of glutamine amide-derived ammonia.(AU)

A method for the isolation of the amide nitrogen of glutamine from biological samples for mass spectrometry

The amide nitrogen from L-glutamine has been isolated from an artificial plasma, in a form suitable for mass spectrometry, by a macromodification of the glutaminase reaction. The prior removal of free ammonia of free ammonia was carried out by alkaline aeration. When this was performed at zero degree for 3 h, spontaneous hydrolysis of glutamine was 1.4 percent. Cross-contamination with nitrogen liberated from the amide group of asparagine can be avoided by preincubation with asparaginase for 2 h and removal of the freed ammonia prior to reacting with glutaminase. Hydrolysis of glutamine during this step is 12 percent. Measurements of enrichment can be made on samples yielding more than æmol of glutamine amide-derived ammonia (AU)

Modification of diamine oxidase activity in vitro by metabolites of asparagine and differences in asparagine decarboxylation in normal and virus-transformed baby hamster kidney cells

The oxidation of putrescine in vitro by pig kidney diamine oxidase (EC 1.4.3.6) was increased in the presence of 2-oxosuccinamic acid and malonamic acid. It was inhibited by 3-aminopropionamide, oxaloacetate and pyruvate. 2-Oxosuccinamate was derived from asparagine in virus-transformed baby hamster kidney (BHK) cells growing in tissue culture. Asparagine was decarboxylated more efficiently by transformed than by normal BHK cells. In BHK cells transformed by polyoma virus (Py BHK), 2-oxosuccinamate is the most likely immediate precursor of the 14 CO2 arising from [U-14C] asparagine, and there was some evidence for its formation in an asparagine-dependent clone of BHK cells before and after their transformation by hamster sarcoma virus (respectivey Asn- and HSV Asn-). The relationship between 2-oxosuccinamate and pyruvate and the possible roles of these two substances in controlling cellular diamine oxidase activity are discussed (AU)