Isolation and identification by gas chromatographic mass spectrometry of the carbonyl-active site of pig kidney diamine oxidase.
An adduct with phenylhydrazine was formed with the purified pig kidney diamine oxidase and in parallel with the l-tyrosine decarboxylase from Streptococcus faecalis. The labeled enzymes were hydrolyzed by chemical hydrolysis and the adducts released by hydrolysis were isolated and identified first in HPLC and successively in GC-MS. Both enzymes gave the same adduct which was identified as the phenylhydrazone of pyridoxal. The isolated adduct had the same retention time as the phenylhydrazone of pyridoxal in HPLC and in gas chromatography and showed the same molecular weight in mass spectrometry when chemical ionization was used and the same fragmentation in mass spectrometry when electronic impact was used. The reported results show that pig kidney diamine oxidase contains pyridoxal in the form of covalently linked pyridoxal phosphate which can be released from the enzyme only by chemical hydrolysis. Pig kidney diamine oxidase is therefore a pyridoxal enzyme such as l-tyrosine decarboxylase as hypothesized in the past but never clearly demonstrated.
Department of Pharmacology, University of Florence, Italy.
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Buffoni
D006631:Amine Oxidase (Copper-Containing) / Q000032:analysis*; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D013293:Enterococcus faecalis / Q000201:enzymology; D008401:Gas Chromatography-Mass Spectrometry; D007668:Kidney / Q000201:enzymology*; D010659:Phenylhydrazines / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000032:analysis; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D014445:Tyrosine Decarboxylase / Q000032:analysis
D010659:Phenylhydrazines; C030299:phenylhydrazine; D011732:Pyridoxal Phosphate; D006631:Amine Oxidase (Copper-Containing); D014445:Tyrosine Decarboxylase