Barley polyamine oxidase: Characterisation and analysis of the cofactor and the N-terminal amino acid sequence

This paper reports the first purification method developed for the isolatio n of an homogeneous polyamine oxidase (PAO) from etiolated barley seedlings . The crude enzyme preparation was obtained after initial precipitation of the extract with protamine sulphate and ammonium sulphate. The enzyme was f urther purified to a final homogeneity (by the criteria of isoelectric focu sing and SDS-PAGE) using techniques of low pressure chromatography followed by two FPLC steps. The purified yellow enzyme showed visible absorption ma xima of a flavoprotein at 380 and 450 nm: the presence of FAD as the cofact or was further confirmed by measuring the fluorescence spectra, Barley PAO is an acidic protein (pI 5.4) containing 3% of neutral sugars: its molecula r mass determined by SDS-PAGE was 56 kDa, whilst gel permeation chromatogra phy revealed the higher value of 76 kDa. The N-terminal amino acid sequence of barley PAO shows a high degree of similarity to that of maize PAO and t o several other flavoprotein oxidases. The polyamines spermine and spermidi ne were the only two substrates of the enzyme with K-m values 4 x 10(-5) an d 3 x 10(-5) M and pH optima of 5.0 and 6.0, respectively. Barley polyamine oxidase is markedly inhibited by acridine dyes and hydrazines, Weak inhibi tion was observed with substrate analogues, aminoaldehydes, metal chelating agents and several other compounds. Copyright (C) 2001 John Wiley & Sons, Ltd.