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GrainGenes Reference Report: PAN-12-166

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Reference	PAN-12-166
Title	Barley polyamine oxidase: Characterisation and analysis of the cofactor and the N-terminal amino acid sequence
Journal	Phytochemical Analysis
Year	2001
Volume	12
Pages	166-173
Author	Radova A [ Show all 7 ]
Abstract	Summary: This paper reports the first purification method developed for the isolation 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 further purified to a final homogeneity (by the criteria of isoelectric focusing and SDS-PAGE) using techniques of low pressure chromatography followed by two FPLC steps. The purified yellow enzyme showed visible absorption maxima of a flavoprotein at 380 and 450 nm: the presence of FAD as the cofactor was further confirmed by measuring the fluorescence spectra. Barley PAO is an acidic protein (pI 5.4) containing 3% of neutral sugars: its molecular mass determined by SDS-PAGE was 56 kDa, whilst gel permeation chromatography 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 to several other flavoprotein oxidases. The polyamines spermine and spermidine were the only two substrates of the enzyme with Km values 4 x 10(-5) and 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 inhibition was observed with substrate analogues, aminoaldehydes, metal chelating agents and several other compounds
Keyword	[ Hide all but 1 of 46 ] acid amino acid amino acid sequence ammonia ammonium barley barley seedlings cell-wall chelating agents chromatography cofactor enzyme enzyme characterisation etiolated barley flavin adenine dinucleotide fluorescence hordeum vulgare inhibition isolation localization maize maximum method molecular masses n-terminal amino acid sequence oat seedlings oxidase oxidation paper permeation ph plant science polyamine polyamine oxidase polyamines protein purification sds page seedling sequence spectrum spermidine spermine substrate sugar zea mays l

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