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 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.
Radová
A
A
Department of Biochemistry, Faculty of Science, Palacký University, Slechtitelů 11, 783 71 Olomouc-Holice, Czech Republic. radova@prfholnt.upol.cz
Sebela
M
M
Galuszka
P
P
Frébort
I
I
Jacobsen
S
S
Faulhammer
H G
HG
Pec
P
P
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000097667:Polyamine Oxidase
D000587:Oxidoreductases Acting on CH-NH Group Donors; D000097667:Polyamine Oxidase