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.

FAD-containing polyamine oxidases: a timely challenge for researchers in biochemistry and physiology of plants.

Recent investigations on plant polyamine oxidase (PAO) are reviewed. The enzyme belongs to a new class of flavoenzymes with similar structural features including, among others, monoamine oxidase. Plant PAOs catalyse the oxidation of the polyamine substrates spermidine and spermine. The reaction products are propane-1,3-diamine and 1-pyrroline or 1-(3-aminopropyl)pyrrolinium, respectively, along with hydrogen peroxide. Plant PAOs are predominantly localised in the cell wall. Purification procedures and molecular properties of several plant PAOs are compared. A special attention is being paid to the recently solved crystal structure of the maize enzyme and its implications for the substrate binding and catalytic mechanism. Substrate specificity and inhibitors of plant PAOs are also described. The potential roles for PAO-generated H(2)O(2) in lignin biosynthesis and cell wall cross-linking reactions, which may regulate growth and contribute to cell defence, are discussed.

Characterisation of a homogeneous plant aminoaldehyde dehydrogenase.

According to our knowledge, this is the first purification method developed, enabling isolation of a homogeneous aminoaldehyde dehydrogenase (AMADH) from etiolated pea seedlings. The procedure involved initial purification with precipitants followed by three low pressure chromatographic steps. Partially purified enzyme was further subjected to fast protein liquid chromatography on a Mono Q column and to affinity-interaction chromatography on 5'-AMP Sepharose. Purity of the final enzyme preparation was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and chromatofocusing. Pea AMADH exists as a tetramer of 230 kDa in the native state, a molecular mass of one subunit was determined as 57 kDa. The enzyme was found to be an acidic protein with pI 5.4. AMADH showed a broad substrate specificity utilising various aminoaldehydes (C3-C6) as substrates. The best substrate of pea AMADH was 3-aminopropionaldehyde, the enzyme also efficiently oxidised 4-aminobutyraldehyde and omega-guanidinoanalogues of the aminoaldehydes. Pea AMADH was inhibited by SH reagents, several elementary aldehydes and metal-binding agents. Although AMADH did not oxidise betaine aldehyde at all, the N-terminal amino acid sequence of the enzyme shows a high degree of homology with those of plant betaine aldehyde dehydrogenases (BADHs) of spinach, sugar beet and amaranth. Several conserved amino acids were found in comparison with BADH from cod liver of known crystal structure.