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) f rom etiolated pea seedlings. The procedure involved initial purification wi th precipitants followed by three low pressure chromatographic steps. Parti ally purified enzyme was further subjected to fast protein liquid chromatog raphy on a Mono Q column and to affinity-interaction chromatography on 5'-A MP Sepharose. Purity of the final enzyme preparation was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and chromatofocusing. Pe a AMADH exists as a tetramer of 230 kDa in the native state, a molecular ma ss of one subunit was determined as 57 kDa. The enzyme was found to be an a cidic protein with pI 5.4. AMADH showed a broad substrate specificity utili sing various aminoaldehydes (C3-C6) as substrates. The best substrate of pe a 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 homolo gy with those of plant betaine aldehyde dehydrogenases (BADHs) of spinach, sugar beet and amaranth. Several conserved amino acids were found in compar ison with BADH from cod liver of known crystal structure. (C) 2000 Elsevier Science B.V. All rights reserved.