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.
Sebela
M
M
Department of Biochemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic. sebela@risc.upol.cz
Brauner
F
F
Radová
A
A
Jacobsen
S
S
Havlis
J
J
Galuszka
P
P
Pec
P
P
D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D000445:Aldehyde Oxidoreductases; C019679:aminobutyraldehyde dehydrogenase