Half-site reactivity with p-nitrophenylhydrazine and subunit separation of the dimeric copper-containing amine oxidase from Aspergillus niger.
Structural properties of dimeric (2 x 75 kDa) copper-containing amine oxidase (EC 1.4.3.6) from Aspergillus niger were studied. The enzyme treated with SDS was dissociated into subunits which showed different mobility on polyacrylamide gel without SDS. The separated subunits had no activity but a quinone moiety was detected in both by a redox-cyclic quinone staining. After titration of the enzyme with p-nitrophenylhydrazine, which showed half-site reactivity (1 mole per dimer), and SDS treatment both p-nitro-phenylhydrazone and a remaining quinone moiety were detected in each subunit. It is suggested that the half-site reactivity with phenylhydrazine is caused by conformational changes after binding of the inhibitor to any one of the active sites leading to inaccessibility of the second active site for the inhibitor. The difference in electrophoretic mobility of the separated subunits originates probably from their structural difference likely to occur outside the active site, even if the amino acid sequences of the subunits appear to be identical.
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
I
I
Frébort
H
H
Toyama
K
K
Matsushita
O
O
Adachi
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001234:Aspergillus niger / Q000201:enzymology*; D004396:Coloring Agents; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010659:Phenylhydrazines; D011809:Quinones / Q000032:analysis; D012967:Sodium Dodecyl Sulfate
D004396:Coloring Agents; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D010659:Phenylhydrazines; D011809:Quinones; C005307:4-nitrophenylhydrazine; D012967:Sodium Dodecyl Sulfate; D006631:Amine Oxidase (Copper-Containing)