Polyphenol oxidase from wheat bran is a serpin.
Polyphenol oxidase (PPO; EC 1.10.3.2) was isolated from wheat bran by a procedure that included ammonium sulfate fractionation, batch adsorption by DEAE-cellulofine, CM-cellulofine column chromatography, DEAE-cellulofine column chromatography, preparative isoelectric focusing, adsorption on the membrane of a Vivapure Q Maxi H spin column, and heat treatment. These procedures led to 150-fold purification with 4.2% recovery. The PPO was homogeneous by SDS/PAGE. The relative molecular weight of the PPO was estimated to be 37,000 based on its mobility in SDS/PAGE. The isoelectric point of the PPO was 4.4. The K(m) values of the PPO for caffeic acid, chlorogenic acid, pyrocatechol, 4-methyl catechol and l-DOPA as substrates were 0.077, 0.198, 1.176, 1.667 and 4.545 mM. The PPO was strongly inhibited by tropolone. The K(i) value for tropolone is 2.2 x 10(-7) M. The sequence of the 15 N-terminal amino-acid residues was determined to be ATDVRLSIAHQTRFA, which was identical to those of serpin from Triticum aestivum and protein Z from Hordeum vulgare. The PPO strongly inhibited the activity of trypsin, which is an enzyme of serine proteases; 50% inhibition was observed with 1.5 x 10(-7) M PPO. The K(i) value for PPO is 2.3 x 10(-8) M. The wheat bran PPO should be a very important protein for protecting wheat against disease, virus, insect and herbivore damages by both the activities of PPO and protease inhibitor.
Yamasaki
Yoshiki
Y
Research Institute for Bioresources, Okayama University, Okayama, Japan. yosikiy@rib.okayama-u.ac.jp
Konno
Haruyoshi
H
Noda
Kazuhiko
K
D000595:Amino Acid Sequence; D004156:Catechol Oxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D004043:Dietary Fiber / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015843:Serpins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology / Q000235:genetics; D014334:Tropolone / Q000494:pharmacology
D002614:Chelating Agents; D004043:Dietary Fiber; D010940:Plant Proteins; D015843:Serpins; D012967:Sodium Dodecyl Sulfate; D014334:Tropolone; D004156:Catechol Oxidase