Purification and characterization of three neutral extracellular isoperoxidases from rye leaves.
Rye (Secale cereale L.) seedlings; contain two major flavone glucuronides, luteolin 7-O-diglucuronyl-4'-O-glucuronide (L3GlcUA) (1) and luteolin 7-O-diglucuronide (L2GlcUA) (2) in abundance in the apoplast of primary leaves; express a large number of peroxidase isoenzymes; and release H(2)O(2) into the apoplast during primary leaf development. We purified and characterized three neutral extracellular peroxidase isoenzymes (rPOXs N1, N2, and N3) that can oxidize L2GlcUA as a natural substrate. The isoelectric points and molecular weights of rPOXs N1, N2, and N3 were 6.1, 7.2, and 6.3, and 42, 37, and 51 kDa, respectively. The optimum pH of the rPOXs N1, N2, and N3 were 5.5, 5.5, and 8.5, respectively, and their optimum temperatures ranged from 45 to 50 degrees C for all isoenzymes. rPOXs N1, N2, and N3 recognized flavonoids with 3', 4'-OH groups as potential substrates, but not flavonoids with a glycosylated 4'-OH group or those without a 3'-OH group. The activities on phenol-type substrates were high in the order of guaiacol>catechol>o-cresol for all isoenzymes. rPOXs N1, N2, and N3 exhibited broad reactivity with endogenous hydrogen donors including luteolin glucuronides derived from the apoplast of rye primary leaves.
Murakami
Satoshi
S
Laboratory of Biochemistry and Molecular Biology, Department of Applied Bioresource Science, School of Agriculture, Ibaraki University, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan.
Takahara
Hidenari
H
Shiraiwa
Masakazu
M
D002396:Catechols / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003408:Cresols / Q000378:metabolism; D004586:Electrophoresis; D020719:Glucuronides / Q000737:chemistry / Q000378:metabolism; D006139:Guaiacol / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D047311:Luteolin / Q000378:metabolism; D015394:Molecular Structure; D008970:Molecular Weight; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012434:Secale / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D002396:Catechols; D003408:Cresols; D020719:Glucuronides; D007527:Isoenzymes; D010940:Plant Proteins; D006139:Guaiacol; D006861:Hydrogen Peroxide; D010544:Peroxidases; D047311:Luteolin; C034221:catechol; C034047:2-cresol