Cell-wall proteins from Sitka spruce xylem are selectively insolubilised during formation of dehydrogenation polymers of coniferyl alcohol.
Extracts from the lignifying xylem of Sitka spruce that were enriched in cell-wall-associated glycoproteins contained peroxidase and oxidase activity and readily formed lignin-like water-insoluble dehydrogenation polymers (DHPs) from coniferyl alcohol (CA) when supplied with H2O2. During the formation of DHPs, the abundance of a number of polypeptides in the extracts was diminished. However, these polypeptides were also diminished in control reactions that contained H2O2 but lacked CA. Polypeptides could be recovered from the DHPs by heating in SDS-PAGE sample buffer but no insolubilised polypeptides could be recovered from the + H2O2 reactions. Although most of the DHP-bound polypeptides were easily removed by pre-washing the DHPs, two polypeptides at 125 and 52 kDa remained tightly bound to the DHPs. The abundance of the two DHP-bound polypeptides mirrored the diminution of 120 and 46 kDa polypeptides in the extracts. The N-terminal protein sequences of the 125 and 52 kDa DHP-bound polypeptides were essentially identical to the sequences obtained from the 120 and 46 kDa polypeptides from the extracts, which confirmed that the DHP-bound polypeptides were derived from these soluble polypeptides. The 125-kDa DHP-bound polypeptide yielded an N-terminal protein sequence that was identical to a laccase-type oxidase previously identified in similar extracts from lignifying Sitka xylem. The N-terminal protein sequence of the 46-kDa polypeptide was homologous with a subset of plant peroxidases. The DHPs had tightly bound peroxidase and oxidase activity, which suggested that these polypeptides were active in their insolubilised state. The mechanism and selectivity of insolubilisation of these enzymes is discussed.
McDougall
G J
GJ
Biochemistry and Cell Biology Division, Scottish Crop Research Institute, Invergowrie, Dundee, Scotland, UK. gmcdou@scri.sari.ac.uk
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006859:Hydrogen / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010636:Phenols / Q000737:chemistry*; D011108:Polymers; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014197:Trees / Q000737:chemistry*
D008565:Membrane Proteins; D010636:Phenols; D011108:Polymers; D006859:Hydrogen; C010559:coniferyl alcohol