ELECTROPHORETIC ANALYSIS OF CONIFERYL ALCOHOL OXIDASE AND RELATED LACCASES

Gradient gel electrophoretic methods enabled a distinction to be made between coniferyl alcohol oxidase (CAO) of lignifying cell walls and a pI similar to 9 pine ''laccase'' recently implicated in lignification (Science 1993 260, 672). Following treatment of a partially purified protein mixture from developing xylem of Pinus strobus with 2-[N-morph oline]ethanesulfonic acid (MES) buffer, isoelectric focusing and sodiu m dodecyl sulphate-polyacrylamide gel electrophoresis indicated that C AO had been selectively precipitated by MES and thereby purified to el ectrophoretic homogeneity. Purified CAO was determined to be a cell-wa ll-bound glycoprotein (38% glycan), M(r) 107 500, pI 7.6, pH and tempe rature optima 6.3 and 30 degrees C, respectively. By graphite-furnace atomic-absorption analysis, CAO contained one copper atom per protein molecule. Proteins obtained from lignifying cambial derivatives of con ifers (family Pinaceae) and from Rhus typhina bark were compared with CAO and the pI similar to 9 pine ''laccase'' following electrophoresis and Western blotting. For Abies balsamea, Larix laricina, Picea ruben s, Pinus banksiana, Pinus taeda, and R. typhina, the isoelectric point s of oxidatively active bands were identical to those of purified CAO. In addition, for all species only the pI 7.6 band was immunoreactive with antibodies against periodate-deglycosylated CAO.