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 approximately 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-morpholine]ethanesulfonic acid (MES) buffer, isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that CAO had been selectively precipitated by MES and thereby purified to electrophoretic homogeneity. Purified CAO was determined to be a cell-wall-bound glycoprotein (38% glycan), M(r) 107,500, pI 7.6, pH and temperature 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 conifers (family Pinaceae) and from Rhus typhina bark were compared with CAO and the pI approximately 9 pine "laccase" following electrophoresis and Western blotting. For Abies balsamea, Larix laricina, Picea rubens, Pinus banksiana, Pinus taeda, and R. typhina, the isoelectric points 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.
Faculty of Forestry and Environmental Management, University of New Brunswick, Fredericton, Canada.
P
P
Udagama-Randeniya
R
R
Savidge
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D000906:Antibodies; D003300:Copper / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D015151:Immunoblotting; D007525:Isoelectric Focusing / Q000379:methods; D042845:Laccase; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D010947:Plants, Toxic; D013045:Species Specificity; D013054:Spectrophotometry, Atomic / Q000379:methods; D012251:Toxicodendron / Q000201:enzymology*; D014197:Trees / Q000201:enzymology*
D000906:Antibodies; D003300:Copper; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D042845:Laccase