Enzyme-gold affinity labelling of cellulose.
The enzyme-linked colloidal gold affinity labelling technique was tested as a method to localize cellulose on thin sections of plant cell walls and slime mold spores. Commercially available cellulase from cultures of Trichoderma reesei, the main components being cellobiohydrolase I and II (CBH I, CBH II) and endoglucanase (EG), was linked to colloidal gold by using standard techniques and applied as a dilute, buffered suspension to thin sections. After brief exposure, e.g., 15-30 minutes, cellulose exposed on the surface of sections was labelled with the enzyme-gold complex. Poststaining did not appear to have a deleterious effect on the labelled sections. The specificity of labelling was demonstrated by its complete inhibition when carboxymethylcellulose was incorporated in the labelling mixture, by lack of labelling of 1,4-beta-mannans or 1,3-beta-xylans in noncellulosic walls of marine algae, by lack of labelling of 1,4-beta-glucans in chitin, by much lower labelling density when done at 4 degrees C, and by lack of labelling when sections were predigested with cellulase. Labelling with the crude commercial cellulase was compared to labelling with purified CBH I-, CBH II-, and EG-linked colloidal gold, and the labelling pattern was similar. This method was found useful on conventionally fixed material and required no special preparation other than the use of inert (Ni or Au) grids and 0.5% gelatin to reduce nonspecific binding of the gold complex. Labelling was similar in the several embedding resins tested: LR White, Lowicryl K4M, Epon 812, and Spurr's.(ABSTRACT TRUNCATED AT 250 WORDS)
Berg
R H
RH
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611.
Erdos
G W
GW
Gritzali
M
M
Brown
R D
RD
D000345:Affinity Labels; D002473:Cell Wall / Q000032:analysis; D002480:Cellulase; D002482:Cellulose / Q000032:analysis*; D003080:Cold Temperature; D006046:Gold; D009235:Myxomycetes / Q000032:analysis; D010944:Plants / Q000032:analysis; D013170:Spores / Q000032:analysis; D013379:Substrate Specificity
D000345:Affinity Labels; D006046:Gold; D002482:Cellulose; D002480:Cellulase