Detection of enzymes active on various beta-1,3-glucans after denaturing polyacrylamide gel electrophoresis.
Enzymes were assayed for glucanase activity after denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing beta-1,3-glucans embedded as substrate. Lentinan, curdlan, paramylon, baker's yeast alkali-insoluble glucan, baker's yeast alkali-soluble glucan and carboxymethyl (CM)-pachyman were compared to oligomeric laminarin, which is the usual substrate for assaying beta-1,3-glucanase activities. Detecting enzyme activities by aniline blue fluorescent staining was also compared with the staining of released reducing sugars by 2,3,5-triphenyltetrazolium chloride (TTC). For the nonreduced proteins, the Driselase extract exhibited one major band at 32.5 kDa and one less intense band at 23 kDa for most substrates with the two detection procedures. No Lyticase enzyme was detected in either detection procedures for all tested substrates. For barley enzymes, no activity was revealed after aniline blue staining while one undescribed 19 kDa glucanase activity was best shown after TTC staining with curdlan, paramylon and CM-pachyman as substrates. In the case of reduced proteins, the Lyticase extract yielded three bands (33, 36 and 46 kDa) on several substrates with both detection procedures. This was the same for the barley leaf extract (32, 36 and 39 kDa). The Driselase extract showed one 42 kDa band. Many enzymes active on beta-1,3-glucans are thus best revealed when proteins are denatured and reduced and when protein renaturation after SDS-PAGE involves a pH 8.0 treatment and the inclusion of 1 mM cysteine in buffers. However, some enzymes are only detected when proteins are denatured without reduction. Finally, the use of various polymeric beta-1,3-glucan substrates different from oligomeric laminarin is necessary to detect new types of enzymes such as the 19 kDa barley glucanase.
Trudel
J
J
Département de phytologie, Faculté des sciences de l'agriculture et de l'alimentation, Université Laval, Québec, Canada.
Grenier
J
J
Asselin
A
A
D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D043326:Glucan 1,3-beta-Glucosidase; D005936:Glucans / Q000378:metabolism*; D008623:Mercaptoethanol; D011489:Protein Denaturation; D013379:Substrate Specificity; D047071:beta-Glucans; D001617:beta-Glucosidase / Q000378:metabolism*
D005936:Glucans; D047071:beta-Glucans; D008623:Mercaptoethanol; C033363:beta-1,3-glucan; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase