ß-glucosidase zymograms in denaturing (SDS) gels --Asim Esen and Gunay Gungor We performed a systematic search for concentrations of protein denaturing agents that inactivate maize ß-glucosidase activity. In the course of these studies, it was discovered that the enzyme retained about 50% of its activity after treatment with or in the presence of the anionic detergent sodium dodecyl sulfate (SDS), a potent denaturant, when used at or around 1% concentrations. When coleoptile extracts or purified enzyme preparations were incubated in the presence of SDS concentrations varying from 0 to 1.6%, electrophoresed through 6-8% polyacrylamide gels and stained for activity, zymogram patterns similar to those of untreated samples were obtained (Fig. 1a). However, the intensity of enzyme bands decreased as SDS concentration increased to 0.8% or higher. In view of these results, controls and SDS-treated samples were subjected to standard SDS-PAGE through 10-12% gels using the method of Laemmli and the resulting gels were stained for enzyme activity. Surprisingly, a zone of activity including 1 to 6 bands, depending on sample age and buffer pH and composition, in the 60 kD region of the gel was obtained (Fig. 1b). Figure 1 shows representative zymograms obtained when the control and SDS-treated samples were electrophoresed through a 6% native (Fig. 1a) and a 10% SDS gel (Fig. 1b). Multiple ß-glucosidase bands are visible in both zymograms. In this case, the source of the heterogeneity is thought to be artifactual due to the activity of an endogenous SH-proteinase because it occurred independently of SDS treatment and the samples were handled under conditions (e.g., extraction and storage in a pH 5 buffer containing 2-mercaptoethanol) promoting the activity of such proteinase prior to SDS treatment and electrophoresis. In subsequent experiments (results not shown), a single band was obtained when the sample was not exposed to the reducing agent. Furthermore, it was shown that the zymograms of the maize inbreds classified as null at the Glu locus contained ß-glucosidase bands after their coleoptile extracts were treated with SDS. These results suggest that the 60 kD polypeptide, the ß-glucosidase monomer, shows full enzymatic activity and, thus, the in vivo form of the functional enzyme is a monomer, not dimer as assumed on the basis of zymogram patterns of hybrid genotypes. In addition, the results clearly show that the maize inbreds that are classified as null for Glu encoded ß-glucosidase activity are not null. The enzyme of these so-called null genotypes appears to occur as large aggregates that fail to enter the gel and cannot be detected by standard zymogram techniques.

Figure 1. ß-glucosidase zymograms of a maize coleoptile extract after treatment with SDS prior to electrophoresis. a, 6% nondenaturing (native) gel. b, 10% denaturing (SDS) gel. The enzyme was extracted with 25 mM NaAc buffer, pH 5.0, containing 35 mM 2-mercaptoethanol. Lane 1, control (no SDS added). Lanes 2-6, after adding SDS to a final concentration of 0.1 to 1.6%. 2, 0.1%; 3, 0.2%; 4, 0.4%; 5,0.8%; 6, 1.6%.
 

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