Virginia Polytech. Institute & State Univ.

Proteolytic activity in coleoptile extracts causes artifactual ß-glucosidase multiplicity --Asim Esen and Cumhur Cokmus Maize ß-glucosidases (ß-D-glucoside glucohydrolase, EC isolated from inbred lines display multiple electrophoretic variants. These variants appear in a time-dependent manner during storage, most notably at acidic pHs (below pH 6.0) and in the presence of a reducing agent. The enzyme was extracted from the coleoptiles of 6-day-old seedlings with a variety of aqueous buffers and assayed for activity. Zymograms were obtained by incubating the gels with the synthetic substrate 6-bromo-2-naphthyl ß-D-glucopyranoside after electrophoresis. The pHs of the crude enzyme preparations (extracts) were adjusted to vary from 3.0 to 10.0, and the preparations were incubated at different temperatures (-30, 4, 25, and 37 C) for varying lengths of time in the presence and absence of the reducing agent 2-mercaptoethanol (2-ME) and assayed for activity and changes in zymogram patterns. The results showed that the enzyme lost activity at higher temperatures (25 and 37 C) and at pHs below 4 and above 9. Similarly, the number of charge variants resolved by electrophoresis increased during incubation, especially at higher temperatures. These novel variants had increased anodal mobilities, and could be resolved into 3 to 5 distinct bands after incubation at pHs between 4 and 6 (Fig. 1a, lanes 1-6). When polypeptide profiles of the extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after the above-mentioned treatments, it was observed that the staining intensity of individual polypeptides decreased and there was a complete loss of some polypeptides at pHs between 4 and 6 in a time-dependent manner under reducing conditions, especially after incubation at 25 and 37 C (Fig. 1b, lanes 1-7). An example of the results is shown in Fig. 1. Figure 1a shows the changes in ß-glucosidase zymograms of the coleoptile extract from inbred K55 made with 25 mM sodium acetate buffer (pH 5.0) containing 35 mM 2-ME. The extract (final pH 5.2) was incubated at two different temperatures (25 and 37 C) for 6, 12, and 24 hours, respectively. It is evident that zones (bands) of ß-glucosidase ac-tivity increase in number, and novel anodal bands appear as cathodal bands disappear or decrease in intensity in a time- and temperature-dependent manner (Fig. 1a, lanes 2-6). When the pH of the same extract was adjusted to 7.0 before treatments, the shifts from cathodic to anodic zones of activity slowed substantially and the anodic shift produced mostly a diffuse zone of activity instead of distinct bands (Fig. 1a, lanes 9-14). At both pHs, the highest enzyme activity and the least anodal shift in zymograms was in the samples stored at 4 C for 24 hours (Fig. 1a, lanes 1 and 8). Figure 1b shows the changes in polypeptide profiles of the same samples used to develop the zymograms in Figure 1a. It is apparent that the cause of the changes in zymograms at pH 5.2 is proteolysis because the size and intensity of the ß-glucosidase polypeptide (60 kD band, arrow) decreases in a time- and temperature-dependent manner (Fig. 1b, lanes 2-7). Moreover, essentially complete proteolysis of polypeptides other than the ß-glucosidase monomer is evident from (1) the absence of any bands in lanes 1-7 (Fig. 1b), and (2) the presence of a diffuse, low molecular weight Coomassie blue stained zone at the anodic end (bottom) of the gel. In contrast, little or no proteolysis is evident in samples whose pH was adjusted to 7.0 (Fig. 1b, lanes 8-11), except some slight proteolysis at 37 C (Fig. 1b, lanes 12-14). We were able to inhibit the proteinase active at acidic pHs with antipain, leupeptin and the alkylating agent iodoacetic acid, but not the one active at neutral or alkaline pHs. Based on these data, it was concluded that maize ß-glucosidase multiplicity observed in zymograms of inbred lines is an artifact of proteolysis. An SH-proteinase active at acidic pHs and requiring a reducing agent (e.g., 2-ME) for activation was primarily responsible for artifactual ß-glucosidase multiplicity. Another class (undefined) of proteolytic activity was found to be responsible for producing charge multiplicity above pH 6; but this multiplicity appeared mostly as a broad and diffuse zone instead of distinct bands. Using buffers of neutral or alkaline pH or excluding reducing agents from the extraction medium and performing all the operations at 0-4 C appear to be effective strategies against the SH-proteinase.

Figure 1. Time-course of the changes in ß-glucosidase zymograms (a) and polypeptide patterns (b) after extraction with 25 mM Na-Acetate, pH 5 / 35 mM 2-ME. The extract (pH 5.2) was incubated at 25 and 37 C for 6, 12, and 24 hrs. Lane 1, 4 C control (24 hrs); lanes 2-4, after 6, 12, and 24 hrs, respectively, at 25 C; lanes 5-7, after 6, 12, and 24 hrs, respectively, at 37 C. Lanes 8-14, after the pH of the extract was adjusted from 5.2 to 7. Lane 8, 4 C control (24 hrs); lanes 9-11, after 6, 12, and 24 hrs, respectively, at 25 C; lanes 12-14, after 6, 12, and 24 hrs, respectively, at 37 C. The last lane (unnumbered) in b includes marker proteins ranging in size from 14 to 66 kD. The arrow on right points to the 60 kD polypeptide (ß-glucosidase monomer).

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