An activity stain was developed to detect sucrose synthase (SS) in starch gels. Sucrose synthase (EC 2.4.1.13) catalyzes the reversible reaction of sucrose with UDP to produce UDP-glucose and fructose. The stain is based on a triple enzyme coupling mechanism. The production of fructose is coupled to hexokinase (HK) followed by phosphoglucoisomerase (PGI), and glucose-6-P dehydrogenase (G6PDH). The last enzyme is coupled to the tetrazolium stain.
Crude endosperm and embryo extracts from 25-day-old W22 kernels were obtained by grinding tissue with extraction buffer (Tris-HC1, 0.1 M, pH 7.5) in a 1:1 ratio. The homogenate was clarified by centrifugation at 16,000g for 30 min at 4 C. Immunoprecipitations were carried out according to Chourey (Mol. Gen. Genet. 184:372, 1981). An equal volume of antibody was added to the crude extract; PEG (8000 MW) was also added to a final concentration of 0.5%. The mixture was incubated for 4h at 4 C, and then centrifuged at 16,000g for 30 min at 4 C. Electrophoretic separation was performed in a discontinuous buffer system in 10% starch gels for 3.5h at 4 C at a constant voltage of 10.8V/cm gel. The gel buffer was histidine-HC1, 5mM, pH 7.0 (adjusted with NaOH); the electrode buffer was Tris 135mM/citric acid 43mM, pH 7.0. The staining solution contained sucrose 100mM, UDP 0.75mM, ATP 2.36mM, HK IU ml, PGI 0.5U/ml, G6PDH 0.3U/ml, NADP+ 0.04mM, MTT 0.48mM, PMS 0.13mM, MgCl2 20mM, and Tris 0.1M, pH 7.5. It should be noted that ADP, an alternative for UDP as a substrate, is also present in this stain, due to the HK reaction. Gels were stained at room temperature, and SS bands appeared after about 1h.
Four criteria were used to determine the identity of the sucrose synthase bands in the gels: substrate specificity, tissue specificity, a deletion stock, and immunoprecipitation. The use of crude extracts in combination with a 3-enzyme coupling system leads also to the production of bands other than those representing SS activity. Modifications of the SS stain were used to account for all bands in the gel. Similar banding patterns were obtained when UDP was replaced with ADP; however, the 2 bands closest to the origin appeared at a much slower rate (Fig. 1). This would be expected for maize SS activity because the enzyme prefers UDP as a substrate. The stain can also detect invertase activity since this enzyme produces both fructose and glucose. Removal of PGI and UDP eliminated only the two bands closest to the origin, indicating that they corresponded to SS bands; invertase bands appeared only after prolonged incubation (12h). Finally, exclusion of all the coupling enzymes, ATP, and UDP yielded only the 4 most anodal bands. This is further evidence indicating that the bands closest to the origin represent zones of SS activity.
There are two loci which encode proteins with SS activity in the seed. While the product of the Sh locus is found only in the endosperm, the other isozyme is present in both endosperm and embryo. Accordingly, the Sh-W22 endosperm extract (Fig. 1, lane a) had 2 bands corresponding to the 2 different enzymes, whereas the embryo extract (lane b) had only the most anodal of the 2 bands, SS-2. Furthermore, the endosperm extract of the sh deletion mutant, sh bz-m4, lacking most, if not all, of the coding region of the Sh locus, did not produce the SS-1 isozyme (lane c). Finally, endosperm extracts treated with extraction buffer or nonspecific antiserum (lanes d and e, respectively) had both isozymes, whereas treatment with antibody raised against the Sh protein (lane f) produced only the SS-2 band with markedly reduced activity. Embryo extracts incubated in extraction buffer (lane g) or nonspecific antiserum. (lane h) showed the SS-2 band, however, incubation in the antibody (lane i) eliminated the band. Reduction in activity of the 4 most anodal bands in the immunoprecipitation treatments was due to dilution of the samples.
To our knowledge, this is the first report of an activity stain for sucrose synthase. Prior to this, it was only possible to measure overall SS activity in a sample. It will now be possible to examine and compare activities of the two isozymes.
Figure 1. Gel stained for SS activity. Lanes a and b are crude extracts of Sh-W22 endosperm and embryo, respectively. Lane c is crude endosperm extract from sh-W22, bz-m4. Lanes d-f are Sh-W22 endosperm extracts incubated in extraction buffer, nonspecific antiserum, and Sh specific antibody, respectively. Lanes g-i are Sh-W22 embryo extracts incubated in extraction buffer, nonspecific antiserum, and antibody, respectively.
B.L. Bournival, C.E. Vallejos, PS. Chourey and L.C. Hannah
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