Null alleles at the Glu locus code for ß-glucosidase activity and immunoreactive protein --Asim Esen and Cumhur Cokmus Maize ß-glucosidase (ß-D-glucoside glucohydrolase, EC 3.2.1.21) was extracted from coleoptiles of fifteen genotypes (three normals, 10 nulls, and two hybrids) in two fractions, the buffer-soluble and the buffer-insoluble (extracted with SDS-sample buffer). The enzyme activity was measured spectrophotometrically in the soluble fraction and also studied on zymograms after native gel electrophoresis and isoelectric focusing. The enzyme was purified from a normal genotype by ion exchange chromatography and preparative electrophoresis. Antisera were raised in four rabbits, and the soluble and the insoluble extracts of each genotype were analyzed for a cross-reacting material by ELISA and immunoblotting. The results showed that extracts from both the normal and the null genotypes had ß-glucosidase activity, and the activity measured spectrophotometrically was two to ten-fold higher in normals than in nulls (Table 1). Zymograms (not shown) of the null genotypes were devoid of distinct bands that were present in those of normals and hybrids from crosses between normals and nulls. Zymograms of both the normal and the null genotypes had a diffuse, smeared zone of activity at the cathodic end of native gels. A cross-reacting antigen was present in extracts of both genotypes when assayed by ELISA and a 60 kD polypeptide (ß-glucosidase monomer, Figs. 1a and 1c, arrow) was detected by four different monospecific ß-glucosidase antisera on

Table 1. ß-glucosidase activity in coleoptile extracts of selected maize lines and hybrids.

Figure 1. a. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12%T, 4%C) profiles of coleoptile extracts (soluble protein). b. Immunoblot of a developed with antiserum (R681) to ß-glucosidase. c. SDS-PAGE profiles of coleoptile pellet extracts (insoluble protein, extracted with SDS-sample buffer). d. Immunoblot of c developed with antiserum (R681) to ß-glucosidase. In both gels and blots, the cathode is at top. 1, F6; 2, CG10; 3, OH51A; 4, CO125; 5, CH586-12; 6, CH592-46; 7, CH593-32; 8, CH701-30; 9, MICH77-6; 10, CI.90A; 11, OH7B; 12, OH7BxH95; 13, H95; 14, H99xH95; 15, H99. Note the presence of an immunoreactive 60 kD polypeptide (ß-glucosidase monomer) in both null and normal genotypes and its increased amount in the insoluble fraction from null genotypes.

Western blots by immunostaining (Figs. 1b and 1d). Moreover, six out of seven null genotypes had a larger amount of their 60 kD polypeptide in the insoluble fraction (Fig. 1d) than in the soluble fraction (Fig. 1b). These data show that both the null and the normal genotypes have similar amounts of the enzyme protein, but the enzyme occurs mostly as insoluble or poorly soluble polymers in nulls and does not enter the gel; thus, it is not detected by zymogram techniques. The monogenic inheritance reported for the null alleles of the Glu locus is likely to be for a factor encoded by another locus which affects directly or indirectly the solubility of the enzyme by increasing its polymerization into large quaternary structures.

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