Maize genotypes classified as null at theglu locus have β-glucosidase activity and immunoreactive protein |
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Authors: | Asim Esen Cumbur Cokmus |
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Institution: | (1) Department of Biology, Virginia Polytechnic Institute and State University, 24061 Blacksburg, Virginia;(2) Department of Biology, Ankara University, Faculty of Sciences, 06100 Besevler, Ankara, Turkey |
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Abstract: | Maize β-glucosidase (β-d-glucoside glucohydrolase; EC 3.2.1.21) was extracted from coleoptiles of 15 maize genotypes (3 normals, 10 nulls, and 2 hybrids)
in two fractions, the soluble and the insoluble. 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 anion-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 β-glucosidase activity, and the activity measured spectrophotometrically
was 2- to 10-fold higher in normals than in nulls. Zymograms 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 (β-glucosidase monomer) was detected by four different
monospecific β-glucosidase antisera on Western blots by immunostaining. Moreover, six of seven null genotypes had a larger
amount of their 60-kD polypeptide in the insoluble fraction than in the soluble fraction. 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 the monogenic inheritance reported for the null alleles of theglu 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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Keywords: | Zea mays Gramineae β -glucosidase null allelism immunoblotting electrophoresis |
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