Levels of starch branching enzyme IIb and gene dosage at the ae locus

Branching enzyme (a-1,4-glucan: a -1,4-glucan-6-glycosyI transferase; E.C. 2.4.1.18) is one of the enzymes directly involved in the synthesis of starch. Branching enzyme modifies the developing a -1,4 polyglucan by the hydrolysis of an internal a -1,4 bond, followed by subsequent reattachment of the small a -1,4 glucan chain to the remaining or another a -1,4 glucan chain by an a -1,6 bond. This creates branch points as well as additional non-reducing ends where further synthesis of a -1,4 glucan chains can occur. Multiple forms of branching enzyme in developing maize kernel have been detected using DEAE-cellulose chromatography (C. D. Boyer and J. Preiss, 1978, Carbohyd. Res. 61:321). In addition, studies have shown the DEAE-cellulose profile of branching enzymes from kernels homozygous for amylose-extender (ae) to have no detectable quantities of branching enzyme IIb, while branching enzymes I and IIa remain unaltered (Boyer and Preiss, 1978, Biochem. Biophys. Res. Comm. 80:169). We have now examined the levels of branching enzyme from maize kernels with endosperm with 0, 1, 2 and 3 doses of the recessive ae allele.

Characterization of branching enzyme levels present in 22-day-old maize kernels (inbred W64A) involved partial purification consisting of homogenization, centrifugation, precipitation with 40% ammonium sulfate, and DEAE-cellulose chromatography. All procedures were performed at 4 C. DEAE-cellulose fractions were assayed for branching enzyme activity according to the rate of a -glucan formation from glucose-1-phosphate by phosphorylase a, and further characterized by the branching of amylose. Levels of individual branching enzyme fractions were corrected for differential rates of recovery of total branching enzyme activity. Results showed an inverse relationship between increasing dosage of the recessive ae allele and branching enzyme IIb levels in the endosperm:
 
Dose of recessive ae allele Relative activity Activity/kernel (µmoles/min) Activity/gm F.W. (µmoles/min)
3 0 0 0
2 12.2 0.21 0.99
1 35.8 1.03 5.15
0 49.8 1.41 6.43

Levels and properties of branching enzymes I and IIa did not vary as a function of ae dosage. Despite different recoveries of activity in different enzyme preparations, we still obtain a fairly linear relationship of enzyme level and dose of the dominant ae allele. These results are consistent with the possibility that ae is the structural gene for branching enzyme IIb.

Karen D. Hedman and Charles D. Boyer


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