A "b-glucosidase aggregating factor" (BGAF)
is present in "null" genotypes
--Neval H. Erturk and Asim Esen
Maize ß-glucosidase catalyzes the hydrolysis of DIMBOA-glucoside, an abundant physiological substrate whose aglycone is believed to be involved in the defense of young plant parts against pests. The enzyme occurs as large aggregates in some maize inbreds. Such inbreds were originally thought to have a null allele at the enzyme locus. However the immunological and biochemical studies in our laboratory showed that these inbreds have enzyme activity and an immunoreactive enzyme monomer. We have recently discovered that "null" genotypes have a factor, ß-glucosidase aggregating factor (BGAF), which causes the aggregation and poor solubility of the enzyme in such genotypes. The existence and activity of BGAF were demonstrated as follows: (1) Equal amounts of 4- to 5-day-old frozen, etiolated shoots from a normal (K55) and a "null" (H95) genotype were mixed, homogenized, and extracted together and the enzyme activity in the supernatant was assayed. It was observed that activity in the supernatant fluid of these mixed extractions was decreased to a level barely detectable in spectrophotometric assays (data not shown), instead of being equal to the arithmetic mean of the H95 and K55 extracts made separately. (2) K55 and H95 shoots were homogenized separately and the homogenates were mixed in different ratios and extracted together, and assayed for enzyme activity. The results showed that the enzyme activity in supernatants decreased linearly as the amount of the H95 homogenate in the mixture increased (Fig. 1). (3) Shoot extracts of K55 and H95 were mixed and then assayed; the activity in the mixture was the arithmetic mean of that found in each of the two extracts. However, when the mixtures were incubated overnight and the activity was assayed in supernatants after the centrifugation, a reduction in enzyme activity was observed as the amount of the H95 extract in the mixture increased (Fig. 2). Moreover, the activity lost from the supernatant upon centrifugation was recovered quantitatively from the pellet by extraction with a buffer containing 0.5% SDS (Fig. 2). (4) The pellets of the H95 ("null") homogenates were suspended with the K55 supernatant of known activity level and incubated for 30 min., and activity was assayed in the K55 supernatant before and after incubation. Data showed that enzyme activity in the K55 supernatant decreased as the amount of the H95 pellet used for incubation increased. All these data suggest that a substance (i.e. BGAF) is present in "null" inbreds and it interacts with ß-glucosidase and causes its aggregation. BGAF is present both in the supernatant and pellet fractions of homogenates from "null" genotypes.
In order to study the effect of pH on the extractability and activity of ß-glucosidase and BGAF, freeze-dried K55 (normal) and H95 ("null") whole shoot powders were extracted in separate tubes four times with the following buffers of indicated pH: sodium citrate (pH 3), sodium acetate (pH 4 and 5), MES (pH 6), HEPES (pH 7), Tris-HCl (pH 8), CHES (pH 9), and sodium carbonate-bicarbonate (pH 10 and 11). All extractions were made on ice for 30 min each. Four extracts of each genotype made with the same buffer were saved separately for assays. Because the first and second extracts contained about 90-95% of the total extractable activity at a given pH, they were pooled (1:1 volume ratio) and reassayed for enzyme activity and protein so that the activity extracted at each pH could also be expressed as specific activity. The results (Figs. 3-4) show that the amount of enzyme and protein extractable increased with pH, being lowest or negligible at pH 3 and highest at pH 11, increasing in the pH range 4 to 11. When the highest total extractable activity at pH 11 is set as 100%, relative extractabilities were 52% at pH 4 and increased from 74% at pH 6 to 93% at pH 10 in the case of K55 (Fig. 3). However, in terms of specific activity (expressed here as A410 units or the absorbance of pNP produced/mg protein), the pH 4 and 5 extracts had the highest activities (92 and 89 units, respectively) and decreased from 55 at pH 6 to 36 at pH 11 in the case of the K55 extracts (Fig. 3). As for the H95 ('null') extracts, the following striking differences were observed in comparison to those of K55: (1) The amount of total extractable ß-glucosidase activity but not protein was about 3 to 20 times lower, depending on pH, resulting in drastic decreases (3.5 to 15 units/mg protein) in specific activities (Fig. 4). (2) The pool of the first two extracts contained a lower percentage (80 and 67%, respectively) of the total extractable activity while the corresponding values ranged from 97 to 89% in the pH range 4 to 9 in K55 (Fig. 3). (3) Most surprisingly, extractability of the enzyme increased with pH only in the pH range 7 to 11 (Fig. 4). Relative extractabilities in the pH range 7 to 10 increased from 42 to 61%, in stark contrast to the corresponding values of 86 to 93% obtained for K55 in the same pH range. The real surprise was the decrease of relative extractability to 19% at pH 5 and to 35% at pH 6 after 40% at pH 4 (Fig. 4). The most plausible interpretation of these results is that BGAF is solely responsible for the differences between K55 and H95. It appears that BGAF activity is highest at and around pH 5, and it decreases as pH increases from 7 to 11. It seems that at pH 10 to 11, either BGAF is gradually solubilized and extracted or inactivated as exposure to high pH continues, as would be the case with repeated extractions. Studies focusing on isolation and further characterization of BGAF are in progress.
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