b-glucosidase occurs as large aggregates in "null" genotypes of maize
--Asim Esen

In maize, the catalytically active form of °-glucosidase is a dimer. Genotypes whose zymograms are devoid of detectable enzyme bands and thus are thought to be homozygous for a null allele at the Glu1 locus are known. This is puzzling in view of the data implicating the enzyme in important functions that are critical to plant growth and development. We have shown that both null and normal genotypes have similar amounts of the enzyme protein and activity, but the enzyme occurs mostly as insoluble or poorly soluble aggregates in nulls and does not enter the gel; thus, it is not detected by zymogram techniques (Esen and Cokmus, 1990).

Further studies were carried out on °b-glucosidase isolated from "null" genotypes in order to determine the nature of the interactions responsible for aggregation as well as the sizes of enzyme aggregates. Coleoptile extracts from inbreds K55 (normal) and H95 (null) were made with 50mM sodium acetate buffer, pH 5, and subjected to size fractionation through a column of Sephacryl HR 300 (exclusion limit, 1.5 million Daltons) using appropriate calibration standards. Column fractions were assayed for enzyme activity spectrophotometrically and for the enzyme monomer by SDS-PAGE. The gel filtration data show that nearly 80% of °b-glucosidase activity in extracts of H95 appeared in the flow-through fraction suggesting that it had a molecular mass equal to or greater than 1.5 million Daltons while only about 20% of the activity eluted as a dimer (120,000 Daltons) (Table 1). In contrast, nearly all of the activity in extracts of K55 eluted from the column as a dimer. These data clearly indicate that the enzyme in extracts from "null" genotypes occurs as large aggregates.

Table 1. Distribution of °b-glucosidase activity in normal and "null" phenotypes.
 

Phenotype	Est. Mol. Wt. (Daltons)
	120,000	Ñ1.5 million
Normal (K55)	98%	2%
Null (H95)	22	78%

Estimated by gel filtration (Sephacryl HR 300)

In order to determine the nature of the interaction responsible for aggregation, the enzyme was extracted from null inbreds with buffers containing SDS, or SDS was added to the supernatant fluid after extraction without SDS in buffer. These samples were electrophoresed through native, IEF and SDS-gels. All of the 8 different "null" genotypes tested yielded zymograms with active bands in all of these gel systems after extracts were added or made with SDS-containing buffers. Figure 1 provides evidence for the fact that the enzyme from a typical "null" (H95) does not enter the gel until SDS is either added to a final concentration of 0.5% to the extract or extraction is made with a buffer containing at or above 0.5% SDS. These results suggest that the basis of interaction leading to aggregation is very likely to be hydrophobic because it could be disrupted by SDS, releasing catalytically active dimeric enzyme. However, the question of whether or not the aggregates are formed between enzyme molecules after interaction with one another or between enzyme molecules and other protein or nonprotein components is not known and under investigation. Likewise whether the aggregates are formed as an artifact during extraction or they occur also in vivo is worthy of further investigation.

On the basis of the data above it can be concluded that none of the maize genotypes classified as "null" is truly null, and apparent null phenotype is due to formation of large quaternary associations of the enzyme which can be dissociated with SDS. Thus the observed monogenic inheritance for "null" phenotype appears to be for an allele of another locus whose product either interacts with the enzyme or causes it to aggregate in vivo or during isolation.

Figure 1. Zymogram (6% native PAGE; anode at bottom) showing the entry of °b-glucosidase from a null genotype (H95) into the gel after SDS was added to the extract or the extraction buffer at a final concentration of 0.5% or higher. Note that enzyme aggregates remain in the stacking gel and form a band at the top of the resolving gel.

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