Methods for Determining Relative Average Number of Channels per Maize Starch Granule and Digestion of Raw Granules of Mutant Maize Cultivars by Amyloglucosidase

Abstract

Six methods were investigated for determining the relative average numbers of channels per granule in populations of starch granules. The first method involved digestion of raw starch granules with amyloglucosidase. There were differences in the rates of digestion of starch of the same mutation in two different inbred‐line backgrounds, but we concluded that a better method was needed. Three methods involved measuring either all channel proteins or one specific protein. Extraction of granules using 70% 2‐propanol containing 1% 2‐mercaptoethanol (ME) was successful in removing the majority of external surface proteins (<31 kDa). Further extraction of starch granules using 0.1% SDS containing 0.2% ME, pH 8, removed channel proteins without extracting matrix proteins. By measuring SDS‐PAGE band intensities of protein clusters at 31–97.4 kDa or a marker protein at ≈42 kDa, methods to quantify relative average degrees of channelization (RADC) in different maize backgrounds were developed. Using either method, Oh43 had the highest RADC and B73 had the lowest RADC among the inbred lines examined. Another method involves extraction of total proteins using 2% SDS containing 0.2% ME from granules with the majority of surface proteins removed. The extract is subjected to SDS‐PAGE and Western blotting, followed by location and quantification of the actin band using antibodies to maize pollen actin. These four methods give somewhat different results for six inbred lines of corn with a relatively narrow range of RADC. In an attempt to relate them to the actual number of observed pores (channel openings to the surface), granules were treated with α‐amylase to enlarge the pores, with and without prior treatment with a protease. We concluded that this treatment revealed something about the nature of the granules and the action of α‐amylase on them, but it was not an indication of the number of channels present. The outcome of this research provides evidence that the number of channels in corn starch granules varies with the genetic makeup of the parent plant. We concluded that measuring granule actin is the preferred method.

The Architecture, Nature, and Mystery of Starch Granules. Part 1: A Concise History of Early Investigations and Certain Granule Parts

“…[ 41 ] published evidence that there were (in cassava starch granules) short radial channels of amorphous starch that spanned only a few layers. Also, in studies of the attack of glucoamylase on native maize starch granules, it appears that many more “bore holes” are formed than there are visible pores on the surface of the granules (Widya et al., [ 300 ] and earlier indications), indicating that there may be weak spots on the surface that are not openings to the type of channels found by BeMiller et al., but perhaps to the type of channel containing amorphous starch proposed by Gallant et al.…”