PubMed · 6873452
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A Sarko, H C Wu, C T Chuah. 1983. Multiple-helical glucans.. https://pubmed.ncbi.nlm.nih.gov/6873452/
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Amylose was prepared by enzymatic polymerization of alpha-D-glucose 1-phosphate dipotassium catalyzed by a phosphorylase using two kinds of the primers derived from maltopentaose, and then it was chemically bonded to silica gel to be used as a chiral stationary phase (CSP) in high-performance liquid chromatography. In method I, maltopentaose was first lactonized and allowed to react with (3-aminopropyl)triethoxysilane to form an amide bond. Amylose chains with a desired chain length and a narrow molecular weight distribution were then constructed by the enzymatic polymerization. The resulting amylose bearing a trialkoxysilyl group at the terminal was allowed to react with silica gel for immobilization. In method II, maltopentaose was first oxidized to form a potassium gluconate at the reducing terminal. After the enzymatic polymerization was performed with the potassium gluconate, the amylose end was lactonized to be immobilized to 3-aminopropyl-silanized silica gel through amide bond formation. Two amylose-conjugated silica gels thus obtained were treated with a large excess of 3,5-dimethylphenyl isocyanate to convert hydroxy groups of amylose to corresponding carbamate residues. The CSP derived through method II was superior in chiral recognition to the CSP derived from method I and showed better resolving power and higher durability against solvents such as tetrahydrofuran compared with a coated-type CSP. Influences of degree of polymerization of amylose, the spacer length between amylose and silica gel, and mobile phase compositions on chiral recognition were investigated.
The wild-type gene encoding granule-bound starch synthase (GBSS) is capable of both complementing the amylose-free (amf) potato mutant and inhibiting the endogenous GBSS gene expression in wild-type potato. Co-suppression of the endogenous GBSS gene, easily visualised by staining the starch with iodine, occurred when the full-size GBSS sequence (genomic), GBSS cDNA or even the mutant amf allele were introduced into the wild-type potato. Conversely, introduction of the GBSS promoter sequence alone, did not result in co-suppression in the 80 analysed transformants. Neither the orientation of the GBSS gene with respect to kanamycin resistance nor the presence of an enhancer influenced the frequency of plants showing a co-suppression phenotype. After crossing a partially complemented amf mutant with a homozygous wild-type plant, the F1 offspring segregated into plant phenotypes with normal and decreased expression of the GBSS gene. This decreased expression correlated with the presence of a linked block of five T-DNA inserts which was previously shown to be correlated with partial complementation of the amf mutant. This crossing experiment indicates that co-suppression can cause inhibition of gene expression of both inserted and endogenous wild-type GBSS genes. The frequency of partially complemented amf plants was equal to the frequency of co-suppressed wild types when a construct, with an enhancer in front of the GBSS promoter, was used (pWAM 101E). This might suggest that partial complementation of the amf genotype caused by unstable expression of the transgene can be overcome by inserting an enhancer in front of the GBSS promoter.
Ultracentrifugation was used to clarify seven amylose fractions obtained by aqueous leaching of corn starch by 5 degrees C steps over a temperature range of 65-95 degrees C. Each fraction was characterized by its hydrodynamic radius R-(H), as determined by dynamic light scattering--and by the macromolecular features (M-(W), the weight-average molecular weight; R-(G), the z-average gyration radius, MWD, the molecular weight distribution) determined by size-exclusion chromatography coupled on line with multi-angle laser light scattering using 0.1 M KOH as solvent. This procedure provided new insights into the leaching process. In particular, a key temperature (85 degrees C) was noted, after which amylose M-(W) increased markedly from 8 x 10(4) - 1.4 x 10(5) to 3 x 10(5) - 3.5 x 10(5) g.mol(-1). Molecular weight dependences of macromolecular sizes were consistent with the behaviour of an expanded chain in good solvent with in particular a hydrodynamic coefficient v within the 0.6-0.7 range for the R(G) approximately M(v) relation. Finally, a method was developed to fit corresponding experimental molecular weight distributions with mathematical distributions of two populations. This enable us to determine that the molecular weight distribution for each leached amylose fraction was at least bimodal and to calculate the corresponding M(W) to each component.