The elongation of amylose and amylopectin chains in isolated starch granules

The aim of this work was to investigate the conditions required for amylose synthesis in starch granules. Although the major granule-bound isoform of starch synthase - GBSSI - catalyses the synthesis of amylose in vivo, ¹⁴C from ADP[¹⁴C]glucose was incorporated primarily into a specific subset of amylopectin chains when supplied to starch granules isolated from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers. Incubation of granules with soluble extracts of these organs revealed that the extracts contained compounds that increased the incorporation of ¹⁴C into amylose. These compounds were rendered inactive by treatment of the extracts with α-glucosidase, suggesting that they were malto-oligosaccharides. Consistent with this idea, provision of pure malto-oligosaccharides to isolated granules resulted in a dramatic shift in the pattern of incorporation of ¹⁴C, from amylopectin chains to amylose molecules. Comparison of the pattern of incorporation in granules from wild-type peas and lam mutant peas which lack GBSSI showed that this effect of malto-oligosaccharides was specifically on GBSSI. The significance of these results for understanding of the synthesis of amylose and amylopectin in storage organs is discussed.     

Biological activity of gibberellin analogues

In order to determine the significance of the C-6 carboxyl group for the biological activity gibberellin A₃, 6-epigibberellin A₃, 7-norgibberellin A₃, 6-methyl-7-norgibberellin A₃, and 7-homogibberellin A₃ were studied using dwarf pea, dwarf maize, dwarf rice, dwarf barley and -amylase bioassays. All gibberellin A₃(GA₃)derivatives tested were considerably less active than GA₃. In all biossays, 6-epi-GA₃ showed a low activity of the same order, whereas 6-methyl-7-nor-GA₃ was inactive. Surprisingly, 7-nor-GA₃ had some activity in the dwarf rice (root application), dwarf barley, and -amylase bioassay, in contrary to its low potency in the dwarf pea, dwarf maize, and dwarf rice (micro drop) bioassay. 7-Homo-GA₃ was primarily active in the dwarf maize, dwarf barley and dwarf rice bioassay. It also caused antigibberellin effects in dwarf rice. The results demonstrate that the C-6 carboxyl group is not absolutely essential for biological activity of gibberellins. The different activities of 7-nor-GA₃ observed in the various test systems may indicate that the C-6 carboxyl group is a structural requirement more for uptake and/or transport processes than for receptor affinity.Abbreviation GA₃ gibberellic acid     

Location-dependent variations in the material properties of the anterior cruciate ligament.

Our recent anterior drawer studies in human cadaveric knees [Guan and Butler, Adv. Bioengng 17, 5 (1990); Guan et al., Trans. orthop. Res. Soc. 16, 589 (1991)] have suggested that anterior bundles of the anterior cruciate ligament (ACL) develop higher load-related material properties than posterior bundles. This was confirmed when we reevaluated the axial failure data for these bundle-bone specimens from an earlier study [Butler et al., J. Biomechanics 19, 425-432 (1986)]. The purpose of this study was to determine, in a larger data set, if anteromedial and anterolateral bundles of the anterior cruciate ligament exhibit significantly larger load-related material properties than the posterior ligament bundles. Seven ACL-bone units from seven donors (the three tissues from the original study plus four new ones) were subdivided into three subunits, preserving the bone insertions. The subunits were failed in tension at a constant strain rate (100% s-1) and four material properties were compared within and between donors. The anterior bundles developed significantly larger moduli, maximum stresses, and strain energy densities to maximum stress than the posterior subunits. Moduli for the anterior vs posterior subunits averaged 284 MPa vs 155 MPa, maximum stresses averaged 38 MPa vs 15 MPa, and strain energy densities averaged 2.7 N m cc-1 vs 1.1 N m cc-1, respectively. No significant differences were found, however, among strains to maximum stress or between any of the other properties for the two anterior subunits. These results are important to the design of ligament replacements and suggest new experiments designed to distinguish in vivo force levels in these ACL bands, a possible reason for the material differences.     

The two-stranded alpha-helical coiled-coil is an ideal model for studying protein stability and subunit interactions.

We have designed de novo a two-stranded alpha-helical coiled-coil which consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment. Their structure is stabilized by interchain hydrophobic interactions from hydrophobes at positions "a" and "d" of a repeating heptad sequence. The formation and stability of the coiled-coil is dependent on peptide concentration due to the monomer-dimer equilibrium. In contrast, that coiled-coil containing an inter-helical disulfide bond does not show any concentration dependence in the guanidine hydrochloride denaturation experiments as expected. Replacement of one large hydrophobic Leu residue in each chain with Ala significantly decreases coiled-coil stability in both the reduced and oxidized coiled-coils [decreases in transition midpoint of 1.6M (2.3-0.7) and 2.4M (5.3-2.9), respectively]. A large pH dependence on coiled-coil stability is observed over the pH range 4 to 7 (transition midpoints at pH 4, 5, 5.5, 6 and 7 were 3.8, 3.2, 2.0, 1.2 and 0.7M, respectively). The increasing stability with decreasing pH correlates with the protonation of the Glu acid side-chains and reduction of intrachain repulsions between Glu-Glu side-chains in positions i, i + 3 or i, i + 4 along each alpha-helix of the coiled-coil. In addition, coiled-coil stability increases with increasing ionic strength.