beta]-Glucan Synthesis in the Cotton Fiber (IV. In Vitro Assembly of the Cellulose I Allomorph) |
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Authors: | Kudlicka K Brown R M Li L Lee J H Shin H Kuga S |
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Institution: | Department of Botany, The University of Texas at Austin, Austin, Texas 78713-7640. |
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Abstract: | In vitro assembly of cellulose from plasma membrane extracts of the cotton (Gossypium hirsutum) fiber was enriched by a combination of 3-(N-morpholino)propanesulfonic acid extraction buffer and two independent digitonin solubilization steps consisting of 0.05% digitonin (SE1) followed by 1% digitonin (SE2). Glucan synthase activity assays revealed that, although the SE2 fraction possessed higher activity, only 8.6% of the in vitro product survived acetic/nitric acid treatment. On the other hand, the SE1 fraction was less active, but 32.1% of the total glucan in vitro product was resistant to acetic/nitric acid. In vitro products synthesized from the SE1 fraction contained beta]-1,3-glucan and fibrillar cellulose I, whereas the SE2 fraction produced beta]-1,3-glucan and cellulose II. Both celluloses assembled in vitro were labeled with cellobiohydrolase I-gold complex, and the electron diffraction patterns of both products from SE1 and SE2 revealed cellulose I and cellulose II, respectively. Contamination of native cellulose was ruled out by extensive evidence from autoradiography of the ethanol-insoluble and acetic/nitric acid-insoluble materials, including three different controls. |
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