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 (SE,) followed by 1% digitonin (SE,). Glucan synthase activity assays revealed that, although the SE, fraction possessed higher activity, only 8.6% of the in vitro product survived acetic/nitric acid treatment. On the other hand, the SE, 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 SE, fraction contained ß-1,3-glucan and fibrillar cellulose I, whereas the SE, fraction produced ß-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 SE, and SE, 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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