Β-glucan synthesis in the cotton fiber: IV. In vitro assembly of the cellulose I allomorph

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 β-1,3-glucan and fibrillar cellulose I, whereas the SE₂ fraction produced β-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.