BETA-GLUCAN SYNTHESIS IN THE COTTON FIBER .4. IN VITRO ASSEMBLY OF THE CELLULOSE I ALLOMORPH

摘要

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(1)) followed by 1% digitonin (SE(2)). Glucan synthase activity assays revealed that, although the SE(2) fraction possessed higher activity, only 8.6% of the in vitro product survived acetic/ nitric acid treatment. On the other hand, the SE(1) fraction was less active, but 32.1% of the total glucan in vitro product was resistant to aceticitric acid. In vitro products synthesized from the SE(1) fraction contained beta-1,3-glucan and fibrillar cellulose I, whereas the SE(2) 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 SE(1) and SE(2) revealed cellulose I and cellulose II, respectively. Contamination of native cellulose was ruled out by extensive evidence from autoradiography of the ethanol-insoluble and aceticitric acid-insoluble materials, including three different controls. [References: 46]