P - Glucan Synthesis in the Cotton Fiber ’ 1 . ldentification of p - 1 , 4 - and p - 1 , 3 - Glucans Synthesized in Vitro

In vitro B-glucan products were synthesized by digitonin-solubilized enzyme preparations from plasma membrane-enriched fractions of cotton (Cossypium hirsutum) fiber cells. The reaction mixture favoring &1,4-glucan synthesis included the following effedors: MgZ+, Caz+, cellobiose, cyclic-3’:5‘-CMP, and digitonin. The ethanol insoluble fraction from this reaction contained &1,4glucan and 8-1,3-glucan in an approximate ratio of 25:69. Approximately 16% of the &1,4-glucan was resistant to the acetic/nitric acid reagent. The x-ray diffraction pattern of the treated produd favoring &l,4-glucan synthesis strongly resembled that of cellulose II. On the basis of methylation analysis, the acetic/nitric acid reagent-insoluble glucan produd was found to be exclusively 81,4-linked. Enzymic hydrolysis confirmed that the produd was hydrolyzed only by cellobiohydrolase 1. Autoradiography proved that the produd was synthesized in vitro. l h e degree of polymerization (DP) of the in vitro product was estimated by nitration and size exclusion chromatography; there were two average DPs of 59 (70%) and 396 (30%) for the @-1,3-glucanase-treated sample, and an average DP of 141 for the acetic/nitric acid reagent-insoluble produd. On the basis of product analysis, the positive identification of in vitro-synthesized cellulose was established.