Cellulose biosynthesis by enzymes from the hypocotyl of the mung bean (Phaseolus aureus), as judged by the extraction technique of Updegraff (1969)

The numbers refer to the chapters concerned. 1. Current knowledge on cellulose biosynthesis was summarised. The section is divided into four main parts concerned with; A. the importance of cellulose; B. the structure of cellulose; C . cellulose biosynthesis, and; D. the cellular location of cellulose biosynthesis. Where relevant, cellulose biosynthesis in the cellulosic bacterium Acetobacten xylinum and algal systems was included. 2. This chapter presents the rationale behind this work and the criteria used to assess the cellulosic nature of the synthesised products. 3. The materials and methods used in this work were described. 4. The incorporation of radioactivity from GDP-[U-14C]-G, UDP-[U-14G]-G, [U-14C]-sucrose, [U-14C]-glucose and [U-14C]-glucose-1-phosphate into the water-and chloroform: methanol (3:2 v/v)-insoluble products was investigated. It was found that the sugar-nucleotides were the most efficient substrates, that the incorporation of radioactivity from 1.0 mM UDP-[U-14c]-G into these products appeared to be substrate-activated, and that the products synthesised from UDP-[U-14C]-G at this concentration contained beta (1-3) linked glucose. The distribution of the enzymic activity utilising the sugar-nucleotides between wall and particulate fractions and the effect of storage of the enzymes at -20°C was also investigated. 5. The alpha-cellulose extraction was explained. Radioactivity from GDP-[U-14]-G, GDP-[U-14C]-M and UDP-[U-14C]-G was incorporated into alpha-cellulose. GDP-[U-14C]-M was found to be an efficient substrate and GDPM stimulated the incorporation of radioactivity from GDP-[U-14C]-G into this fraction. 6. The Updegraff extraction was introduced. Both UDP-[U-14C]-G and GDP-[U-14C]-G acted as substrates for Updegraff cellulose (U-cellulose) synthesis. The effect of prolonged periods of extraction of the water-and chloroform : m ethanol (3:2 v/v)-insoluble products in the Updegraff reagent and the effect of retaining the enzymes at -20 °C was studied. 7. The incorporation of radioactive glucose from GDP- [U-14C]-G (particularly at higher concentrations than that traditionally used by previous researchers) and GDP- [U-14C]-M into U-cellulose was studied. The factors involved in the cessation of U-cellulose synthesis from GDP-[U-14C]-G were investigated and discussed. The products appear to be glucomannan (on the basis of a kinetic study of the effect of GDPM on U-cellulose synthesis from GDP-[U-14C]-G, structural and gel-filtration studies) and possibly mannan (on the basis of a time course study on the synthesis of U-cell- ulose from 102 muM GDP- [U-14C]-M) and glucan (based on a time-course study of U-cellulose synthesis from 1.0 mM GDP-[U-14C]-G, the effect of E D T A on U-cellulose synthesis from GDP- [U-14Cl-G and GDP-[U-14C]-M, and a structural study). There was some indication of the presence of a substrate-activated enzyme which synthesised pure glucan from GDP-[U-14C]-G. The non-glucan products could not be solubilised even after prolonged periods of extraction in Updegraff reagent. 8. The incorporation of radioactive glucose from 1-5 muM UDP-[U-14C]-G into U-cellulose was further investigated. The synthesis of U-cellulose from 1 muM UDP-[U-14C]-G showed no distinct pH optimum in the range 4-11 and the factors involved in the cessation of the reaction were discussed. Gel filtration of the water and chloroform : methanol (3:2 v/v)-insoluble products suggested that the majority of the products had a molecular weight of less than 7 x 10. 9. The.general conclusions on this work and their possible significance for cellulose biosynthesis were evaluated. In particular, a hypothesis is presented by which the purportedly negative results obtained by previous researchers working on GDPG as a precursor of cellulose may be reconciled with cellulose biosynthesis