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. 1993 Apr;101(4):1131–1142. doi: 10.1104/pp.101.4.1131

[beta]-Glucan Synthesis in the Cotton Fiber (I. Identification of [beta]-1,4- and [beta]-1,3-Glucans Synthesized in Vitro).

K Okuda 1, L Li 1, K Kudlicka 1, S Kuga 1, R M Brown Jr 1
PMCID: PMC160630  PMID: 12231764

Abstract

In vitro [beta]-glucan products were synthesized by digitonin-solubilized enzyme preparations from plasma membrane-enriched fractions of cotton (Gossypium hirsutum) fiber cells. The reaction mixture favoring [beta]-1,4-glucan synthesis included the following effectors: Mg2+, Ca2+, cellobiose, cyclic-3[prime]:5[prime]-GMP, and digitonin. The ethanol insoluble fraction from this reaction contained [beta]-1,4-glucan and [beta]-1,3-glucan in an approximate ratio of 25:69. Approximately 16% of the [beta]-1,4-glucan was resistant to the acetic/nitric acid reagent. The x-ray diffraction pattern of the treated product favoring [beta]-1,4-glucan synthesis strongly resembled that of cellulose II. On the basis of methylation analysis, the acetic/nitric acid reagent-insoluble glucan product was found to be exclusively [beta]-1,4-linked. Enzymic hydrolysis confirmed that the product was hydrolyzed only by cellobiohydrolase I. Autoradiography proved that the product was synthesized in vitro. The 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 [beta]-1,3-glucanase-treated sample, and an average DP of 141 for the acetic/nitric acid reagent-insoluble product. On the basis of product analysis, the positive identification of in vitro-synthesized cellulose was established.

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Selected References

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  1. Anderson M. A., Stone B. A. A new substrate for investigating the specificity of beta-glucan hydrolases. FEBS Lett. 1975 Apr 1;52(2):202–207. doi: 10.1016/0014-5793(75)80806-4. [DOI] [PubMed] [Google Scholar]
  2. Brown R. M., Jr Cellulose microfibril assembly and orientation: recent developments. J Cell Sci Suppl. 1985;2:13–32. doi: 10.1242/jcs.1985.supplement_2.2. [DOI] [PubMed] [Google Scholar]
  3. Bureau T. E., Brown R. M. In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6985–6989. doi: 10.1073/pnas.84.20.6985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hayashi T., Read S. M., Bussell J., Thelen M., Lin F. C., Brown R. M., Delmer D. P. UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product. Plant Physiol. 1987 Apr;83(4):1054–1062. doi: 10.1104/pp.83.4.1054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Heiniger U., Delmer D. P. UDP-glucose: Glucan Synthetase in Developing Cotton Fibers: II. Structure of the Reaction Product. Plant Physiol. 1977 Apr;59(4):719–723. doi: 10.1104/pp.59.4.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kopriwa B. M. A reliable, standardized method for ultrastructural electron microscopic radioautography. Histochemie. 1973 Oct 3;37(1):1–17. doi: 10.1007/BF00306855. [DOI] [PubMed] [Google Scholar]
  7. Li L., Brown R. M., Jr [beta]-Glucan Synthesis in the Cotton Fiber (II. Regulation and Kinetic Properties of [beta]-Glucan Synthases. Plant Physiol. 1993 Apr;101(4):1143–1148. doi: 10.1104/pp.101.4.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Li L., Drake R. R., Jr, Clement S., Brown R. M., Jr [beta]-Glucan Synthesis in the Cotton Fiber (III. Identification of UDP-Glucose-Binding Subunits of [beta]-Glucan Synthases by Photoaffinity Labeling with [[beta]-32P]5[prime]-N3-UDP-Glucose. Plant Physiol. 1993 Apr;101(4):1149–1156. doi: 10.1104/pp.101.4.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lin F. C., Brown R. M., Jr, Cooper J. B., Delmer D. P. Synthesis of Fibrils in Vitro by a Solubilized Cellulose Synthase from Acetobacter xylinum. Science. 1985 Nov 15;230(4727):822–825. doi: 10.1126/science.230.4727.822. [DOI] [PubMed] [Google Scholar]
  10. Lin F. C., Brown R. M., Jr, Drake R. R., Jr, Haley B. E. Identification of the uridine 5'-diphosphoglucose (UDP-Glc) binding subunit of cellulose synthase in Acetobacter xylinum using the photoaffinity probe 5-azido-UDP-Glc. J Biol Chem. 1990 Mar 25;265(9):4782–4784. [PubMed] [Google Scholar]
  11. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  12. Meikle P. J., Ng K. F., Johnson E., Hoogenraad N. J., Stone B. A. The beta-glucan synthase from Lolium multiflorum. Detergent solubilization, purification using monoclonal antibodies, and photoaffinity labeling with a novel photoreactive pyrimidine analogue of uridine 5'-diphosphoglucose. J Biol Chem. 1991 Nov 25;266(33):22569–22581. [PubMed] [Google Scholar]
  13. Saxena I. M., Lin F. C., Brown R. M., Jr Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum. Plant Mol Biol. 1990 Nov;15(5):673–683. doi: 10.1007/BF00016118. [DOI] [PubMed] [Google Scholar]
  14. Saxena I. M., Lin F. C., Brown R. M., Jr Identification of a new gene in an operon for cellulose biosynthesis in Acetobacter xylinum. Plant Mol Biol. 1991 Jun;16(6):947–954. doi: 10.1007/BF00016067. [DOI] [PubMed] [Google Scholar]
  15. Updegraff D. M. Semimicro determination of cellulose in biological materials. Anal Biochem. 1969 Dec;32(3):420–424. doi: 10.1016/s0003-2697(69)80009-6. [DOI] [PubMed] [Google Scholar]
  16. Wong H. C., Fear A. L., Calhoon R. D., Eichinger G. H., Mayer R., Amikam D., Benziman M., Gelfand D. H., Meade J. H., Emerick A. W. Genetic organization of the cellulose synthase operon in Acetobacter xylinum. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8130–8134. doi: 10.1073/pnas.87.20.8130. [DOI] [PMC free article] [PubMed] [Google Scholar]

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