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. 1989 May;90(1):101–108. doi: 10.1104/pp.90.1.101

UDP-Glucose: (1,3)-β-Glucan Synthase from Daucus carota L. 1

Characterization, Photoaffinity Labeling, and Solubilization

Stephen G Lawson 1,2, Theresa L Mason 1,2, Robert D Sabin 1,2, Margaret E Sloan 1,2,2, Richard R Drake 1,2, Boyd E Haley 1,2, Bruce P Wasserman 1,2
PMCID: PMC1061683  PMID: 16666718

Abstract

The membrane-bound UDP-glucose-β-(1,3)-glucan synthase from Daucus carota L. was characterized and a solubilization procedure was developed. The enzyme exhibited maximal activity in the presence of 0.75 millimolar Ca2+, 0.5 millimolar EGTA, and 5 millimolar cellobiose at pH 7.5 and 30°C at 1 millimolar UDPG. Reaction products were confirmed to be (1,3)-linked glucan. Polypeptides of 150, 57, and 43 kilodaltons were labeled with the photoactivatible affinity label 5-azido-uridine 5′-β-[32P] diphosphateglucose. Labeling of the 150 and 57 kilodalton polypeptides was completely protected against by 1 millimolar non-radioactive UDPG suggesting that one or both of these polypeptides may represent the UDPG binding subunit of glucan synthase. Carrot glucan synthase was solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in the absence of divalent cations and chelators; however, the percentage of enzyme which could be solubilized showed variability with membrane source. With microsomal membranes, up to 80% of the enzyme was released with 0.7% CHAPS. Solubilized enzyme was stable for at least 9 hours at 4°C. When more highly purified membrane fractions were isolated from sucrose step gradients a slightly different picture emerged. Activity from the 20/30% interface (Golgi and tonoplast enriched) was readily solubilized and expressed. Activity from the 30/40% interface (plasma membrane enriched) was also solubilized; however, it was necessary to add heat inactivated microsomes to assay mixtures for full activity to be expressed. A requirement for endogenous activators is suggested.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boss W. F., Mott R. L. Effects of divalent cations and polyethylene glycol on the membrane fluidity of protoplast. Plant Physiol. 1980 Nov;66(5):835–837. doi: 10.1104/pp.66.5.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boss W. F., Ruesink A. W. Isolation and Characterization of Concanavalin A-labeled Plasma Membranes of Carrot Protoplasts. Plant Physiol. 1979 Dec;64(6):1005–1011. doi: 10.1104/pp.64.6.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Callaghan T., Ross P., Weinberger-Ohana P., Garden G., Benziman M. beta-Glucoside Activators of Mung Bean UDP-Glucose: beta-Glucan Synthase : I. Identification of an Endogenous beta-Linked Glucolipid Activator. Plant Physiol. 1988 Apr;86(4):1099–1103. doi: 10.1104/pp.86.4.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Della-Penna D., Christoffersen R. E., Bennett A. B. Biotinylated proteins as molecular weight standards on Western blots. Anal Biochem. 1986 Feb 1;152(2):329–332. doi: 10.1016/0003-2697(86)90417-3. [DOI] [PubMed] [Google Scholar]
  5. Eiberger L. L., Wasserman B. P. Partial Purification of Digitonin-Solubilized beta-Glucan Synthase from Red Beet Root. Plant Physiol. 1987 Apr;83(4):982–987. doi: 10.1104/pp.83.4.982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Evans R. K., Haley B. E. Synthesis and biological properties of 5-azido-2'-deoxyuridine 5'-triphosphate, a photoactive nucleotide suitable for making light-sensitive DNA. Biochemistry. 1987 Jan 13;26(1):269–276. doi: 10.1021/bi00375a037. [DOI] [PubMed] [Google Scholar]
  7. Girard V., Maclachlan G. Modulation of Pea Membrane beta-Glucan Synthase Activity by Calcium, Polycation, Endogenous Protease, and Protease Inhibitor. Plant Physiol. 1987 Sep;85(1):131–136. doi: 10.1104/pp.85.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Harris P. J., Henry R. J., Blakeney A. B., Stone B. A. An improved procedure for the methylation analysis of oligosaccharides and polysaccharides. Carbohydr Res. 1984 Apr 2;127(1):59–73. doi: 10.1016/0008-6215(84)85106-x. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Henry R. J., Schibeci A., Stone B. A. Localization of beta-glucan synthases on the membranes of cultured Lolium multiflorum (ryegrass) endosperm cells. Biochem J. 1983 Mar 1;209(3):627–633. doi: 10.1042/bj2090627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Henry R. J., Stone B. A. Factors Influencing beta-Glucan Synthesis by Particulate Enzymes from Suspension-Cultured Lolium multiflorum Endosperm Cells. Plant Physiol. 1982 Mar;69(3):632–636. doi: 10.1104/pp.69.3.632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Henry R. J., Stone B. A. Solubilization of beta-glucan synthases from the membranes of cultured ryegrass endosperm cells. Biochem J. 1982 Jun 1;203(3):629–636. doi: 10.1042/bj2030629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Owens J. R., Haley B. E. A study of adenosine 3'-5' cyclic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3'-5' cyclic monophosphate, a photoaffinity probe. J Supramol Struct. 1976;5(1):91–102. doi: 10.1002/jss.400050110. [DOI] [PubMed] [Google Scholar]
  15. Parente J. P., Cardon P., Leroy Y., Montreuil J., Fournet B., Ricart G. A convenient method for methylation of glycoprotein glycans in small amounts by using lithium methylsulfinyl carbanion. Carbohydr Res. 1985 Aug 15;141(1):41–47. doi: 10.1016/s0008-6215(00)90753-5. [DOI] [PubMed] [Google Scholar]
  16. Porzio M. A., Pearson A. M. Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochim Biophys Acta. 1977 Jan 25;490(1):27–34. doi: 10.1016/0005-2795(77)90102-7. [DOI] [PubMed] [Google Scholar]
  17. Press G. A., Glazer H. S., Wasserman T. H., Aronberg D. J., Lee J. K., Sagel S. S. Thoracic wall involvement by Hodgkin disease and non-Hodgkin lymphoma: CT evaluation. Radiology. 1985 Oct;157(1):195–198. doi: 10.1148/radiology.157.1.4034966. [DOI] [PubMed] [Google Scholar]
  18. Read S. M., Delmer D. P. Inhibition of Mung Bean UDP-Glucose: (1-->3)-beta-Glucan Synthase by UDP-Pyridoxal: Evidence for an Active-Site Amino Group. Plant Physiol. 1987 Dec;85(4):1008–1015. doi: 10.1104/pp.85.4.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sloan M. E., Rodis P., Wasserman B. P. CHAPS Solubilization and Functional Reconstitution of beta-Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue. Plant Physiol. 1987 Oct;85(2):516–522. doi: 10.1104/pp.85.2.516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Villemez C. L., Franz G., Hassid W. Z. Biosynthesis of Alkali Insoluble Polysaccharide from UDP-d-Glucose with Particulate Enzyme Preparations from Phaseolus aureus. Plant Physiol. 1967 Sep;42(9):1219–1223. doi: 10.1104/pp.42.9.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]

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