Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1987 Oct;85(2):516–522. doi: 10.1104/pp.85.2.516

CHAPS Solubilization and Functional Reconstitution of β-Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue 1

Margaret E Sloan 1,2, Panayotis Rodis 1,3, Bruce P Wasserman 1
PMCID: PMC1054287  PMID: 16665729

Abstract

A method for the solubilization and reconstitution of red beet (1,3)-β-d-glucan synthase with the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) was developed. Glucan synthase was effectively solubilized from microsomal or plasma membranes by 0.6% CHAPS in the presence of EGTA and EDTA. Chelators were found essential for effective solubilization and divalent cations inhibitory. A preextraction of membranes with 0.3% CHAPS and 5 millimolar Mg2+ prior to the solubilization step was found to remove protein contaminants and increase the specific activity of the solubilized enzyme. Conditions for recovering activity from Sepharose 4B gel filtration columns were defined. Addition of phospholipids and low levels of CHAPS in column elution buffers resulted in complete functional reconstitution with 100% recovery of added activity. Specific activities were increased 20- to 22-fold over microsomes. Active vesicles were recovered by centrifugation. These results provide independent and direct confirmation of the enzyme's requirement for a phospholipid environment.

Full text

PDF
516

Selected References

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

  1. Aloni Y., Cohen R., Benziman M., Delmer D. Solubilization of the UDP-glucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase (cellulose synthase) from Acetobacter xylinum. A comparison of regulatory properties with those of the membrane-bound form of the enzyme. J Biol Chem. 1983 Apr 10;258(7):4419–4423. [PubMed] [Google Scholar]
  2. Briskin D. P., Poole R. J. Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet. Plant Physiol. 1983 Feb;71(2):350–355. doi: 10.1104/pp.71.2.350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cook N. J., Zeilinger C., Koch K. W., Kaupp U. B. Solubilization and functional reconstitution of the cGMP-dependent cation channel from bovine rod outer segments. J Biol Chem. 1986 Dec 25;261(36):17033–17039. [PubMed] [Google Scholar]
  4. Delmer D. P. Biosynthesis of cellulose. Adv Carbohydr Chem Biochem. 1983;41:105–153. doi: 10.1016/s0065-2318(08)60057-8. [DOI] [PubMed] [Google Scholar]
  5. Delmer D. P., Cooper G., Alexander D., Cooper J., Hayashi T., Nitsche C., Thelen M. New approaches to the study of cellulose biosynthesis. J Cell Sci Suppl. 1985;2:33–50. doi: 10.1242/jcs.1985.supplement_2.3. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Gorissen H., Aerts G., Ilien B., Laduron P. Solubilization of muscarinic acetylcholine receptors from mammalian brain: an analytical approach. Anal Biochem. 1981 Feb;111(1):33–34. doi: 10.1016/0003-2697(81)90224-4. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Hjelmeland L. M. A nondenaturing zwitterionic detergent for membrane biochemistry: design and synthesis. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6368–6370. doi: 10.1073/pnas.77.11.6368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hjelmeland L. M., Chrambach A. Solubilization of functional membrane proteins. Methods Enzymol. 1984;104:305–318. doi: 10.1016/s0076-6879(84)04097-0. [DOI] [PubMed] [Google Scholar]
  12. Langley K. E., Kennedy E. P. Partial purification and properties of CTP:phosphatidic acid cytidylyltransferase from membranes of Escherichia coli. J Bacteriol. 1978 Oct;136(1):85–95. doi: 10.1128/jb.136.1.85-95.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Manolson M. F., Rea P. A., Poole R. J. Identification of 3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate- and N,N'-dicyclohexylcarbodiimide-binding subunits of a higher plant H+-translocating tonoplast ATPase. J Biol Chem. 1985 Oct 5;260(22):12273–12279. [PubMed] [Google Scholar]
  14. Morrow D. L., Lucas W. J. (1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization. Plant Physiol. 1986 May;81(1):171–176. doi: 10.1104/pp.81.1.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Perlin D. S., Kasamo K., Brooker R. J., Slayman C. W. Electrogenic H+ translocation by the plasma membrane ATPase of Neurospora. Studies on plasma membrane vesicles and reconstituted enzyme. J Biol Chem. 1984 Jun 25;259(12):7884–7892. [PubMed] [Google Scholar]
  16. 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]
  17. Rivnay B., Metzger H. Reconstitution of the receptor for immunoglobulin E into liposomes. Conditions for incorporation of the receptor into vesicles. J Biol Chem. 1982 Nov 10;257(21):12800–12808. [PubMed] [Google Scholar]
  18. Smith M. M., Stone B. A. Beta-glucan synthesis by cell-free extracts from Lolium multiflorum endosperm. Biochim Biophys Acta. 1973 Jun 20;313(1):72–94. doi: 10.1016/0304-4165(73)90189-x. [DOI] [PubMed] [Google Scholar]
  19. Thelen M. P., Delmer D. P. Gel-Electrophoretic Separation, Detection, and Characterization of Plant and Bacterial UDP-Glucose Glucosyltransferases. Plant Physiol. 1986 Jul;81(3):913–918. doi: 10.1104/pp.81.3.913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wagner S. L., Dean W. L., Gray R. D. Effect of a zwitterionic detergent on the state of aggregation and catalytic activity of cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase. J Biol Chem. 1984 Feb 25;259(4):2390–2395. [PubMed] [Google Scholar]
  21. Wasserman B. P., McCarthy K. J. Regulation of Plasma Membrane beta-Glucan Synthase from Red Beet Root by Phospholipids : Reactivation of Triton X-100 Extracted Glucan Synthase by Phospholipids. Plant Physiol. 1986 Oct;82(2):396–400. doi: 10.1104/pp.82.2.396. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES