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. 1978 Feb;61(2):259–265. doi: 10.1104/pp.61.2.259

Purification and Comparative Properties of Microsomal and Glyoxysomal Malate Synthase from Castor Bean Endosperm 1,2

Linda Bowden 1, J Michael Lord 1
PMCID: PMC1091844  PMID: 16660272

Abstract

Sucrose density gradient centrifugation was employed to separate microsomes, mitochondria, and glyoxysomes from homogenates prepared from castor bean (Ricinus communis) endosperm. In the case of tissue removed from young seedlings, a significant proportion of the characteristic glyoxysomal enzyme malate synthase was recovered in the microsomal fraction. Malate synthase was purified from both isolated microsomes and glyoxysomes by a procedure involving osmotic shock, KCI solubilization, and sucrose density gradient centrifugation. All physical and catalytic properties examined were identical for the enzyme isolated from both organelle fractions. These properties include a molecular weight of 575,000, with a single subunit type of molecular weight 64,000, a pH optimum of 8, apparent Km for acetyl-CoA of 10 μm and glyoxylate of 2 mm. Microsomal and glyoxysomal malate synthases showed identical responses to various inhibitors. Adenine nucleotides were competitive inhibitors with respect to acetyl-CoA, and oxalate (Ki 110 μm) and glycolate (Ki 150 μm) were competitive inhibitors with respect to glyoxylate. Antiserum raised in rabbits against purified glyoxysomal malate synthase was used to confirm serological identity between the microsomal and glyoxysomal enzymes, and was capable of specifically precipitating 35S-labeled malate synthase from KCI extracts of both microsomes and glyoxysomes isolated from [35S]methionine-labeled endosperm tissue.

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Selected References

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

  1. Beevers H. Glyoxysomes of castor bean endosperm and their relation to gluconeogenesis. Ann N Y Acad Sci. 1969 Dec 19;168(2):313–324. doi: 10.1111/j.1749-6632.1969.tb43118.x. [DOI] [PubMed] [Google Scholar]
  2. Bieglmayer C., Graf J., Ruis H. Membranes of glyoxysomes from castor-bean endosperm. Enzymes bound to purified-membrane preparations. Eur J Biochem. 1973 Sep 3;37(3):553–562. doi: 10.1111/j.1432-1033.1973.tb03018.x. [DOI] [PubMed] [Google Scholar]
  3. Bowden L., Lord J. M. Development of phospholipid synthesizing enzymes in castor bean endosperm. FEBS Lett. 1975 Jan 1;49(3):369–371. doi: 10.1016/0014-5793(75)80787-3. [DOI] [PubMed] [Google Scholar]
  4. Bowden L., Lord J. M. Similarities in the polypeptide composition of glyoxysomal and endoplasmic-reticulum membranes from castor-bean endosperm. Biochem J. 1976 Feb 15;154(2):491–499. doi: 10.1042/bj1540491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bowden L., Lord J. M. The cellular origin of glyoxysomal proteins in germinating castor-bean endosperm. Biochem J. 1976 Feb 15;154(2):501–506. doi: 10.1042/bj1540501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Breidenbach R. W. Characterization of some glyoxysomal proteins. Ann N Y Acad Sci. 1969 Dec 19;168(2):342–347. doi: 10.1111/j.1749-6632.1969.tb43120.x. [DOI] [PubMed] [Google Scholar]
  7. Brown R. H., Lord J. M., Merrett M. J. Fractionation of the proteins of plant microbodies. Biochem J. 1974 Dec;144(3):559–566. doi: 10.1042/bj1440559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cook J. R. Properties of partially purified malate synthase for Euglena gracilis. J Protozool. 1970 May;17(2):232–235. doi: 10.1111/j.1550-7408.1970.tb02362.x. [DOI] [PubMed] [Google Scholar]
  9. DIXON G. H., KORNBERG H. L., LUND P. Purification and properties of malate synthetase. Biochim Biophys Acta. 1960 Jul 1;41:217–233. doi: 10.1016/0006-3002(60)90004-4. [DOI] [PubMed] [Google Scholar]
  10. Donaldson R. P. Membrane lipid metabolism in germinating castor bean endosperm. Plant Physiol. 1976 Apr;57(4):510–515. doi: 10.1104/pp.57.4.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Donaldson R. P., Tolbert N. E., Schnarrenberger C. A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue. Arch Biochem Biophys. 1972 Sep;152(1):199–215. doi: 10.1016/0003-9861(72)90208-1. [DOI] [PubMed] [Google Scholar]
  12. Gonzalez E., Beevers H. Role of the endoplasmic reticulum in glyoxysome formation in castor bean endosperm. Plant Physiol. 1976 Mar;57(3):406–409. doi: 10.1104/pp.57.3.406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Huang A. H., Beevers H. Localization of enzymes within microbodies. J Cell Biol. 1973 Aug;58(2):379–389. doi: 10.1083/jcb.58.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kagawa T., Lord J. M., Beevers H. The origin and turnover of organelle membranes in castor bean endosperm. Plant Physiol. 1973 Jan;51(1):61–65. doi: 10.1104/pp.51.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. Lazarow P. B., de Duve C. The synthesis and turnover of rat liver peroxisomes. V. Intracellular pathway of catalase synthesis. J Cell Biol. 1973 Nov;59(2 Pt 1):507–524. doi: 10.1083/jcb.59.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lord J. M., Kagawa T., Moore T. S., Beevers H. Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm. J Cell Biol. 1973 Jun;57(3):659–667. doi: 10.1083/jcb.57.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. MARTIN R. G., AMES B. N. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J Biol Chem. 1961 May;236:1372–1379. [PubMed] [Google Scholar]
  19. Moore T. S., Lord J. M., Kagawa T., Beevers H. Enzymes of phospholipid metabolism in the endoplasmic reticulum of castor bean endosperm. Plant Physiol. 1973 Jul;52(1):50–53. doi: 10.1104/pp.52.1.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Seghal P. P., Tanis R. J., Naylor A. W. A low molecular-weight form of urease. Biochem Biophys Res Commun. 1965 Sep 8;20(5):550–554. doi: 10.1016/0006-291x(65)90433-x. [DOI] [PubMed] [Google Scholar]
  21. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  22. YAMAMOTO Y., BEEVERS H. Purification and properties of malate synthetase from castor beans. Biochim Biophys Acta. 1961 Mar 18;48:20–25. doi: 10.1016/0006-3002(61)90510-8. [DOI] [PubMed] [Google Scholar]

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