Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Sep 15;358(Pt 3):773–781. doi: 10.1042/0264-6021:3580773

Lipolytic activity of ricin from Ricinus sanguineus and Ricinus communis on neutral lipids.

S Lombard 1, M E Helmy 1, G Piéroni 1
PMCID: PMC1222111  PMID: 11535138

Abstract

The present study was carried out with a view of determining ricin lipolytic activity on neutral lipids in emulsion and in a membrane-like model. Using 2,3-dimercapto-1-propanol tributyrate (BAL-TC(4)) as substrate, the lipolytic activity of ricin was found to be proportional to ricin and substrate concentrations, with an apparent K(m) (K(m,app)) of 2.4 mM, a k(cat) of 200 min(-1) and a specific activity of 1.0 unit/mg of protein. This work was extended to p-nitrophenyl (pNP) fatty acid esters containing two to twelve carbon atoms. Maximum lipolytic activity was registered on pNP decanoate (pNPC(10)), with a K(m,app) of 3.5 mM, a k(cat) of 173 min(-1) and a specific activity of 3.5 units/mg of protein. Ricin lipolytic activity is pH and galactose dependent, with a maximum at pH 7.0 in the presence of 0.2 M galactose. Using the monolayer technique with dicaprin as substrate, ricin showed a lipolytic activity proportional to the ricin concentration at 20 mN/m, which is dependent on the surface pressure of the lipid monolayer and is detectable up to 30 mN/m, a surface pressure that is of the same order of magnitude as that of natural cell membranes. The methods based on pNPC(10) and BAL-TC(4) hydrolysis are simple and reproducible; thus they can be used for routine studies of ricin lipolytic activity. Ricin from Ricinus communis and R. sanguineus were treated with diethyl p-nitrophenylphosphate, an irreversible serine esterase inhibitor, and their lipolytic activities on BAL-TC(4) and pNPC(10), and cytotoxic activity, were concurrently recorded. A reduction in lipolytic activity was accompanied by a decrease in cytotoxicity on Caco2 cells. These data support the idea that the lipolytic activity associated with ricin is relevant to a lipase whose activity is pH and galactose dependent, sensitive to diethyl p-nitrophenylphosphate, and that a lipolytic step may be involved in the process of cell poisoning by ricin. Both colorimetric tests used in this study are sensitive enough to be helpful in the detection of possible lipolytic activities associated with other cytotoxins or lectins.

Full Text

The Full Text of this article is available as a PDF (181.1 KB).

Selected References

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

  1. Agapov I. I., Tonevitsky A. G., Shamshiev A. T., Pohl E., Pohl P., Palmer R. A., Kirpichnikov M. P. The role of structural domains in RIP II toxin model membrane binding. FEBS Lett. 1997 Jan 27;402(1):91–93. doi: 10.1016/s0014-5793(96)01452-4. [DOI] [PubMed] [Google Scholar]
  2. Araki T., Funatsu G. The complete amino acid sequence of the B-chain of ricin E isolated from small-grain castor bean seeds. Ricin E is a gene recombination product of ricin D and Ricinus communis agglutinin. Biochim Biophys Acta. 1987 Jan 30;911(2):191–200. doi: 10.1016/0167-4838(87)90008-2. [DOI] [PubMed] [Google Scholar]
  3. Argent R. H., Roberts L. M., Lord J. M. Proteolytic processing of ricin A chain is not required for cytotoxicity. Biochem Biophys Res Commun. 1997 Dec 29;241(3):617–621. doi: 10.1006/bbrc.1997.7796. [DOI] [PubMed] [Google Scholar]
  4. Arias F. J., Rojo M. A., Ferreras J. M., Iglesias R., Muñoz R., Soriano F., Méndez E., Barbieri L., Girbés T. Isolation and characterization of two new N-glycosidase type-1 ribosome-inactivating proteins, unrelated in amino-acid sequence, from Petrocoptis species. Planta. 1994;194(4):487–491. doi: 10.1007/BF00714460. [DOI] [PubMed] [Google Scholar]
  5. Barbieri L., Battelli M. G., Stirpe F. Ribosome-inactivating proteins from plants. Biochim Biophys Acta. 1993 Dec 21;1154(3-4):237–282. doi: 10.1016/0304-4157(93)90002-6. [DOI] [PubMed] [Google Scholar]
  6. Boraston A. B., Tomme P., Amandoron E. A., Kilburn D. G. A novel mechanism of xylan binding by a lectin-like module from Streptomyces lividans xylanase 10A. Biochem J. 2000 Sep 15;350(Pt 3):933–941. [PMC free article] [PubMed] [Google Scholar]
  7. Cory A. H., Owen T. C., Barltrop J. A., Cory J. G. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun. 1991 Jul;3(7):207–212. doi: 10.3727/095535491820873191. [DOI] [PubMed] [Google Scholar]
  8. Demel R. A., Geurts van Kessel W. S., Zwaal R. F., Roelofsen B., van Deenen L. L. Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers. Biochim Biophys Acta. 1975 Sep 16;406(1):97–107. doi: 10.1016/0005-2736(75)90045-0. [DOI] [PubMed] [Google Scholar]
  9. Ersson B., Aspberg K., Porath J. The phytohemagglutinin from sunn hemp seeds (Crotalaria juncea). Purification by biospecific affinity chromatography. Biochim Biophys Acta. 1973 Jun 15;310(2):446–452. doi: 10.1016/0005-2795(73)90128-1. [DOI] [PubMed] [Google Scholar]
  10. Frankel A. E., Burbage C., Fu T., Tagge E., Chandler J., Willingham M. C. Ricin toxin contains at least three galactose-binding sites located in B chain subdomains 1 alpha, 1 beta, and 2 gamma. Biochemistry. 1996 Nov 26;35(47):14749–14756. doi: 10.1021/bi960798s. [DOI] [PubMed] [Google Scholar]
  11. Fu T., Burbage C., Tagge E. P., Brothers T., Willingham M. C., Frankel A. E. Ricin toxin contains three lectin sites which contribute to its in vivo toxicity. Int J Immunopharmacol. 1996 Dec;18(12):685–692. doi: 10.1016/s0192-0561(97)85550-6. [DOI] [PubMed] [Google Scholar]
  12. Furukawa I., Kurooka S., Arisue K., Kohda K., Hayashi C. Assays of serum lipase by the "BALB-DTNB method" mechanized for use with discrete and continuous-flow analyzers. Clin Chem. 1982 Jan;28(1):110–113. [PubMed] [Google Scholar]
  13. Girbés T., Ferreras J. M., Iglesias R., Citores L., De Torre C., Carbajales M. L., Jiménez P., De Benito F. M., Muñoz R. Recent advances in the uses and applications of ribosome-inactivating proteins from plants. Cell Mol Biol (Noisy-le-grand) 1996 Jun;42(4):461–471. [PubMed] [Google Scholar]
  14. Halling K. C., Halling A. C., Murray E. E., Ladin B. F., Houston L. L., Weaver R. F. Genomic cloning and characterization of a ricin gene from Ricinus communis. Nucleic Acids Res. 1985 Nov 25;13(22):8019–8033. doi: 10.1093/nar/13.22.8019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Helmy M., Lombard S., Piéroni G. Ricin RCA60: evidence of its phospholipase activity. Biochem Biophys Res Commun. 1999 May 10;258(2):252–255. doi: 10.1006/bbrc.1999.0618. [DOI] [PubMed] [Google Scholar]
  16. Hirabayashi J., Ubukata T., Kasai K. Purification and molecular characterization of a novel 16-kDa galectin from the nematode Caenorhabditis elegans. J Biol Chem. 1996 Feb 2;271(5):2497–2505. doi: 10.1074/jbc.271.5.2497. [DOI] [PubMed] [Google Scholar]
  17. Johnston D. S., Chapman D. The properties of brain galactocerebroside monolayers. Biochim Biophys Acta. 1988 Jan 13;937(1):10–22. doi: 10.1016/0005-2736(88)90222-2. [DOI] [PubMed] [Google Scholar]
  18. Lamb F. I., Roberts L. M., Lord J. M. Nucleotide sequence of cloned cDNA coding for preproricin. Eur J Biochem. 1985 Apr 15;148(2):265–270. doi: 10.1111/j.1432-1033.1985.tb08834.x. [DOI] [PubMed] [Google Scholar]
  19. Liu C. L., Tsai C. C., Lin S. C., Wang L. I., Hsu C. I., Hwang M. J., Lin J. Y. Primary structure and function analysis of the Abrus precatorius agglutinin A chain by site-directed mutagenesis. Pro(199) Of amphiphilic alpha-helix H impairs protein synthesis inhibitory activity. J Biol Chem. 2000 Jan 21;275(3):1897–1901. doi: 10.1074/jbc.275.3.1897. [DOI] [PubMed] [Google Scholar]
  20. Lord J. M., Roberts L. M., Robertus J. D. Ricin: structure, mode of action, and some current applications. FASEB J. 1994 Feb;8(2):201–208. [PubMed] [Google Scholar]
  21. Macbeth M. R., Wool I. G. Characterization of in vitro and in vivo mutations in non-conserved nucleotides in the ribosomal RNA recognition domain for the ribotoxins ricin and sarcin and the translation elongation factors. J Mol Biol. 1999 Jan 15;285(2):567–580. doi: 10.1006/jmbi.1998.2337. [DOI] [PubMed] [Google Scholar]
  22. Maylié M. F., Charles M., Desnuelle P. Action of organophosphates and sulfonyl halides on porcine pancreatic lipase. Biochim Biophys Acta. 1972 Jul 13;276(1):162–175. doi: 10.1016/0005-2744(72)90017-4. [DOI] [PubMed] [Google Scholar]
  23. Moulin A., Teissère M., Bernard C., Piéroni G. Lipases of the euphorbiaceae family: purification of a lipase from Euphorbia characias latex and structure-function relationships with the B chain of ricin. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11328–11332. doi: 10.1073/pnas.91.24.11328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nicolson G. L., Blaustein J. The interaction of Ricinus communis agglutinin with normal and tumor cell surfaces. Biochim Biophys Acta. 1972 May 9;266(2):543–547. doi: 10.1016/0005-2736(72)90109-5. [DOI] [PubMed] [Google Scholar]
  25. Pieroni G., Verger R. Hydrolysis of mixed monomolecular films of triglyceride/lecithin by pancreatic lipase. J Biol Chem. 1979 Oct 25;254(20):10090–10094. [PubMed] [Google Scholar]
  26. Pohl P., Antonenko Y. N., Evtodienko V. Y., Pohl E. E., Saparov S. M., Agapov I. I., Tonevitsky A. G. Membrane fusion mediated by ricin and viscumin. Biochim Biophys Acta. 1998 Apr 22;1371(1):11–16. doi: 10.1016/s0005-2736(98)00024-8. [DOI] [PubMed] [Google Scholar]
  27. Roberts L. M., Lamb F. I., Pappin D. J., Lord J. M. The primary sequence of Ricinus communis agglutinin. Comparison with ricin. J Biol Chem. 1985 Dec 15;260(29):15682–15686. [PubMed] [Google Scholar]
  28. Roy D. C., Griffin J. D., Belvin M., Blättler W. A., Lambert J. M., Ritz J. Anti-MY9-blocked-ricin: an immunotoxin for selective targeting of acute myeloid leukemia cells. Blood. 1991 Jun 1;77(11):2404–2412. [PubMed] [Google Scholar]
  29. Sandvig K., Olsnes S. Entry of the toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells. I. Requirement for calcium. J Biol Chem. 1982 Jul 10;257(13):7495–7503. [PubMed] [Google Scholar]
  30. Sandvig K., Olsnes S. Entry of the toxic proteins abrin, modeccin, ricin, and diphtheria toxin into cells. II. Effect of pH, metabolic inhibitors, and ionophores and evidence for toxin penetration from endocytotic vesicles. J Biol Chem. 1982 Jul 10;257(13):7504–7513. [PubMed] [Google Scholar]
  31. Sandvig K., van Deurs B. Endocytosis and intracellular transport of ricin: recent discoveries. FEBS Lett. 1999 Jun 4;452(1-2):67–70. doi: 10.1016/s0014-5793(99)00529-3. [DOI] [PubMed] [Google Scholar]
  32. Simpson J. C., Roberts L. M., Römisch K., Davey J., Wolf D. H., Lord J. M. Ricin A chain utilises the endoplasmic reticulum-associated protein degradation pathway to enter the cytosol of yeast. FEBS Lett. 1999 Oct 1;459(1):80–84. doi: 10.1016/s0014-5793(99)01222-3. [DOI] [PubMed] [Google Scholar]
  33. Steeves R. M., Denton M. E., Barnard F. C., Henry A., Lambert J. M. Identification of three oligosaccharide binding sites in ricin. Biochemistry. 1999 Sep 7;38(36):11677–11685. doi: 10.1021/bi990493o. [DOI] [PubMed] [Google Scholar]
  34. Van Damme E. J., Barre A., Rougé P., Van Leuven F., Peumans W. J. The NeuAc(alpha-2,6)-Gal/GalNAc-binding lectin from elderberry (Sambucus nigra) bark, a type-2 ribosome-inactivating protein with an unusual specificity and structure. Eur J Biochem. 1996 Jan 15;235(1-2):128–137. doi: 10.1111/j.1432-1033.1996.00128.x. [DOI] [PubMed] [Google Scholar]
  35. Van Damme E. J., Hao Q., Charels D., Barre A., Rougé P., Van Leuven F., Peumans W. J. Characterization and molecular cloning of two different type 2 ribosome-inactivating proteins from the monocotyledonous plant Polygonatum multiflorum. Eur J Biochem. 2000 May;267(9):2746–2759. doi: 10.1046/j.1432-1327.2000.01295.x. [DOI] [PubMed] [Google Scholar]
  36. Verger R., Mieras M. C., de Haas G. H. Action of phospholipase A at interfaces. J Biol Chem. 1973 Jun 10;248(11):4023–4034. [PubMed] [Google Scholar]
  37. Verger R., Rietsch J., Van Dam-Mieras M. C., de Haas G. H. Comparative studies of lipase and phospholipase A2 acting on substrate monolayers. J Biol Chem. 1976 May 25;251(10):3128–3133. [PubMed] [Google Scholar]
  38. Wawrzynczak E. J., Drake A. F., Watson G. J., Thorpe P. E., Vitetta E. S. Ricin B chain-containing immunotoxins prepared with heat-denatured B chain lacking galactose-binding ability potentiate the cytotoxicity of a cell-reactive ricin A chain immunotoxin. Biochim Biophys Acta. 1988 Aug 19;971(1):55–62. doi: 10.1016/0167-4889(88)90161-9. [DOI] [PubMed] [Google Scholar]
  39. Wood K. A., Lord J. M., Wawrzynczak E. J., Piatak M. Preproabrin: genomic cloning, characterisation and the expression of the A-chain in Escherichia coli. Eur J Biochem. 1991 Jun 15;198(3):723–732. doi: 10.1111/j.1432-1033.1991.tb16072.x. [DOI] [PubMed] [Google Scholar]
  40. Yamamoto K., Maruyama I. N., Osawa T. Cyborg lectins: novel leguminous lectins with unique specificities. J Biochem. 2000 Jan;127(1):137–142. doi: 10.1093/oxfordjournals.jbchem.a022575. [DOI] [PubMed] [Google Scholar]
  41. Youle R. J., Neville D. M., Jr Kinetics of protein synthesis inactivation by ricin-anti-Thy 1.1 monoclonal antibody hybrids. Role of the ricin B subunit demonstrated by reconstitution. J Biol Chem. 1982 Feb 25;257(4):1598–1601. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES