ANALYSIS OF LOW MOLECULAR WEIGHT THIOLS PRESENT IN A HUMAN PROSTATE CANCER CELL LINE (LNCAP) USING 4-DIMETHYLAMINOAZOBENZENE-4'-MALEIMIDE (DABMA) AS A LABELLING AGENT: APPARENT GLUTATHIONE CONTENT.

ANALYSIS OF LOW MOLECULAR WEIGHT THIOLS PRESENT IN A HUMAN PROSTATE CANCER CELL LINE (LNCAP) USING 4-DIMETHYLAMINOAZOBENZENE-4'-MALEIMIDE (DABMA) AS A LABELLING AGENT: APPARENT GLUTATHIONE CONTENT.

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Author(s)

Author(s): Michael Gronow

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694 1128 Volume 1 - Nov 2012

Abstract

Low molecular weight thiols play important roles in cell homeostasis, drug metabolism, cancer formation and progression. The major component in this fraction is commonly assumed to be the tripeptide glutathione but there have been reports of the presence of other unknown thiols in tumour cells. To investigate this further the composition of the non protein sulphydryl components of the acid soluble fraction (ASF) of LNCap cells (a lymph node human prostate cancer) have been investigated after labelling with 4-dimethylaminoazobenzene-4’-maleimide (DABMA) at pH 5.5.A preliminary isolation of the adducts formed was achieved using reverse phase chromatography on C-18 silica gel (ODS-AQ) giving three crude fractions. The first fraction contained the glutathione adduct and this could be separated from the other thiol adducts by flash chromatography on silica gel followed by ion exchange chromatography on a cellulose anion exchanger.The molar extinction coefficient of the glutathione derivative at the λ max of 455nm (pH5.5) was estimated by amino acid analysis to be 26,600 M-1 cm-1 (DABMA in methanol 32,000 M-1 cm-1). Using this value it was calculated that, in the total ASF, not more than 40% of the total measured thiol could be attributed to glutathione.Other thiol adducts were shown to be present in variable amounts; separation and analysis proved difficult but using HPLC and TLC techniques a number of unknown components were shown to be present. Amino acid analysis revealed that they did not contain cysteine or peptide material and UV/visible spectra ruled out the presence of nucleic acid derivatives. MS and NMR analyses indicated that unknown water soluble thiols are present which are probably lipid in nature. Further analysis of one isolated adduct indicated the presence of 5-mercapto-pentanol.

Keywords

Thiols, acid soluble fraction, glutathione, prostate cancer cells, analysis, maleimide adducts, silica gel chromatography, HPLC, TLC

References

  1. M.Pompella,G.Banhegyi, M.Wellman-Rosseau, (Eds.) Thiol metabolism and redox regulation of cellular functions IOS Press; NATO Science Series1 : Life and Behavourial Sciences 347, 2002
  2. T.A.Connors, Protection against the toxicity of alkylating agents by thiols: the mechanism of protection and its relevance to cancer chemotherapy a review
  3. Eur.J.Cancer 2 (1966) 293-305
  4. I.A.Cotgreave, R.G.Gerdes Recent trends in glutathione biochemistry – glutathione-protein interactions: a molecular link between oxidative stress and cell proliferation Biochem.Biophys.Res.Comms. 242 (1998) 1- 9
  5. A.Bindoli, J.M.Fukuto, H.J.Forman, Thiol chemistry in peroxidase catalysis and redox signaling, Antioxidants & redox signaling 10 (9) (2008) 1549-1564
  6. D.J.McConkey, Biochemical determinants of apoptosis and necrosis, Toxicol.Lett. 99 (1998) 157-168
  7. M.Valko, C.J.Rhodes, J.Monocol, M.Izakovic, M.Mazur, Free radicals, metals, and antioxidants in oxidative stress-induced cancer, Chem. Biol. Interact. 160 (2006) 1-40
  8. M. Gronow, Studies on the Non-Protein Thiols of a Human Prostatic Cancer
  9. Cell Line: Glutathione Content, Cancers, 2(2), (2010) 1092-1106
  10. E. Friedmann, D. H. Marrian, I. Simon-Reuss, Reaction of maleimides with thiols by mixing equimolar neutral solutions gives stable derivatives: Antimitotic action of maleimide and related substances, Brit. J. Pharmacol., 4, (1949) 105-108
  11. M.R.F.Ashworth, The determination of sulphur-containing groups, in vol.2 Analytical Methods for Thiol Groups, Academic Press, New York, 1976, pp.151-156
  12. J-Y Chang, R Knecht, D G Braun, A new method for the selective isolation of cysteine containing peptides – specific labelling of the thiol group with a hydrophobic chromophore. Biochem. J. 211, (1983) 163-171
  13. G.L. Ellman, Tissue Sulfhydryl Groups, Arch.Biochem. Biophys., 82 (1959) 70-77
  14. M.Friedman The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides and Proteins, , 292-293, Pergamon, New York 1973 pp.114-118
  15. J.D.Hayes, D.J.Pulford, The glutathione S transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance, Critical reviews in Biochemistry and Molecular Biology 30 (6) (1995) 445-599
  16. A.Meister, M.A.Anderson, Glutathione Ann Rev.Biochem., 52 (1983) 711
  17. L. Packer (Ed.) “Biothiols”, Methods in Enzymology; 251 Part A & 252 Part B Academic Press, 1995
  18. G.Calcutt, D. Doxey, The apparent glutathione content of some normal tissues and some animal tumours. Brit.J.Cancer 16 (1962) 562-569
  19. J.J.Eady, T.Orta, M.F.Dennis, M.R.L.Stratford, J.H. Peacock, (1995) GSH determination by the Tietze enzymatic recycling assay and its relationship to cellular radiation response, Brit.J.Cancer 72, 1089-1095
  20. M.Linder, R.Deschenes, (2007) Palmitoylation: policing protein stability and traffic. Nature 8, 74-84
  21. M.Fukasawa, O.Varlamov, W.S. Eng, T.H.Söllner, J.E.Rothman, (2004) Localization and activity of the SNARE Ykt6 determined by its regulatory domain and palmitoylation. Proc. Natl. Acad. Sci. U S A. 101(14), 4815-20

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