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. 1976 Mar 1;68(3):440–450. doi: 10.1083/jcb.68.3.440

Effects of caffeine and other methylxanthines on the development and metabolism of sea urchin eggs. Involvement of NADP and glutathione

PMCID: PMC2109668  PMID: 16015

Abstract

Methylxanthines (MX) inhibit cell division in sea urchin and clam eggs. This inhibitory effect is not mediated via cAMP. MX also inhibit respiration in marine eggs, at concentrations which inhibit cleavage. Studies showed that no changes occurred in ATP and ADP levels in the presence of inhibitory concentrations of MX, indicating an extra- mitochondrial site of action for the drug. Subsequent studies revealed decreased levels of NADP+ and NADPH, when eggs were incubated with inhibitory concentrations of MX, but no change in levels of NAD+ and NADH. MX did not affect the pentose phosphate shunt pathway and did not have any effect on the enzyme NAD+ -kinase. Further studies showed a marked inhibitory effect on the glutathione reductase activity of MX- treated eggs. Reduced glutathione (GSH) could reverse the cleavage inhibitory effect of MX. Moreover, diamide, a thiol-oxidizing agent specific for GSH in living cells, caused inhibition of cell division in sea urchin eggs. Diamide added to eggs containing mitotic apparatus (MA) could prevent cleavage by causing a dissolution of the formed MA. Both MX and diamide inhibit a Ca2+-activated ATPase in whole eggs. The enzyme can be reactivated by sulfhydryl reducing agents added in the assay mixture. In addition, diamide causes an inhibition of microtubule polymerization, reversible with dithioerythritol. All experimental evidence so far suggests that inhibition of mitosis in sea urchin eggs by MX is mediated by perturbations of the in vivo thiol-disulfide status of target systems, with a primary effect on glutathione levels.

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Selected References

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  1. Amos W. B., Routledge L. M., Yew F. F. Calcium-binding proteins in a vorticellid contractile organelle. J Cell Sci. 1975 Oct;19(1):203–213. doi: 10.1242/jcs.19.1.203. [DOI] [PubMed] [Google Scholar]
  2. Blomquist C. H. Partial purification and characterization of nicotinamide adenine dinucleotide kinase from sea urchin eggs. J Biol Chem. 1973 Oct 25;248(20):7044–7048. [PubMed] [Google Scholar]
  3. CLOWES G. H. A., KELTCH A. K., STRITTMATTER C. F., WALTERS C. P. Action of nitro- and halophenols upon oxygen consumption and phosphorylation by a cell-free particulate system from arbacia eggs. J Gen Physiol. 1950 May 20;33(5):555–561. doi: 10.1085/jgp.33.5.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dikstein S. Stimulability, adenosine triphosphatases and their control by cellular redox processes. Naturwissenschaften. 1971 Sep;58(9):439–443. doi: 10.1007/BF00624617. [DOI] [PubMed] [Google Scholar]
  5. Eipper B. A. Properties of rat brain tubulin. J Biol Chem. 1974 Mar 10;249(5):1407–1416. [PubMed] [Google Scholar]
  6. Fahey R. C., Brody S., Mikolajczyk S. D. Changes in the glutathione thiol-disulfide status of Neurospora crassa conidia during germination and aging. J Bacteriol. 1975 Jan;121(1):144–151. doi: 10.1128/jb.121.1.144-151.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fredborg A., Lindahl P. E. Decreased glutathione-reductase activity in the mitotic Ehrlich ascites-tumour cell. Exp Cell Res. 1970 Mar;59(3):452–456. doi: 10.1016/0014-4827(70)90653-1. [DOI] [PubMed] [Google Scholar]
  8. Habig W. H., Pabst M. J., Fleischner G., Gatmaitan Z., Arias I. M., Jakoby W. B. The identity of glutathione S-transferase B with ligandin, a major binding protein of liver. Proc Natl Acad Sci U S A. 1974 Oct;71(10):3879–3882. doi: 10.1073/pnas.71.10.3879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harris J. W., Patt H. M. Non-protein sulfhydryl content and cell-cycle dynamics of Ehrlich ascites tumor. Exp Cell Res. 1969 Jul;56(1):134–141. doi: 10.1016/0014-4827(69)90406-6. [DOI] [PubMed] [Google Scholar]
  10. Hosoda S., Nakamura W. Role of glutathione in regulation of hexose monophosphate pathway in Ehrlich ascites tumor cells. Biochim Biophys Acta. 1970 Oct 27;222(1):53–64. doi: 10.1016/0304-4165(70)90350-8. [DOI] [PubMed] [Google Scholar]
  11. ITZHAKI R. F., GILL D. M. A MICRO-BIURET METHOD FOR ESTIMATING PROTEINS. Anal Biochem. 1964 Dec;9:401–410. doi: 10.1016/0003-2697(64)90200-3. [DOI] [PubMed] [Google Scholar]
  12. Ii I., Sakai H. Glutathione reductase in the sea urchin egg. I. Purification and general properties. Biochim Biophys Acta. 1974 May 20;350(1):141–150. doi: 10.1016/0005-2744(74)90212-5. [DOI] [PubMed] [Google Scholar]
  13. Ii I., Sakai I. Glutathione reductase in the sea urchin egg. II. Cleavage-associating fluctuation of the activity and its possible regulatory mechanism. Biochim Biophys Acta. 1974 May 20;350(1):151–161. doi: 10.1016/0005-2744(74)90213-7. [DOI] [PubMed] [Google Scholar]
  14. Kimura I. Further evidence of the similarity of microtubule protein from mitotic apparatus and sperm tail of the sea urchin, as a substrate in thiol-disulfide exchange reaction. Exp Cell Res. 1973 Jun;79(2):445–446. doi: 10.1016/0014-4827(73)90464-3. [DOI] [PubMed] [Google Scholar]
  15. Kinoshita S., Yazaki I. The behaviour and localization of intracellular relaxing system during cleavage in the sea urchin egg. Exp Cell Res. 1967 Sep;47(3):449–458. doi: 10.1016/0014-4827(67)90003-1. [DOI] [PubMed] [Google Scholar]
  16. Klein P., Robbins E. An ultrasensitive assay for soluble sulfhydryl and its application to the study of glutathione levels during the HeLa life cycle. J Cell Biol. 1970 Jul;46(1):165–168. doi: 10.1083/jcb.46.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kosower N. S., Vanderhoff G. A., Kosower E. M. Glutathione. 8. The effects of glutathione disulfide on initiation of protein synthesis. Biochim Biophys Acta. 1972 Jul 31;272(4):623–637. [PubMed] [Google Scholar]
  18. Kuriyama R., Sakai H. Role of tubulin-SH groups in polymerization to microtubules. Functional-SH groups in tubulin for polymerization. J Biochem. 1974 Sep;76(3):651–654. doi: 10.1093/oxfordjournals.jbchem.a130609. [DOI] [PubMed] [Google Scholar]
  19. Lee J. C., Frigon R. P., Timasheff S. N. The chemical characterization of calf brain microtubule protein subunits. J Biol Chem. 1973 Oct 25;248(20):7253–7262. [PubMed] [Google Scholar]
  20. Mabuchi I. ATPase in the cortical layer of sea urchin egg: its properties and interaction with cortex protein. Biochim Biophys Acta. 1973 Feb 28;297(2):317–332. doi: 10.1016/0304-4165(73)90079-2. [DOI] [PubMed] [Google Scholar]
  21. Maran M., Himelstein R., Dikstein S. Vorticella--a model for chemopharmacodynamic action on smooth muscle. Comp Gen Pharmacol. 1972 Sep;3(11):363–370. doi: 10.1016/0010-4035(72)90016-x. [DOI] [PubMed] [Google Scholar]
  22. NASATIR M., STERN H. Changes in the activities of aldolase and D-glyceraldehyde-3-phosphate dehydrogenase during the mitotic cycle in microspores of Lilium longiflorum. J Biophys Biochem Cytol. 1959 Oct;6:189–192. doi: 10.1083/jcb.6.2.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nath J., Rebhun L. I. Studies on cyclic AMP levels and phosphodiesterase activity in developing sea urchin eggs. Effects of puromycin, 6-dimethylamino purine and aminophylline. Exp Cell Res. 1973 Mar 15;77(1):319–322. doi: 10.1016/0014-4827(73)90583-1. [DOI] [PubMed] [Google Scholar]
  24. Nath J., Rebhun L. I. Studies on cyclic AMP phosphodiesterase in sea urchin eggs. Biochim Biophys Acta. 1974 Dec 29;370(2):498–509. doi: 10.1016/0005-2744(74)90111-9. [DOI] [PubMed] [Google Scholar]
  25. Nath J., Rebhun L. I. Studies on the uptake and metabolism of adenosine 3':5'-cyclic monophosphate and N6,O2-dibutyryl 3':5'-cyclic adenosine monophosphate in sea urchin eggs. Exp Cell Res. 1973 Nov;82(1):73–78. doi: 10.1016/0014-4827(73)90246-2. [DOI] [PubMed] [Google Scholar]
  26. Okazaki Y., Mabuchi I., Kimura I., Sakai H. Binding sites of -SH reagents in dividing sea urchin egg. Exp Cell Res. 1973 Dec;82(2):325–334. doi: 10.1016/0014-4827(73)90349-2. [DOI] [PubMed] [Google Scholar]
  27. Petzelt C. Ca 2+ -activated APTase during the cell cycle of the sea urchin Strongylocentrotus purpuratus. Exp Cell Res. 1972 Feb;70(2):333–339. doi: 10.1016/0014-4827(72)90144-9. [DOI] [PubMed] [Google Scholar]
  28. Petzelt C., von Ledebur-Villiger M. Ca2+-stimulated ATPase during the early development of parthenogenetically activated eggs of the sea urchin Paracentrotus lividus. Exp Cell Res. 1973 Sep;81(1):87–94. doi: 10.1016/0014-4827(73)90114-6. [DOI] [PubMed] [Google Scholar]
  29. Rappaport R. Cytokinesis in animal cells. Int Rev Cytol. 1971;31:169–213. doi: 10.1016/s0074-7696(08)60059-5. [DOI] [PubMed] [Google Scholar]
  30. Rebhun L. I., White D., Sander G., Ivy N. Cleavage inhibition in marine eggs by puromycin and 6-dimethylaminopurine. Exp Cell Res. 1973 Mar 15;77(1):312–318. doi: 10.1016/0014-4827(73)90582-x. [DOI] [PubMed] [Google Scholar]
  31. SAKAI H., DAN K. Studies on sulfhydryl groups during cell division of sea urchin egg. I. Glutatione. Exp Cell Res. 1959 Jan;16(1):24–41. doi: 10.1016/0014-4827(59)90192-2. [DOI] [PubMed] [Google Scholar]
  32. Stephens R. E., Kane R. E. Some properties of hyalin: the calcium-insoluble protein of the hyaline layer of the sea urchin egg. J Cell Biol. 1970 Mar;44(3):611–617. doi: 10.1083/jcb.44.3.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Szent-Györgyi A., Együd L. G., McLaughlin J. A. Keto-aldehydes and cell division. Science. 1967 Feb 3;155(3762):539–541. doi: 10.1126/science.155.3762.539. [DOI] [PubMed] [Google Scholar]
  34. Tietze F. Disulfide reduction in rat liver. I. Evidence for the presence of nonspecific nucleotide-dependent disulfide reductase and GSH-disulfide transhydrogenase activities in the high-speed supernatant fraction. Arch Biochem Biophys. 1970 May;138(1):177–188. doi: 10.1016/0003-9861(70)90297-3. [DOI] [PubMed] [Google Scholar]
  35. Zehavi-Willner T., Kosower E. M., Hunt T., Kosower N. S. Glutathione. V. The effects of the thiol-oxidizing agent diamide on initiation and translation in rabbit reticulocytes. Biochim Biophys Acta. 1971 Jan 1;228(1):245–251. [PubMed] [Google Scholar]
  36. Zotin A. I., Milman L. S., Faustov V. S. ATP level and cleavage of sea urchin eggs Strongylocentrotus dröbachiensis (O. F. Müller). Exp Cell Res. 1965 Sep;39(2):567–576. doi: 10.1016/0014-4827(65)90058-3. [DOI] [PubMed] [Google Scholar]

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