Skip to main content
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1984 Oct;74(4):1132–1142. doi: 10.1172/JCI111522

Isolation of a sodium transport inhibitory factor, inhibitin, from cultured leukemic promyelocytes.

K Morgan, M A Mir
PMCID: PMC425279  PMID: 6090500

Abstract

Previous studies have shown that leukemic blood contains a factor that has an inhibitory effect on bidirectional sodium transport in erythrocytes. This study was designed to isolate this factor from cultured leukemic promyelocytes. An extract from the promyelocytes reduced significantly (P less than 0.001) the ouabain-insensitive sodium efflux rate, from 0.096 +/- 0.009 to 0.056 +/- 0.003 SD. Using the inhibition of ouabain-insensitive sodium transport in erythrocytes as an assay to identify the factor, we ran the crude promyelocyte extract through Sephadex G-25 and G-10, with an intermediate ion-exchange step on DE-32, and finally subjected the active fraction to reverse-phase high-performance liquid chromatography. The specific inhibitory activity of the final fraction was 180-fold higher than that of the crude promyelocyte extract. The inhibitory activity could be destroyed by acid hydrolysis and by enzymatic digestion with several proteases but not by heating at 80 degrees C for 30 min; these characteristics suggest that the active factor, called inhibitin, is a peptide. Inhibitin is released by immature myeloid cells but not by differentiated white cells or by leukemic lymphocytes. It has no effect on potassium influx but inhibits sodium/sodium exchange in erythrocytes.

Full text

PDF
1132

Selected References

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

  1. Chikkappa G., Corcino J., Greenerg M. L., Herert V. Correation between vario blood white cell pools and the serum B12-binding capaities. Blood. 1971 Feb;37(2):142–151. [PubMed] [Google Scholar]
  2. Duhm J., Becker B. F. Studies on lithium transport across the red cell membrane. V. On the nature of the Na+-dependent Li+ countertransport system of mammalian erythrocytes. J Membr Biol. 1979 Dec 31;51(3-4):263–286. doi: 10.1007/BF01869087. [DOI] [PubMed] [Google Scholar]
  3. Dunn M. J. Ouabain-uninhibited Na+ Transport in human erythrocytes: the effects of triflocin. Biochim Biophys Acta. 1972 Feb 11;255(2):567–571. doi: 10.1016/0005-2736(72)90160-5. [DOI] [PubMed] [Google Scholar]
  4. Dunn M. J. The effects of transport inhibitors on sodium outflux and influx in red blood cells: evidence for exchange diffusion. J Clin Invest. 1970 Oct;49(10):1804–1814. doi: 10.1172/JCI106398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fink M. E., Finch S. C. Serum muramidase and granulocyte turnover. Proc Soc Exp Biol Med. 1968 Feb;127(2):365–367. doi: 10.3181/00379727-127-32692. [DOI] [PubMed] [Google Scholar]
  6. Gallagher R., Collins S., Trujillo J., McCredie K., Ahearn M., Tsai S., Metzgar R., Aulakh G., Ting R., Ruscetti F. Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood. 1979 Sep;54(3):713–733. [PubMed] [Google Scholar]
  7. Ghezzo F., Pegoraro L. Effects of retinoic acid on the fibrinolytic activity of HL 60 human promyelocytic leukemia cells. Experientia. 1981 Apr 15;37(4):425–426. doi: 10.1007/BF01959900. [DOI] [PubMed] [Google Scholar]
  8. Hartree E. F. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972 Aug;48(2):422–427. doi: 10.1016/0003-2697(72)90094-2. [DOI] [PubMed] [Google Scholar]
  9. Kinsella J. L., Aronson P. S. Interaction of NH4+ and Li+ with the renal microvillus membrane Na+-H+ exchanger. Am J Physiol. 1981 Nov;241(5):C220–C226. doi: 10.1152/ajpcell.1981.241.5.C220. [DOI] [PubMed] [Google Scholar]
  10. Krystosek A., Sachs L. Control of lysozyme induction in the differentiation of myeloid leukemic cells. Cell. 1976 Dec;9(4 Pt 2):675–684. doi: 10.1016/0092-8674(76)90131-8. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. MEYER L. M., CRONKITE E. P., MILLER I. F., MULZAC C. W., JONES I. Co60 vitamin B12 binding capacity of human leukocytes. Blood. 1962 Feb;19:229–235. [PubMed] [Google Scholar]
  13. Mayer K. D., Starkey B. J. Simpler flame photometric determination of erythrocyte sodium and potassium: the reference range for apparently healthy adults. Clin Chem. 1977 Feb;23(2 Pt 1):275–278. [PubMed] [Google Scholar]
  14. Mir M. A., Bobinski H. Altered membrane sodium transport and the presence of a plasma ouabain-like inhibitory factor in acute myeloid leukaemia. Clin Sci Mol Med. 1975 Mar;48(3):213–218. doi: 10.1042/cs0480213. [DOI] [PubMed] [Google Scholar]
  15. Mir M. A., Brabin B., Tang O. T., Leyland M. J., Delamore I. W. Hypokalaemia in acute myeloid leukaemia. Ann Intern Med. 1975 Jan;82(1):54–57. doi: 10.7326/0003-4819-82-1-54. [DOI] [PubMed] [Google Scholar]
  16. Mir M. A., Delamore I. W. Hyponatraemia syndrome in acute myeloid leukaemia. Br Med J. 1974 Jan 12;1(5897):52–55. doi: 10.1136/bmj.1.5897.52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mir M. A., Delamore I. W. Metabolic disorders in acute myeloid leukaemia. Br J Haematol. 1978 Sep;40(1):79–92. doi: 10.1111/j.1365-2141.1978.tb03641.x. [DOI] [PubMed] [Google Scholar]
  18. Mir M. A. Effects of myeloid leukaemic blast cell extract on sodium transport in human erythrocytes. Clin Sci (Lond) 1983 Jan;64(1):79–83. doi: 10.1042/cs0640079. [DOI] [PubMed] [Google Scholar]
  19. Mir M. A. Evidence for inhibitory and anti-ouabain-like factors in leukaemic blood. Clin Sci (Lond) 1981 Oct;61(4):391–397. doi: 10.1042/cs0610391. [DOI] [PubMed] [Google Scholar]
  20. Morgan K., Mir M. A. A passive sodium transport inhibitory factor (inhibitin) released from leukaemic promyelocytes in culture. Clin Sci (Lond) 1984 Mar;66(3):365–368. doi: 10.1042/cs0660365. [DOI] [PubMed] [Google Scholar]
  21. Muggia F. M., Heinemann H. O., Farhangi M., Osserman E. F. Lysozymuria and renal tubular dysfunction in monocytic and myelomonocytic leukemia. Am J Med. 1969 Sep;47(3):351–366. doi: 10.1016/0002-9343(69)90219-8. [DOI] [PubMed] [Google Scholar]
  22. Murer H., Hopfer U., Kinne R. Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney. Biochem J. 1976 Mar 15;154(3):597–604. [PMC free article] [PubMed] [Google Scholar]
  23. Pandey G. N., Sarkadi B., Haas M., Gunn R. B., Davis J. M., Tosteson D. C. Lithium transport pathways in human red blood cells. J Gen Physiol. 1978 Aug;72(2):233–247. doi: 10.1085/jgp.72.2.233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sachs J. R., Welt L. G. The concentration dependence of active potassium transport in the human red blood cell. J Clin Invest. 1967 Jan;46(1):65–76. doi: 10.1172/JCI105512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sarkadi B., Alifimoff J. K., Gunn R. B., Tosteson D. C. Kinetics and stoichiometry of Na-dependent Li transport in human red blood cells. J Gen Physiol. 1978 Aug;72(2):249–265. doi: 10.1085/jgp.72.2.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Simons K., Weber T. The vitamin B12-binding protein in human leukocytes. Biochim Biophys Acta. 1966 Mar 28;117(1):201–208. doi: 10.1016/0304-4165(66)90167-x. [DOI] [PubMed] [Google Scholar]
  27. Villamil M. F., Rettori V., Kleeman C. R. Sodium transport by red blood cells in uremia. J Lab Clin Med. 1968 Aug;72(2):308–317. [PubMed] [Google Scholar]
  28. Wiley J. S., Cooper R. A. A furosemide-sensitive cotransport of sodium plus potassium in the human red cell. J Clin Invest. 1974 Mar;53(3):745–755. doi: 10.1172/JCI107613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yamada M., Mori M., Sugimura T. Purification and characterization of small molecular weight myeloperoxidase from human promyelocytic leukemia HL-60 cells. Biochemistry. 1981 Feb 17;20(4):766–771. doi: 10.1021/bi00507a018. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES