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. 1968 May 1;37(2):394–411. doi: 10.1083/jcb.37.2.394

STUDIES ON LYSOSOMES

XI. Characterization of a Hydrolase-Rich Fraction from Human Lymphocytes

Günter Brittinger 1, Rochelle Hirschhorn 1, Steven D Douglas 1, Gerald Weissmann 1
PMCID: PMC2107405  PMID: 5656398

Abstract

Pure suspensions of human lymphocytes were separated from peripheral blood by means of nylon wool, homogenized in 0.34 M sucrose-0.01 M EDTA solution, and fractionated by differential centrifugation. The bulk of acid hydrolase activity was found to be concentrated in a 20,000 g x 20 min granular fraction, whereas nuclear, debris, and supernatant fractions contained lesser concentrations of hydrolases. Acid hydrolase activity present in the granular fraction showed appropriate "latency" as judged by its dose-dependent release into the 20,000 g x 20 min supernatant after exposure to membrane-disruptive agents such as streptolysin S, filipin, and lysolecithin. Heparin proved to be necessary in the suspending medium so that reproducible homogenization and cell fractionation could be obtained. Even excessive contamination of lymphocyte suspensions with platelets did not appreciably alter the acid hydrolase activity of lymphocyte homogenates or the distribution of enzymes in subcellular fractions. Discontinuous density-gradient centrifugation of a 500 g x 10 min supernatant, containing both acid hydrolase-rich organelles and mitochondria, resulted in partial resolution of hydrolase-rich organelles from mitochondria. Fine structural studies of the intact lymphocytes showed the presence of acid phosphatase-positive, membrane-bounded organelles. Electron microscopy of the "large granule" (20,000 g x 20 min) fraction of such lymphocytes demonstrated 80–90% mitochondria, 5–10% platelets, and 5–10% membrane-bounded acid phosphatase-positive structures. The data indicate the presence in human peripheral blood lymphocytes of acid hydrolase-rich granules which possess many of the biochemical and structural characteristics of lysosomes in other tissues.

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Selected References

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  1. ALLISON A. C., MALLUCCI L. LYSOSOMES IN DIVIDING CELLS, WITH SPECIAL REFERENCE TO LYMPHOCYTES. Lancet. 1964 Dec 26;2(7374):1371–1373. doi: 10.1016/s0140-6736(64)91162-6. [DOI] [PubMed] [Google Scholar]
  2. BESSIS M., THIERY J. P. Electron microscopy of human white blood cells and their stem cells. Int Rev Cytol. 1961;12:199–241. doi: 10.1016/s0074-7696(08)60541-0. [DOI] [PubMed] [Google Scholar]
  3. Baudhuin P., Evrard P., Berthet J. Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles. J Cell Biol. 1967 Jan;32(1):181–191. doi: 10.1083/jcb.32.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bowers W. E., Finkenstaedt J. T., de Duve C. Lysosomes in lymphoid tissue. I. The measurement of hydrolytic activities in whole homogenates. J Cell Biol. 1967 Feb;32(2):325–337. doi: 10.1083/jcb.32.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bowers W. E., de Duve C. Lysosomes in lymphoid tissue. II. Intracellular distribution of acid hydrolases. J Cell Biol. 1967 Feb;32(2):339–348. doi: 10.1083/jcb.32.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bowers W. E., de Duve C. Lysosomes in lymphoid tissue. III. Influence of various treatments of the animals on the distribution of acid hydrolases. J Cell Biol. 1967 Feb;32(2):349–364. doi: 10.1083/jcb.32.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. COHN Z. A., BENSON B. THE DIFFERENTIATION OF MONONUCLEAR PHAGOCYTES. MORPHOLOGY, CYTOCHEMISTRY, AND BIOCHEMISTRY. J Exp Med. 1965 Jan 1;121:153–170. doi: 10.1084/jem.121.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. COHN Z. A., HIRSCH J. G. The isolation and properties of the specific cytoplasmic granules of rabbit polymorphonuclear leucocytes. J Exp Med. 1960 Dec 1;112:983–1004. doi: 10.1084/jem.112.6.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DE DUVE C., PRESSMAN B. C., GIANETTO R., WATTIAUX R., APPELMANS F. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J. 1955 Aug;60(4):604–617. doi: 10.1042/bj0600604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. De Duve C., Wattiaux R. Functions of lysosomes. Annu Rev Physiol. 1966;28:435–492. doi: 10.1146/annurev.ph.28.030166.002251. [DOI] [PubMed] [Google Scholar]
  11. Dioguardi N., Ideo G., Mannucci P. M., Fiorelli G. Lactate dehydrogenase (LDH), glutamic oxalacetic transaminase (GOT) and malate dehydrogenase (MDH) isoenzymes in lymphocytes from foetal and adult thymus, spleen and from peripheral blood. Enzymol Biol Clin (Basel) 1966;6(4):324–338. doi: 10.1159/000457867. [DOI] [PubMed] [Google Scholar]
  12. Douglas S. D., Borjeson J., Chessin L. N. Studies on human lymphocytes in vitro. IV. Comparative fine structural features of the established Burkitt lymphoma cell lines AL-1, EB-2 and phytomitogen-transformed lymphocytes. J Immunol. 1967 Aug;99(2):340–346. [PubMed] [Google Scholar]
  13. Douglas S. D., Hoffman P. F., Borjeson J., Chessin L. N. Studies on human peripheral blood lymphocytes in vitro. 3. Fine structural features of lymphocyte transformation by pokeweed mitogen. J Immunol. 1967 Jan;98(1):17–30. [PubMed] [Google Scholar]
  14. Ericsson J. L., Trump B. F. Observations on the application to electron microscopy on the lead phosphate technique for the demonstration of acid phosphatase. Histochemie. 1965 Mar 5;4(6):470–487. doi: 10.1007/BF00281900. [DOI] [PubMed] [Google Scholar]
  15. KALNITSKY G., HUMMEL J. P., DIERKS C. [Some factors which affect the enzymatic digestion of ribonucleic acid]. J Biol Chem. 1959 Jun;234(6):1512–1516. [PubMed] [Google Scholar]
  16. KEISER H., WEISSMANN G., BERNHEIMER A. W. STUDIES ON LYSOSOMES. IV. SOLUBILIZATION OF ENZYMES DURING MITOCHONDRIAL SWELLING AND DISRUPTION OF LYSOSOMES BY STREPTOLYSIN S AND OTHER HEMOLYTIC AGENTS. J Cell Biol. 1964 Jul;22:101–113. doi: 10.1083/jcb.22.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. LUFT J. H. Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol. 1961 Feb;9:409–414. doi: 10.1083/jcb.9.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marcus A. J., Zucker-Franklin D., Safier L. B., Ullman H. L. Studies on human platelet granules and membranes. J Clin Invest. 1966 Jan;45(1):14–28. doi: 10.1172/JCI105318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Parker J. W., Wakasa H., Lukes R. J. The morphologic and cytochemical demonstration of lysosomes in lymphocytes incubated with phytohemagglutinin by electron microscopy. Lab Invest. 1965 Oct;14(10):1736–1743. [PubMed] [Google Scholar]
  21. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. ROTH J. S. Ribonuclease. II. Activators and inhibitors for ribonuclease. Arch Biochem Biophys. 1953 Jun;44(2):265–270. doi: 10.1016/0003-9861(53)90043-5. [DOI] [PubMed] [Google Scholar]
  23. SABATINI D. D., BENSCH K., BARRNETT R. J. Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol. 1963 Apr;17:19–58. doi: 10.1083/jcb.17.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Seeman P. M., Palade G. E. Acid phosphatase localization in rabbit eosinophils. J Cell Biol. 1967 Sep;34(3):745–756. doi: 10.1083/jcb.34.3.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tanaka Y., Liddy T. J. Lipids and acid phosphatase in cultured lymphocytes of peripheral blood. A histochemical and electron microscopic study. Lab Invest. 1966 Feb;15(2):455–463. [PubMed] [Google Scholar]
  26. VALENTINE W. N., BECK W. S. Biochemical studies on leucocytes. I. Phosphatase activity in health, leucocytosis, and myelocytic leucemia. J Lab Clin Med. 1951 Jul;38(1):39–55. [PubMed] [Google Scholar]
  27. WATSON M. L. Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol. 1958 Jul 25;4(4):475–478. doi: 10.1083/jcb.4.4.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. WEISSMANN G., BECHER B., THOMAS L. STUDIES ON LYSOSOMES. V. THE EFFECTS OF STREPTOLYSINS AND OTHER HEMOLYTIC AGENTS ON ISOLATED LEUCOCYTE GRANULES. J Cell Biol. 1964 Jul;22:115–126. doi: 10.1083/jcb.22.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Weissmann G., Hirschhorn R., Pras M., Sessa G., Bevans V. A. Studies of lysosomes. 8. The effect of polyene antibiotics on lysosomes. Biochem Pharmacol. 1967 Jun;16(6):1057–1069. doi: 10.1016/0006-2952(67)90279-1. [DOI] [PubMed] [Google Scholar]

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