TY - JOUR
T1 - Histamine-releasing activity. V. Characterization and purification using high-performance liquid chromatography
AU - Lett-Brown, Michael A.
AU - Thueson, David O.
AU - Plank, Diane E.
AU - Duffy, Lawrence
AU - Grant, J. Andrew
N1 - Funding Information:
* This work, supported by Grant 5ROl-Al-12621 from the National Institute of Allergy and Infectious Diseases, was presented, in part, at the 67th Annual Meeting of the Federation of American Societies for Experimental Biology, Chicago, April 1983. ’ To whom correspondence should be addressed at University of Texas Medical Branch, Department of Internal Medicine, Allergy Section, Clinical Sciences Building 409, Galveston, Tex. 77550. 3 Abbreviations used: BCA, basophil chemotactic activity; DrO, deuterium oxide; Hepes, e(Zhydroxyethylk 1-piperazineethanesulfonic acid HACM, Hepes-buffered saline containing 0.3 mg/ml human serum albumin, 2 nul4 CaClr, and 1 mM MgC12; HPLC, high-performance liquid chromatography; HRA, histamine-releasing activity; TDL, thoracic duct lymphocytes.
PY - 1984/9
Y1 - 1984/9
N2 - The production of large quantities of the lymphokine(s) histamine-releasing activity (HRA) and its partial purification by Sephadex G-75 and ion-exchange chromatography on carboxymethyl (CM) Sepharose 6B have been detailed (M. A. Lett-Brown, D. O. Thueson, D. E. Plank, M. P. Langford, and J. A. Grant, Cell. Immunol. 87, 434-444, 1984). Two peaks of activity (HRA I and II) were recovered. Preparations of HRA have now been analyzed by high-performance liquid chromatography (HPLC). Thoracic duct lymphocytes stimulated with 200 U/ml streptokinase were used as a source of HRA. Gel-filtration HPLC on a TSK 3000 column separated HRA into two peaks of activity (10,000-20,000 and 1300 Da). Reverse-phase high-performance liquid chromatography using a Nucleosil C-8 column showed that HRA II (the activity eluted at a conductivity of 18-20 mmho on the CM-Sepharose column) eluted as a single sharp peak, the main protein contaminant being cytochrome c, the carrier protein added to enhance the yield of HRA. High-performance liquid chromatography was found to be a useful analytical tool and may be suitable for the large-scale purification of HRA.
AB - The production of large quantities of the lymphokine(s) histamine-releasing activity (HRA) and its partial purification by Sephadex G-75 and ion-exchange chromatography on carboxymethyl (CM) Sepharose 6B have been detailed (M. A. Lett-Brown, D. O. Thueson, D. E. Plank, M. P. Langford, and J. A. Grant, Cell. Immunol. 87, 434-444, 1984). Two peaks of activity (HRA I and II) were recovered. Preparations of HRA have now been analyzed by high-performance liquid chromatography (HPLC). Thoracic duct lymphocytes stimulated with 200 U/ml streptokinase were used as a source of HRA. Gel-filtration HPLC on a TSK 3000 column separated HRA into two peaks of activity (10,000-20,000 and 1300 Da). Reverse-phase high-performance liquid chromatography using a Nucleosil C-8 column showed that HRA II (the activity eluted at a conductivity of 18-20 mmho on the CM-Sepharose column) eluted as a single sharp peak, the main protein contaminant being cytochrome c, the carrier protein added to enhance the yield of HRA. High-performance liquid chromatography was found to be a useful analytical tool and may be suitable for the large-scale purification of HRA.
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U2 - 10.1016/0008-8749(84)90013-3
DO - 10.1016/0008-8749(84)90013-3
M3 - Article
C2 - 6205771
AN - SCOPUS:0021233871
SN - 0008-8749
VL - 87
SP - 445
EP - 451
JO - Cellular Immunology
JF - Cellular Immunology
IS - 2
ER -