Purification of mast cell proteases from murine skin.
Different subpopulations of mast cells are characterized by their abundant contents of either tryptase or in addition chymase. These two neutral proteases are found in mast cells and may thus hold a key to the understanding of mast cell dependent reactions. Such studies are however hampered by the lack of readily available supplies of chymase. We have therefore studied the simultaneous purification of both proteases from hairless moro hr/hr mouse skin, using a sequence of salt extractions and chromatographic separations. After three steps of extraction, a 13-fold purification with an 82% yield was obtained for tryptase and a 15-fold purification with a 90% yield for chymase. Further one step purification on conventional sephadex, sephacryl and octyl sepharose columns was unsatisfactory because of further protein contamination of the fractions. Heparin affinity chromatography caused a high loss of tryptase and residual protein contamination. Gradient elution on a benzamidine sepharose 6B column resulted however in a single, low yield (17.9%) tryptase peak and a broader, high yield (>90%) chymase peak, and a 34% yield high purity fraction. The proteases thus purified exhibited their typical inhibitor profile. On Western blot analysis and on autoradiography in the presence of the serine protease inhibitor diisopropylfluorophosphate (DFP), only one 28 kD molecule with chymase activity was identified, whereas a broad 32-38 kD band of tryptase monomers was noted. Taken together, these data show that, after salt extraction and a single benzamidine affinity chromatography step, both mast cell chymase and tryptase can be separated and in case of chymase also highly purified, allowing thus for the study of biological activities of this molecule.
Algermissen
B
B
Department of Dermatology, Charité, Humboldt University, Berlin, Germany.
Laubscher
J C
JC
Bauer
F
F
Henz
B M
BM
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002845:Chromatography / Q000379:methods; D053818:Chymases; D004591:Electrophoresis, Polyacrylamide Gel; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D008812:Mice, Hairless; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification*; D012867:Skin / Q000166:cytology* / Q000201:enzymology*; D053802:Tryptases
C509190:Tpsb2 protein, mouse; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; C509229:Cma2 protein, mouse; C509222:Tpsab1 protein, mouse; D053802:Tryptases