Rapid purification of cationic granule proteases: application to human granzymes.
This report describes a simple scheme for the simultaneous purification of cationic human granzymes A, B, and 3 from human interleukin 2 (IL-2)-activated lymphocytes. The process, which requires approximately 8 h, includes: (1) cell cavitation, (2) two centrifugation steps, (3) four granule solubilization steps, and (4) cation-exchange chromatography. Granule solubilization consists of three extractions with a hypotonic buffer (25 mM NaCl) that contained Triton X-100 followed by a final extraction in hypertonic detergent-free buffer (390 mM NaCl). We recovered approximately 35% of the trypsin-like (tryptase) activities mediated by granzymes A and 3, respectively, and approximately 25% of the asp-ase activity of granzyme B. The granzymes were identified after elution from the Mono S column by Western blot with a polyclonal antibody that reacts with a conserved amino acid sequence (9-16) of lymphoid/myeloid serine proteases. By amino-terminal sequencing, eluted granzyme A and B were indeed homogeneous. Granzyme 3, although highly enriched, appears to be contaminated with an uncharacterized granzyme. Although we have developed this scheme to rapidly isolate the granzymes, the procedure should assist the purification of secretory granule-associated cationic proteins that reside in neutrophils and mast cells as well as other cells that possess secretory function.
Hanna
W L
WL
Section of Rheumatology, Evanston Hospital, Northwestern University Medical School, Illinois 60201.
Zhang
X
X
Turbov
J
J
Winkler
U
U
Hudig
D
D
Froelich
C J
CJ
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D015153:Blotting, Western; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D003594:Cytoplasmic Granules / Q000201:enzymology*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D053804:Granzymes; D006801:Humans; D007038:Hypotonic Solutions; D007376:Interleukin-2 / Q000494:pharmacology; D015979:Killer Cells, Lymphokine-Activated / Q000201:enzymology*; D008213:Lymphocyte Activation / Q000187:drug effects; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification*
D003902:Detergents; D007038:Hypotonic Solutions; D007376:Interleukin-2; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human