Purification and use of granzyme B.
Granzyme B (GrB) is the primary molecular mediator of apoptosis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It is a unique mammalian aspartic acid-cleaving serine protease. On T cell receptor activation, GrB is released from the CTL cytoplasmic granules by exocytosis, enters the target cells and, in the presence of the granule pore-forming protein perforin, it initiates the processing of caspases and apoptosis. GrB apoptosis is also activated by adenovirus, which can effectively replace perforin. Methods for the purification and quantitation of GrB and perforin, and the preparation and titration of adenovirus, are described. In addition, methods for application of these reagents to the initiation of apoptosis in tumor target cells, with several assays for detecting GrB apoptotic activity, are detailed.
Manitoba Institute of Cell Biology, University of Manitoba, Winnepeg, Canada.
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L
Shi
X
X
Yang
C J
CJ
Froelich
A H
AH
Greenberg
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods; D002846:Chromatography, Affinity; D002850:Chromatography, Gel / Q000379:methods; D002853:Chromatography, Liquid; D003594:Cytoplasmic Granules / Q000201:enzymology / Q000648:ultrastructure; D004249:DNA Damage; D053938:DNA Fragmentation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D053804:Granzymes; D006454:Hemoglobins / Q000032:analysis; D006461:Hemolysis; D006801:Humans; D007065:Idoxuridine / Q000032:analysis / Q000493:pharmacokinetics; D007457:Iodine Radioisotopes; D007694:Killer Cells, Natural / Q000201:enzymology*; D007942:Leukemia, Experimental / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D012756:Sheep; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology*; D014407:Tumor Cells, Cultured
D006454:Hemoglobins; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; C509213:GZMB protein, human; D053804:Granzymes; C509215:Gzmb protein, rat; D012697:Serine Endopeptidases; D007065:Idoxuridine