Isolation and biochemical and functional characterization of perforin 1 from cytolytic T-cell granules.
The Ca2+-dependent cytolytic activity of isolated T-lymphocyte granules was purified to apparent homogeneity by high-salt extraction, gel filtration, and ion-exchange chromatography. The lytic activity resided in a 72- to 75-kDa protein of cytolytic granules. Incubation of the isolated protein with erythrocytes in the presence of Ca2+ ions resulted in hemolysis and the formation of membrane lesions of 160 A in diameter, corresponding in size and morphology to membrane lesions formed on target cells by cloned, intact natural killer (NK) and cytolytic T lymphocytes. Hence, the 75-kDa granule protein is identified as monomeric perforin 1 (P1), postulated previously from the analysis of membrane lesions formed during NK and T-cell-mediated cytolysis. P1-mediated hemolysis is Ca2+-dependent and is inhibited by Zn2+ ions. Lysis is accompanied by the polymerization of P1 to membrane-associated tubular complexes (poly-P1) that form large transmembrane pores. P1 causes a rapid membrane depolarization of J774 cells in the presence of Ca2+. Purified P1 also induces transmembrane monovalent and divalent ion flow across lipid vesicles only in the presence of Ca2+. Whole-cell patch-clamp recordings of S49 lymphoma cells show a P1-dependent inward membrane current flow in the presence but not in the absence of Ca2+. The current increase can be dissected as a summation of discrete current events, indicative of formation of functional channels by polymerization of P1.
Podack
E R
ER
Young
J D
JD
Cohn
Z A
ZA
D000818:Animals; D001681:Biological Assay; D002118:Calcium / Q000502:physiology; D002463:Cell Membrane Permeability; D003594:Cytoplasmic Granules / Q000032:analysis; D006461:Hemolysis; D006801:Humans; D007473:Ion Channels; D046911:Macromolecular Substances; D008264:Macrophages / Q000502:physiology; D008562:Membrane Glycoproteins; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000502:physiology; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012756:Sheep; D013602:T-Lymphocytes, Cytotoxic / Q000032:analysis* / Q000276:immunology / Q000648:ultrastructure
D007473:Ion Channels; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; C047558:polyperforin; D054353:Perforin; D002118:Calcium