Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. II. Purification and identification of rat A-NKP 1 and A-NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex.
We have recently described nongranular, cytosolic, high-molecular-weight trypsin-like (A-NKP 1) and chymotrypsin-like (A-NKP 2) proteases of interleukin-2-activated rat natural killer (A-NK) cells. A functional correlation between the inactivation of A-NKP 2 and the inhibition of rat A-NK cell-mediated cytotoxicity was found. Herein we describe the 6,000-fold purification of A-NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin-Sepharose chromatography. We also report the novel finding that A-NKP 2 as well as A-NKP 1, derived from either rat A-NK cells or the rat NK leukemic cell line CRNK-16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK-16-derived proteasomal subunits. The identification, characterization, and purification of A-NKP 1 and A-NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A-NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell-mediated cytotoxicity.
Wasserman
K
K
Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pennsylvania 15213.
Kitson
R P
RP
Rivett
A J
AJ
Sweeney
S T
ST
Gabauer
M K
MK
Herberman
R B
RB
Watkins
S C
SC
Goldfarb
R H
RH
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002500:Centrifugation, Isopycnic; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002918:Chymotrypsin; D003429:Cross Reactions; D003546:Cysteine Endopeptidases / Q000302:isolation & purification*; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007376:Interleukin-2 / Q000494:pharmacology*; D007694:Killer Cells, Natural / Q000187:drug effects* / Q000201:enzymology; D008213:Lymphocyte Activation; D008297:Male; D008854:Microscopy, Electron; D009097:Multienzyme Complexes / Q000302:isolation & purification*; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D011916:Rats, Inbred F344; D014407:Tumor Cells, Cultured
D007376:Interleukin-2; D009097:Multienzyme Complexes; D002918:Chymotrypsin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex