TY - JOUR
T1 - GRP94-associated enzymatic activities
T2 - Resolution by chromatographic fractionation
AU - Reed, Robyn C
AU - Zheng, Tianli
AU - Nicchitta, Christopher V.
PY - 2002/7/12
Y1 - 2002/7/12
N2 - GRP94 (gp96), which performs established functions as a molecular chaperone and immune system modulator, has been reported to display a number of intrinsic enzymatic activities, including ATP hydrolysis, protein phosphorylation, and aminopeptidase. In observing that GRP94 co-purified with bacterialβ-galactosidase through multiple chromatographic steps, we have examined the hypothesis that the reported enzymatic activities of GRP94 may reflect co-purification of contaminant enzymes, rather than intrinsic catalytic functions. In subjecting GRP94 to increasingly stringent chromatographic purification, we report that a GRP94 carboxyl-terminal directed protein kinase activity could be separated from GRP94 by heparin affinity chromatography. Analysis of the kinase substrate specificity indicates that this kinase is distinct from casein kinase H, which is known to copurify with GRP94. Electrophoretically pure GRP94 displayed low, but significant levels of aminopeptidase activity. Further purification of GRP94 GEP94 by anion exchange and heparin affinity chromatography yielded resolution of GRP94 from the aminopeptidase activity. Furthermore, exhaustive trypsinolysis of GRP94 preparations displaying aminopeptidase activity yielded complete proteolysis of GRP94 but did not affect aminopeptidase activity. These results are discussed with respect to current models for GRP94 function and the role of such copurifying (poly)peptides in the generation of GRP94-dependent cellular immune responses.
AB - GRP94 (gp96), which performs established functions as a molecular chaperone and immune system modulator, has been reported to display a number of intrinsic enzymatic activities, including ATP hydrolysis, protein phosphorylation, and aminopeptidase. In observing that GRP94 co-purified with bacterialβ-galactosidase through multiple chromatographic steps, we have examined the hypothesis that the reported enzymatic activities of GRP94 may reflect co-purification of contaminant enzymes, rather than intrinsic catalytic functions. In subjecting GRP94 to increasingly stringent chromatographic purification, we report that a GRP94 carboxyl-terminal directed protein kinase activity could be separated from GRP94 by heparin affinity chromatography. Analysis of the kinase substrate specificity indicates that this kinase is distinct from casein kinase H, which is known to copurify with GRP94. Electrophoretically pure GRP94 displayed low, but significant levels of aminopeptidase activity. Further purification of GRP94 GEP94 by anion exchange and heparin affinity chromatography yielded resolution of GRP94 from the aminopeptidase activity. Furthermore, exhaustive trypsinolysis of GRP94 preparations displaying aminopeptidase activity yielded complete proteolysis of GRP94 but did not affect aminopeptidase activity. These results are discussed with respect to current models for GRP94 function and the role of such copurifying (poly)peptides in the generation of GRP94-dependent cellular immune responses.
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U2 - 10.1074/jbc.M203195200
DO - 10.1074/jbc.M203195200
M3 - Article
C2 - 11983709
AN - SCOPUS:0037067702
SN - 0021-9258
VL - 277
SP - 25082
EP - 25089
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 28
ER -