GRP94-associated enzymatic activities. Resolution by chromatographic fractionation.
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 bacterial beta-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 II, which is known to co-purify with GRP94. Electrophoretically pure GRP94 displayed low, but significant levels of aminopeptidase activity. Further purification of GRP94 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 co-purifying (poly)peptides in the generation of GRP94-dependent cellular immune responses.
Reed
Robyn C
RC
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Zheng
Tianli
T
Nicchitta
Christopher V
CV
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D004285:Dogs; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D013379:Substrate Specificity; D013552:Swine; D001616:beta-Galactosidase / Q000302:isolation & purification / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; C018551:glucose-regulated proteins; D001616:beta-Galactosidase