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GRP94-associated enzymatic activities. Resolution by chromatographic fractionation.

Publication ,  Journal Article
Reed, RC; Zheng, T; Nicchitta, CV
Published in: J Biol Chem
July 12, 2002

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.

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Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

July 12, 2002

Volume

277

Issue

28

Start / End Page

25082 / 25089

Location

United States

Related Subject Headings

  • beta-Galactosidase
  • Swine
  • Substrate Specificity
  • Phosphorylation
  • Mice, Inbred C57BL
  • Mice
  • Membrane Proteins
  • HSP70 Heat-Shock Proteins
  • Dogs
  • Chromatography, Liquid
 

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Reed, R. C., Zheng, T., & Nicchitta, C. V. (2002). GRP94-associated enzymatic activities. Resolution by chromatographic fractionation. J Biol Chem, 277(28), 25082–25089. https://doi.org/10.1074/jbc.M203195200
Reed, Robyn C., Tianli Zheng, and Christopher V. Nicchitta. “GRP94-associated enzymatic activities. Resolution by chromatographic fractionation.J Biol Chem 277, no. 28 (July 12, 2002): 25082–89. https://doi.org/10.1074/jbc.M203195200.
Reed RC, Zheng T, Nicchitta CV. GRP94-associated enzymatic activities. Resolution by chromatographic fractionation. J Biol Chem. 2002 Jul 12;277(28):25082–9.
Reed, Robyn C., et al. “GRP94-associated enzymatic activities. Resolution by chromatographic fractionation.J Biol Chem, vol. 277, no. 28, July 2002, pp. 25082–89. Pubmed, doi:10.1074/jbc.M203195200.
Reed RC, Zheng T, Nicchitta CV. GRP94-associated enzymatic activities. Resolution by chromatographic fractionation. J Biol Chem. 2002 Jul 12;277(28):25082–25089.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

July 12, 2002

Volume

277

Issue

28

Start / End Page

25082 / 25089

Location

United States

Related Subject Headings

  • beta-Galactosidase
  • Swine
  • Substrate Specificity
  • Phosphorylation
  • Mice, Inbred C57BL
  • Mice
  • Membrane Proteins
  • HSP70 Heat-Shock Proteins
  • Dogs
  • Chromatography, Liquid