title: Interaction of endoplasmic reticulum chaperone GRP94 with peptide substrates is adenine nucleotide-independent.
abstract: GRP94, the endoplasmic reticulum paralog of hsp90, has recently been identified as a peptide and adenine nucleotide-binding protein. To determine if adenine nucleotides directly contribute to the regulation of GRP94 peptide binding activity, an in vitro peptide binding assay was developed. Using purified GRP94, we observed specific, saturable, temperature-sensitive binding of the peptide VSV8, a known in vivo ligand. ATP was without effect on VSV8 binding to GRP94, whether present during or subsequent to peptide binding. To evaluate the interaction of GRP94 with adenine nucleotides, the ATP binding and hydrolysis activities were directly assayed. Only negligible binding of ATP to GRP94 was observed. In addition, analysis of the GRP94 adenine nucleotide content indicated that GRP94 did not copurify with bound adenine nucleotides. GRP94 preparations exhibited low ATPase and apparent autophosphorylation activities. Further purification, combined with inhibitor studies, indicated that both activities were the result of trace contamination (<0.1%) with casein kinase II. On the basis of these data, we propose that the peptide binding activity of GRP94 is adenine nucleotide-independent and that ATP binding and hydrolysis are not inherent properties of GRP94.
authors.affiliation: Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: P A
authors.initials: PA
authors.lastname: Wearsch
authors.forename: C V
authors.initials: CV
authors.lastname: Nicchitta
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D010455:Peptides; C018551:glucose-regulated proteins; D000255:Adenosine Triphosphate