Wearsch P A, Nicchitta C V
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 1997 Feb 21;272(8):5152-6. doi: 10.1074/jbc.272.8.5152.
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.
GRP94是hsp90在内质网中的同源蛋白，最近被鉴定为一种肽和腺嘌呤核苷酸结合蛋白。为了确定腺嘌呤核苷酸是否直接参与GRP94肽结合活性的调节，我们开发了一种体外肽结合测定法。使用纯化的GRP94，我们观察到了肽VSV8（一种已知的体内配体）的特异性、可饱和、温度敏感的结合。无论在肽结合期间还是之后存在，ATP对VSV8与GRP94的结合均无影响。为了评估GRP94与腺嘌呤核苷酸的相互作用，我们直接测定了ATP结合和水解活性。仅观察到ATP与GRP94的结合可忽略不计。此外，对GRP94腺嘌呤核苷酸含量的分析表明，GRP94与结合的腺嘌呤核苷酸没有共纯化。GRP94制剂表现出低ATP酶活性和明显的自磷酸化活性。进一步纯化并结合抑制剂研究表明，这两种活性均是酪蛋白激酶II痕量污染（<0.1%）的结果。基于这些数据，我们提出GRP94的肽结合活性不依赖于腺嘌呤核苷酸，并且ATP结合和水解不是GRP94的固有特性。
