Unexpected Phosphoryl Transfer from Asp351 to Fluorescein Attached to Lys515 in Sarcoplasmic Reticulum Ca2+-ATPase

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• 作者：David B. McIntosh ; C&#xe9 ; dric Montigny ; Philippe Champeil
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：June 17, 2008
• 年：2008
• 卷：47
• 期：24
• 页码：6386 - 6393
• 全文大小：6851K
• 年卷期：v.47,no.24(June 17, 2008)
• ISSN：1520-4995

文摘

Sarcoplasmic reticulum Ca²⁺-ATPase is an ion pump whose catalytic cycle includes the transient formation of an acyl phosphate at Asp³⁵¹, and fluorescein isothiocyanate is a covalent inhibitor of ATP binding to this pump, known to specifically derivatize Lys⁵¹⁵ in the nucleotide-binding site. It was previously found that an unusually stable, phosphorylated form of fluorescein-ATPase, with low fluorescence, is obtained following Ca²⁺ loading with acetyl phosphate as energy source and then chelation with EGTA of Ca²⁺ on the cytosolic side. Here we show that the phospho-linkage in this low fluorescent species is stable at alkaline pH, unlike the acyl phosphate at Asp³⁵¹. Moreover, the low fluorescence and stable phosphoryl group track together in primary and secondary tryptic subfragments, separated by SDS−PAGE after denaturation. Finally, normal fluorescence and absorbance are recovered upon treatment with alkaline phosphatase after extensive trypsinolysis. We conclude that the low fluorescent species is the result of the phosphoryl group being transferred from Asp³⁵¹ to the fluorescein moiety during pump reversal, yielding fluorescein monophosphate tethered to Ca²⁺-ATPase.