Synthesis of spin‐labeled 2‐azido‐ATP: Evidence for distinct nucleotide‐binding sites in calcium pump protein from sarcoplasmic reticulum

FEBS Letters

Fleischer, Sidney¹  Jakobs, Pia²  Trommer, Wolfgang E.²  McIntyre, J.Oliver¹  Sauer, Hubertus E.² 

[1] Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235, USA;Fachbereich Chemie, Universität Kaiserslautern, Erwin-Schrödinger-Straße, D-6750 Kaiserslautern, FRG

关键词: Protein;    Ca2+ pump;    ATPase;    Ca2+;    Photoaffinity labeling;    Spin label;    Perdeuteration;    Azido-ATP;    2-;    2N3 (8N3);    2-azido (8-azido) derivative of ATP;    SL-;    spin label [C3′-(2;    2;    5;    5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester)] derivatives;    in which the SL is at the 2′- or 3′-hydroxyl of the ribose;    HPLC;    high-performance liquid chromatography;    TLC;    thin-layer chromatography;    Mes;    4-morpholineethanesulfonic acid;    SR;    sarcoplasmic reticulum;   

DOI  :  10.1016/0014-5793(89)80998-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A spin-labeled and photoreactive derivative of ATP was synthesized with the spin label attached to the 2′- or 3′-position of the ribose moiety and an azido group to C2 of the adenine ring (SL-2N₃-ATP). Irradiation of this compound at 350 nm generates a nitrene, which then reacts with nucleophiles in its vicinity. SL-2N₃-ATP, in the presence of Ca²⁺, was hydrolyzed by the calcium pump protein (Ca²⁺-ATPase) of fast twitch skeletal muscle sarcoplasmic reticulum. The SL-2N₃-ATP—enzyme complex in the absence of Ca²⁺ exhibited strongly immobilized ESR spectra. ESR spectra obtained after covalent incorporation of SL-2N₃-ATP into Ca²⁺-ATPase and removal of freely tumbling SL-2N₃-ATP exhibited motionally constrained species indicative of distinct and possibly adjacent ATP-binding sites. By contrast, with SL-ATP devoid of the azido group or with the corresponding ‘non-cleavable’ β,γ-methylene triphosphate analogue (SL-AMP-PCP), two distinct sites were not as well resolved in the ESR spectra due to spectral overlap with the signal from the freely tumbling fraction even with the enhanced spectral resolution provided by perdeuteration of the spin label. Thus, SL-2N₃-ATP may have general application for ESR studies of ATP-dependent proteins under conditions in which noncovalent interactions are too weak for motionally restricted species to be resolved.

【 授权许可】

Unknown