Hunter G W, Negash S, Squier T C
Biochemistry and Biophysics Section, Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045-2106, USA.
Biochemistry. 1999 Jan 26;38(4):1356-64. doi: 10.1021/bi9822224.
Phospholipids containing phosphoethanolamine (PE) headgroups within biological membranes have been suggested to be important with respect to the functional regulation of membrane proteins, including the Ca-ATPase in sarcoplasmic reticulum (SR). To investigate the role of PE headgroups in modulating the catalytic activity of the Ca-ATPase, we have reconstituted the Ca-ATPase into unilamellar liposomes containing defined amounts of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC). The enzymatic activity of the Ca-ATPase progressively increases upon incorporation of increasing amounts of PE into reconstituted vesicles, and approaches that characteristic of native SR membranes. To identify structural changes that correlate with enzyme activation, we have used frequency-domain phosphorescence spectroscopy to measure the rotational dynamics of erythrosin isothiocyanate covalently bound to Lys464 in the phosphorylation domain of the Ca-ATPase. Progressive increases in the rotational dynamics of the phosphorylation domain result from the incorporation of increasing amounts of DOPE, and correlate with enhanced enzymatic function. These results suggest that PE headgroups induce dynamic structural rearrangements involving the phosphorylation domain that modify the rates of nucleotide utilization. In contrast, no changes in the rotational dynamics of the lipid acyl chains are observed irrespective of the PE content. Therefore, the enhanced ATP hydrolytic activity associated with the incorporation of DOPE into these proteoliposomes is the result of specific noncovalent interactions involving PE phospholipid headgroups and the Ca-ATPase.
生物膜中含有磷酸乙醇胺（PE）头部基团的磷脂，被认为对于膜蛋白的功能调节很重要，包括肌浆网（SR）中的钙ATP酶。为了研究PE头部基团在调节钙ATP酶催化活性中的作用，我们将钙ATP酶重组到含有特定量二油酰磷脂酰乙醇胺（DOPE）和二油酰磷脂酰胆碱（DOPC）的单层脂质体中。随着越来越多的PE掺入重组囊泡中，钙ATP酶的酶活性逐渐增加，并接近天然SR膜的特征活性。为了确定与酶激活相关的结构变化，我们使用频域磷光光谱法来测量与钙ATP酶磷酸化结构域中赖氨酸464共价结合的异硫氰酸赤藓红的旋转动力学。磷酸化结构域旋转动力学的逐渐增加是由于越来越多的DOPE掺入所致，并且与增强的酶功能相关。这些结果表明，PE头部基团诱导涉及磷酸化结构域的动态结构重排，从而改变核苷酸利用的速率。相比之下，无论PE含量如何，脂质酰链的旋转动力学均未观察到变化。因此，将DOPE掺入这些蛋白脂质体中所带来的ATP水解活性增强，是涉及PE磷脂头部基团和钙ATP酶的特定非共价相互作用的结果。
