Barrabin H, Scofano H M, Inesi G
Biochemistry. 1984 Mar 27;23(7):1542-8. doi: 10.1021/bi00302a031.
The stoichiometry of phosphorylation (catalytic) sites in sarcoplasmic reticulum vesicles ( SRV ) and SR ATPase purified by differential solubilization with deoxycholate was found to be 4.77 +/- 0.4 and 6.05 +/- 0.18 nmol/mg of protein, respectively, when phosphorylation was carried out under conditions permitting 32P labeling of nearly all sites. Assuming that each site corresponds to a single 115K ATPase chain, the observed site stoichiometry accounts only for 55% and 70% of the total protein. Failure to obtain higher phosphorylation levels was due to the presence of nonspecific protein contaminants in SRV or to the presence of inactive aggregates in the ATPase purified with deoxycholate. This was demonstrated by dissolving SRV and purified ATPase with lithium dodecyl sulfate, subjecting them to molecular sieve HPLC, and collecting the elution fractions for determination of protein, measurement of 32P-labeled sites, and electrophoretic analysis. In fact, in the specific elution peak containing the 115K ATPase chains, phosphorylation levels were 6.62 +/- 0.33 and 7.03 +/- 0.18 in SRV and purified ATPase, corresponding to 68% and 86% of the protein in the specific elution peak. An alternate purification method was then developed, based on solubilization of SRV with dodecyl octaethylene glycol monoether ( C12E8 ), separation of delipidated ATPase by anion-exchange chromatography, and enzyme reactivation with phosphatidylcholine. This preparation yields 7.3 +/- 0.44 nmol of phosphorylation site/mg of protein of the SRV fraction before HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)
当在允许几乎所有位点进行³²P标记的条件下进行磷酸化时，通过用脱氧胆酸盐进行差异增溶纯化的肌浆网囊泡（SRV）和SR ATP酶中磷酸化（催化）位点的化学计量分别为4.77±0.4和6.05±0.18 nmol/mg蛋白质。假设每个位点对应一条单一的115K ATP酶链，观察到的位点化学计量仅占总蛋白质的55%和70%。未能获得更高的磷酸化水平是由于SRV中存在非特异性蛋白质污染物或用脱氧胆酸盐纯化的ATP酶中存在无活性聚集体。通过用十二烷基硫酸钠溶解SRV和纯化的ATP酶，使其通过分子筛高效液相色谱，并收集洗脱级分以测定蛋白质、测量³²P标记位点和进行电泳分析，证明了这一点。事实上，在含有115K ATP酶链的特定洗脱峰中，SRV和纯化的ATP酶中的磷酸化水平分别为6.62±0.33和7.03±0.18，分别对应特定洗脱峰中蛋白质的68%和86%。然后开发了一种替代纯化方法，该方法基于用十二烷基八乙二醇单醚（C12E8）增溶SRV，通过阴离子交换色谱分离脱脂ATP酶，并用磷脂酰胆碱使酶重新活化。在进行高效液相色谱之前，这种制备方法得到的SRV级分的磷酸化位点为7.3±0.44 nmol/mg蛋白质。（摘要截短于250字）
