Lamers J M, Stinis J T
Biochim Biophys Acta. 1980 Aug 21;624(2):443-59. doi: 10.1016/0005-2795(80)90086-0.
A rat heart sarcolemmal preparation could be obtained in which both 5'-nucleotidase and adenylate cyclase were enriched approx. 9-fold by subjecting a homogenate to a discontinuous sucrose gradient, without the use of a high salt extraction. After incubation of this fraction with Mg[gamma-32P]ATP, the majority of 32P incorporated was present in 24 000- and 9000-dalton protein components. Only when a heart cytosol fraction or a purified cyclic AMP-dependent protein kinase was added, was enhancement of 32P-incorporaton found by addition of cyclic AMP. The 9000- and 24 000-dalton proteins appeared to be interconvertible. The degree of conversion could be affected by changing the temperature during solubilizaion of the membranes in SDS prior to electrophoresis. This suggested that the 24 000-dalton protein does not correspond to phospholamban, first identified by others in canine heart sarcoplasmic reticulum. Moreover, it could be excluded that the 24 000-dalton protein was derived from contaminating myofibrillar troponin I. When the sarcolemmal fraction was preincubated with Ca2+, Mg2+, ATP and oxalate, contaminating sarcoplasmic reticulum vesicles, loaded with calcium oxalate, settled to a greater density in the sucrose gradient. Membrane constituents other than those with enzymatic activity were monitored to confirm the separation between sarcolemmal and sarcoplasmic reticulum membranes: Coomassie blue staining material, sialic acid, cholesterol and phospholipid. The 24 000- and 9000-dalton proteins were equally distributed among the sarolemmal and sarcoplasmic reticulum fractions present in the sucrose gradient. However, the rate of 32P-incorporation in the presence of heart cytosol fraction was much slowr in the sarcoplasmic reticulum than in the sarcolemmal fraction.
可以制备大鼠心脏肌膜制剂，通过将匀浆置于不连续蔗糖梯度中，无需使用高盐提取，5'-核苷酸酶和腺苷酸环化酶均可富集约9倍。用Mg[γ-32P]ATP孵育该组分后，掺入的大部分32P存在于24000道尔顿和9000道尔顿的蛋白质组分中。只有当加入心脏胞质溶胶组分或纯化的环磷酸腺苷依赖性蛋白激酶时，加入环磷酸腺苷才会发现32P掺入增加。9000道尔顿和24000道尔顿的蛋白质似乎可以相互转化。转化程度可能会受到在SDS中电泳前溶解膜时改变温度的影响。这表明24000道尔顿的蛋白质与其他人首先在犬心脏肌浆网中鉴定出的受磷蛋白不对应。此外，可以排除24000道尔顿的蛋白质来源于污染的肌原纤维肌钙蛋白I。当肌膜组分与Ca2+、Mg2+、ATP和草酸盐预孵育时，装载有草酸钙的污染肌浆网囊泡在蔗糖梯度中沉降到更高的密度。监测除具有酶活性的成分以外的膜成分，以确认肌膜和肌浆网膜之间的分离：考马斯亮蓝染色物质、唾液酸、胆固醇和磷脂。24000道尔顿和9000道尔顿的蛋白质在蔗糖梯度中存在的肌膜和肌浆网组分中均匀分布。然而，在存在心脏胞质溶胶组分的情况下，肌浆网中32P掺入的速率比肌膜组分慢得多。
