Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump.
Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump.
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犬心脏肌浆网钙泵的蛋白水解激活。
DOI：
10.1021/bi00367a021
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Kasinathan,C
中科院分区：
文献类型：
--
作者：
Kirchberger,MA;Borchman,D;Kasinathan,C
1986年4月29日收到的修订版摘要：用温和的胰蛋白酶处理主要由肌浆网囊泡组成的犬心脏微粒体，可使琥珀酸促进的钙摄取活化数倍。钙摄取的增加与ATP水解的增加有关。除胰蛋白酶以外的蛋白酶也是有效的，尽管程度较低。胰蛋白酶产生的Ca 2+浓度依赖性曲线的钙摄取向较低的Ca 2+浓度，这是几乎相同的微粒体的磷酸化产生的环AMP依赖性蛋白激酶时，胰蛋白酶和蛋白激酶的最大活化浓度。用胰蛋白酶（1.5±0.1）或蛋白激酶（1.7±0.1）处理微粒体后，Ca 2+依赖性的Hill数（±SD）相似，与未处理对照微粒体的Hill数（分别为1.6±0.1和1.8±0.1）无显著差异。十二烷基硫酸钠-聚丙烯酰胺电泳凝胶的放射自显影表明，与用胰蛋白酶抑制剂灭活的胰蛋白酶预处理的对照微粒体相比，胰蛋白酶处理的微粒体在环AMP依赖性蛋白激酶和[7- 32 P] ATP存在下孵育时，32 P掺入受磷蛋白（Mr 27.3 K）或其假定的单体亚基（M，5.5 K）明显减少。增加胰蛋白酶浓度对钙摄取的激活被磷蛋白磷酸化的减少所抵消。胰蛋白酶处理的微粒体先前硫代磷酸化的存在下，环AMP依赖性蛋白激酶和[7- 35 S]硫代ATP没有导致35 S标记的损失从受磷蛋白，这表明磷酸化的受磷蛋白保护免受胰蛋白酶的攻击。胰蛋白酶处理的微粒体制备的兔快骨骼肌，其中不含受磷蛋白，没有刺激钙的吸收。然而，在有利于（Ca ~（2+）+Mg ~（2+））激活的ATP酶反应的100千道尔顿酰基磷蛋白中间体形成的条件下孵育的兔腹肌和犬心脏微粒体的凝胶放射自显影显示出不同浓度胰蛋白酶的相同效应模式。这些数据表明，胰蛋白酶切割两种钙泵蛋白上的相似位点。因此，胰蛋白酶对心肌微粒体钙摄取的刺激作用似乎不是由于对钙泵蛋白的直接作用。这些数据与受磷蛋白片段与细胞质通讯的模型一致。如果该片段被蛋白水解切割，则钙转运的基础速率增加。钙转运的蛋白水解激活表明这种未磷酸化的胞质片段的存在对钙泵具有抑制作用。
Revised Manuscript Received April 29, 1986 abstract: Mild trypsin treatment of canine cardiac microsomes consisting largely of sarcoplasmic reticulum vesicles produced a severalfold activation of oxalate-facilitatedcalcium uptake. Theincrease in calcium uptake was associated with an increase in ATP hydrolysis. Proteases other than trypsin were also effective although to a lesser degree. Trypsin produced a shift of the Ca2+ concentration dependency curve for calcium uptake toward lower Ca2+ concentrations, which was almost identical with that produced by phosphorylation of microsomes by cyclic AMP dependent protein kinase when the trypsin and the protein kinase were present at maximally activating concentrations. The Hill numbers (±SD) of the Ca2+ dependency after treatment of microsomes with trypsin (1.5±0.1) or protein kinase (1.7±0.1) were similar and were not significantly different from those for untreated control microsomes (1.6±0.1 and 1.8±0.1, respectively). Autoradiograms of sodium dodecyl sulfate-polyacrylamide electrophoretic gels indicate that 32P incorporation into phospholamban (Mr 27.3 K) or its presumed monomeric subunit (M, 5.5 K) was markedly reduced when trypsin-treated microsomes were incubated in the presence of cyclic AMP dependent protein kinase and [7-32P] ATP compared to control microsomes incubated similarly butpretreated with trypsin inhibitor inactivated trypsin. The activation of calcium uptake by increasing concentrations of trypsin was paralleled by the reduction of phosphorylation of phospholamban. Trypsin treatment of microsomes previously thio-phosphorylated in the presence of cyclic AMP dependent protein kinase and [7-35S] thio-ATP did not result in a loss of 35S label from phospholamban, which suggests that phosphorylation of phospholamban protects against trypsin attack. Trypsin treatment of microsomes prepared from rabbit fast skeletal muscle, which does not contain phospholamban, did not stimulate calcium uptake. However, autoradiograms of gels of rabbit skeletalmuscle and canine cardiac microsomes incubated under conditions favorable for the formation of the 100-kilodalton acylphosphoprotein intermediate of the (Ca2++ Mg2+)-activated ATPase reaction showed an identical pattern of effects of different concentrations of trypsin. These data suggest that trypsin cleaves similar sites on both calcium pump proteins. Therefore, the stimulatory effect of trypsin on cardiac microsomal calcium uptake does notappear to be due to a direct effect on the calcium pump protein. The data are consistent with a model in which a segment of phospholamban is in communicationwith the cytoplasm. If the segment is cleaved proteolytically, the basal rate of calcium transport is increased. The proteolytic activation of calcium transport would suggest that the presence of this unphosphorylated cytosolic segment has an inhibitory effect on the calcium pump.