Plank B, Wyskovsky W, Hellmann G, Suko J
Biochim Biophys Acta. 1983 Jul 13;732(1):99-109. doi: 10.1016/0005-2736(83)90191-8.
The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25 degrees C, pH 7.0, in the presence of oxalate and a low free Ca2+ concentration (approx. 0.5 microM) was increased from 0.091 to 0.162 mumol . mg-1 . min-1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca2+ (preincubation with magnesium, ATP and 100 microM CaCl2; approx. 75 microM free Ca2+). Half-maximal activation of calcium uptake occurs under these conditions at 10-20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca2+-, Mg2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 microM Ca2+ and assayed at approx. 10 microM Ca2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine. More than 90% of the incorporated [32P]phosphate is confined to a 9-11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 microM Ca2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 microM Ca2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.
在25℃、pH 7.0、存在草酸盐和低游离钙离子浓度（约0.5微摩尔）的条件下，测定犬心脏肌浆网囊泡的钙转运速率。当在较高游离钙离子浓度（与镁、ATP和100微摩尔氯化钙预孵育；约75微摩尔游离钙离子）存在下进行钙摄取测定之前，先进行钙、钙调蛋白依赖性磷酸化反应时，100纳摩尔钙调蛋白可使该速率从0.091增加至0.162微摩尔·毫克⁻¹·分钟⁻¹。在这些条件下，钙摄取的半最大激活发生在10 - 20纳摩尔钙调蛋白浓度时。肌浆网的钙激活ATP水解酶的钙、镁依赖性转运ATP水解速率，随着钙转运速率的增加，在100纳摩尔钙调蛋白作用下也增加；与钙无关的ATP分解不受影响。肌浆网在约75微摩尔钙离子预孵育后，在约10微摩尔钙离子条件下测定的钙、钙调蛋白依赖性磷酸化，最大可达3纳摩尔/毫克蛋白质，在约8纳摩尔钙调蛋白时达到半最大激活；0.5毫摩尔三氟拉嗪可使其消除。超过90%掺入的[³²P]磷酸盐局限于一种9 - 11千道尔顿的蛋白质，该蛋白质也可被环磷酸腺苷依赖性蛋白激酶的催化亚基磷酸化，很可能代表受磷蛋白的一个亚基。在约75微摩尔钙离子存在但无ATP的情况下，肌浆网囊泡与钙调蛋白预孵育后，100纳摩尔钙调蛋白对在0.5微摩尔钙离子条件下测定的钙摄取速率的刺激作用较小，且与显著程度的钙调蛋白依赖性磷酸化相关。然而，在无钙和ATP的情况下与钙调蛋白预孵育后，对钙摄取和钙调蛋白依赖性磷酸化的刺激作用均不存在，这表明这些过程之间存在因果关系。
