Nakamaru Y, Nomura K
J Biochem. 1977 Feb;81(2):321-8. doi: 10.1093/oxfordjournals.jbchem.a131461.
The interaction of Pi with sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle was studied using bromocresol purple (BCP) as a probe and a dual-wavelength spectrophotometer. Two kinds of absorption-intensity changes controlled by a low concentration of Ca2+ (greater than 10(-6)M) were observed after addition of Pi; an increase phase (in the presence of Ca2+), and a decrease phase (in the presence of EGTA). The increase phase was rapid, Ca2+-dependent, Mg2+-enhanced (depressed by high Mg2+ concentration) and not inhibited by PCMB and was suggested to reflect the formation of an SR-Pi complex. The decrease phase was slower than the increase phase, and was strongly inhibited by the low concentration of Ca2+. It required Mg2+, and was completely inhibited by p-chloromercuribenzoate or deoxycholate. It was suggested to reflect the formation of SR-Pi (phosphorylated protein). ATP inhibited this phase by converting it completely to an SR-MgATP phase. PPi was effective for inducing the decrease phase but PPPi was not. From measurements of these phases, the association constants of the SR-Pi complex and SR-Pi at pH 8.8 in the reaction scheme, SR + Pi in equilibrium SR-Pi in equilibrium SR-Pi, were calculated as 5.4 X 10 M-1 and 1.8 X 10(3) M-1, respectively. From the completely different responses of SR-Pi and SR-Pi observed with BCP a marked difference in the conformations of these enzyme states was suggested.
以溴甲酚紫（BCP）为探针，使用双波长分光光度计研究了无机磷酸（Pi）与从兔骨骼肌分离出的肌浆网（SR）的相互作用。加入Pi后，观察到两种由低浓度Ca2+（大于10^(-6)M）控制的吸收强度变化；一个增加阶段（在Ca2+存在下）和一个减少阶段（在乙二醇双四乙酸（EGTA）存在下）。增加阶段迅速，依赖Ca2+，Mg2+增强（高Mg2+浓度会抑制），且不受对氯汞苯甲酸（PCMB）抑制，提示反映了SR-Pi复合物的形成。减少阶段比增加阶段慢，且受到低浓度Ca2+的强烈抑制。它需要Mg2+，并被对氯汞苯甲酸或脱氧胆酸盐完全抑制。提示反映了SR-Pi（磷酸化蛋白）的形成。ATP通过将其完全转化为SR-MgATP阶段来抑制这个阶段。焦磷酸（PPi）对诱导减少阶段有效，但三磷酸焦磷酸（PPPi）无效。通过对这些阶段的测量，计算出反应体系中pH 8.8时SR-Pi复合物和SR-Pi（在反应式SR + Pi ⇌ SR-Pi ⇌ SR-Pi中）的缔合常数分别为5.4×10 M^(-1)和1.8×10^(3) M^(-1)。从用BCP观察到的SR-Pi和SR-Pi的完全不同反应来看，提示这些酶状态的构象存在显著差异。
