Ogawa Y, Kurebayashi N, Harafuji H
J Biochem. 1986 Nov;100(5):1305-18. doi: 10.1093/oxfordjournals.jbchem.a121837.
In order to obtain a better understanding of the mechanism of the function of fragmented sarcoplasmic reticulum (FSR), we examined the binding of beta,gamma-methylene [3H]adenosine triphosphate (AMPOPCP), an unhydrolyzable ATP analogue, and 45Ca to FSR from bullfrog skeletal muscle. In medium containing 100 mM KCl and 20 mM Tris-maleate (pH 6.80) on ice, FSR has a single class of [3H]AMPOPCP-binding sites which amount to 4.4-8.6 nmol/mg protein (usually about 7 nmol/mg protein). The affinity was in the range of 6.2-12.3 X 10(3) M-1 in the absence of Ca2+. Ca2+ increased the affinity for AMPOPCP without changing the total number of binding sites, whereas Mg2+ decreased it. The change of the affinity is due to the direct effect of Ca2+ and Mg2+ on FSR. The possibility that Mg-AMPOPCP, Ca-AMPOPCP, and free AMPOPCP might have different affinities to FSR is excluded. The extent of Ca2+-induced enhancement in AMPOPCP binding is dependent not only on Ca2+ concentration but also on the concentration of AMPOPCP. The binding sites for AMPOPCP are likely to be the ATP-binding sites on Ca2+-ATPase protein on the basis of several lines of evidence, including competition between ATP, ADP, or AMP. FSR also binds 7-13 nmol Ca/mg protein (usually about 8 nmol/mg protein) with the affinity of 4-14 X 10(4) M-1 in the absence of the nucleotide in a similar medium containing 4 mM MgCl2. The ratio of Ca-binding sites to AMPOPCP-binding sites is mostly 1, but occasionally 2, corresponding to the ratio of Ca accumulated to ATP hydrolyzed by frog FSR. In the presence of a sufficient amount of the nucleotide, the affinity for Ca2+ was also increased. These findings are well explained by the random sequence binding model of Ca2+ and AMPOPCP, which bind to FSR with positive cooperative interaction between them. However, high concentrations of the nucleotide result in a negative cooperative interaction in the nucleotide binding in the presence of Ca2+, whereas no cooperativity is observed in the absence of Ca2+. Stimulation of Ca binding by AMPOPCP is also correspondingly affected. Comparative studies show that rabbit skeletal muscle FSR, in contrast to the frog one, shows negative cooperativity in its interactions with Ca2+ and AMPOPCP under some conditions and that the ratio of Ca-binding sites to AMPOPCP-binding sites is 2, corresponding to the well-known stoichiometry with ATP.
为了更好地理解肌浆网片段（FSR）的功能机制，我们检测了β,γ-亚甲基[3H]三磷酸腺苷（AMPOPCP，一种不可水解的ATP类似物）以及45Ca与牛蛙骨骼肌FSR的结合情况。在含有100 mM KCl和20 mM Tris-马来酸（pH 6.80）的介质中于冰上操作时，FSR具有一类[3H]AMPOPCP结合位点，其数量为4.4 - 8.6 nmol/mg蛋白质（通常约为7 nmol/mg蛋白质）。在无Ca2+时，亲和力范围为6.2 - 12.3×10(3) M-1。Ca2+增加了对AMPOPCP的亲和力，而不改变结合位点的总数，而Mg2+则降低了亲和力。亲和力的变化是由于Ca2+和Mg2+对FSR的直接作用。排除了Mg-AMPOPCP、Ca-AMPOPCP和游离AMPOPCP对FSR可能具有不同亲和力的可能性。Ca2+诱导的AMPOPCP结合增强程度不仅取决于Ca2+浓度，还取决于AMPOPCP的浓度。基于包括ATP、ADP或AMP之间的竞争等多条证据，AMPOPCP的结合位点可能是Ca2+-ATP酶蛋白上的ATP结合位点。在含有4 mM MgCl2的类似介质中，在无核苷酸的情况下，FSR还能以4 - 14×10(4) M-1的亲和力结合7 - 13 nmol Ca/mg蛋白质（通常约为8 nmol/mg蛋白质）。Ca结合位点与AMPOPCP结合位点的比例大多为1，但偶尔为2，这与青蛙FSR积累的Ca与水解的ATP的比例相对应。在存在足够量核苷酸的情况下，对Ca2+的亲和力也会增加。这些发现可以通过Ca2+和AMPOPCP的随机序列结合模型得到很好的解释，它们以正协同相互作用结合到FSR上。然而，在存在Ca2+的情况下，高浓度的核苷酸会导致核苷酸结合中的负协同相互作用，而在无Ca2+时未观察到协同作用。AMPOPCP对Ca结合的刺激也相应受到影响。比较研究表明，与青蛙的FSR不同，兔骨骼肌FSR在某些条件下与Ca2+和AMPOPCP的相互作用中表现出负协同作用，并且Ca结合位点与AMPOPCP结合位点的比例为2，这与众所周知的与ATP的化学计量关系相对应。
