Verjovski-Almeida S, de Meis L
Biochemistry. 1977 Jan 25;16(2):329-34. doi: 10.1021/bi00621a026.
The effect of pH on the Ca2+-binding sites of high and low affinity, located respectively on the outer and inner surfaces of the sarcoplasmic reticulum membrane, was investigated using intact and leaky sarcoplasmic reticulum vesicles. With the use of intact vesicles, different pH profiles of membrane phosphorylation and rates of nucleoside triphosphate hydrolysis were obtained depending on the assay temperature, on the Ca2+ concentration, and on whether ATP or ITP was used as substrate. The different pH profiles were related to the amount of Ca2+ accumualted by the vesicles, i.e., to different degrees of saturation of the inner, low-affinity Ca2+-binding site. With the use of leaky vesicles, the saturation of the two Ca2+-binding sites could be controlled more precisely since the Ca2+ concentration on both sides of the membrane was equal to the Ca2+ concentration of the assay medium. Using leaky vesicles and measuring the rates of nucleotide hydrolysis, nucleotide-phosphate exchange and membrane phosphorylation by nucleotide as an indication of the degree of saturation of the Ca2+-binding sites, we observed that the affinity of both the high- and low-affinity sites increased three to four orders of magnitude when the pH of the assay medium was increased from 6.1 to 8.65.
利用完整的和有泄漏的肌浆网囊泡，研究了pH对分别位于肌浆网膜外表面和内表面的高亲和力和低亲和力Ca2+结合位点的影响。使用完整囊泡时，根据测定温度、Ca2+浓度以及是否使用ATP或ITP作为底物，可获得不同的膜磷酸化pH曲线和核苷三磷酸水解速率。不同的pH曲线与囊泡积累的Ca2+量有关，即与内部低亲和力Ca2+结合位点的不同饱和程度有关。使用有泄漏的囊泡时，由于膜两侧的Ca2+浓度与测定介质的Ca2+浓度相等，因此可以更精确地控制两个Ca2+结合位点的饱和度。通过使用有泄漏的囊泡并测量核苷酸水解、核苷酸-磷酸交换和核苷酸介导的膜磷酸化速率，以此作为Ca2+结合位点饱和程度的指标，我们观察到当测定介质的pH从6.1增加到8.65时，高亲和力和低亲和力位点的亲和力均增加了三到四个数量级。
