de Meis L
Departamento de Bioquimica, Universidade Federal do Rio de Janeiro, Ilha do Fundao, Rio de Janeiro, Brasil.
J Biol Chem. 1991 Mar 25;266(9):5736-42.
The Ca2(+)-ATPase found in the light fraction of sarcoplasmic reticulum vesicles can be phosphorylated by Pi, forming an acylphosphate residue at the catalytic site of the enzyme. This reaction was inhibited by the phenothiazines trifluoperazine, chlorpromazine, imipramine, and fluphenazine and by the beta-adrenergic blocking agents propranolol and alprenolol. The inhibition was reversed by raising either the Pi or the Mg2+ concentration in the medium and was not affected by the presence of K+. Phosphorylation of the Ca2(+)-ATPase by Pi was also inhibited by ruthenium red and spermidine. These compounds compete with Mg2+, but, unlike the phenothiazines, they did not compete with Pi at the catalytic site, and the inhibition was abolished when K+ was included in the assay medium. The efflux of Ca2+ from loaded vesicles was greatly increased by the phenothiazines and by propranolol and alprenolol. In the presence of 200 microM trifluoperazine, the rate of Ca2+ efflux was higher than 3 mumol of Ca2+/mg of protein/10 s. The activation of efflux by these drugs was antagonized by Pi, Mg2+, K+, Ca2+, ADP, dimethyl sulfoxide, ruthenium red, and spermidine. The increase of Ca2+ efflux caused by trifluoperazine was not correlated with binding of the drug to the membrane lipids. It is concluded that the Ca2+ pump can be uncoupled by different drugs, thereby greatly increasing the efflux of Ca2+ through the ATPase. Displacement of these drugs by the natural ligands of the ATPase blocks the efflux through the uncoupled pathway and limits it to a much smaller rate. Thus, the Ca2(+)-ATPase can operate either as a pump (coupled) or as a Ca2+ channel (uncoupled).
肌浆网囊泡轻组分中发现的Ca2(+)-ATP酶可被无机磷酸（Pi）磷酸化，在酶的催化位点形成酰基磷酸残基。该反应受到吩噻嗪类药物三氟拉嗪、氯丙嗪、丙咪嗪和氟奋乃静以及β-肾上腺素能阻滞剂普萘洛尔和阿普洛尔的抑制。通过提高培养基中的Pi或Mg2+浓度可逆转这种抑制作用，且不受K+存在的影响。Pi对Ca2(+)-ATP酶的磷酸化作用也受到钌红和亚精胺的抑制。这些化合物与Mg2+竞争，但与吩噻嗪类不同，它们不在催化位点与Pi竞争，并且当测定培养基中加入K+时抑制作用消失。吩噻嗪类药物以及普萘洛尔和阿普洛尔可大大增加负载囊泡中Ca2+的外流。在存在200微摩尔三氟拉嗪的情况下，Ca2+外流速率高于3微摩尔Ca2+/毫克蛋白质/10秒。这些药物对外流的激活作用受到Pi、Mg2+、K+、Ca2+、ADP、二甲亚砜、钌红和亚精胺的拮抗。三氟拉嗪引起的Ca2+外流增加与药物与膜脂的结合无关。结论是Ca2+泵可被不同药物解偶联，从而通过ATP酶大大增加Ca2+的外流。ATP酶的天然配体取代这些药物可阻断通过解偶联途径的外流，并将其限制在小得多的速率。因此，Ca2(+)-ATP酶既可以作为泵（偶联）发挥作用，也可以作为Ca2+通道（解偶联）发挥作用。
