Kemmer T P, Bayerdörffer E, Will H, Schulz I
Max-Planck-Institut für Biophysik, Frankfurt, Federal Republic of Germany.
J Biol Chem. 1987 Oct 5;262(28):13758-64.
Anion dependence of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase and its phosphorylated intermediate have been characterized in both "intact" and "broken" vesicles from endoplasmic reticulum of rat pancreatic acinar cells using adenosine 5'-[gamma-32P] triphosphate ([gamma-32P]ATP). In intact vesicles (Ca2+ + K+)-Mg2+-ATPase activity was higher in the presence of Cl- or Br- as compared to NO3-, SCN-, cyclamate-, SO4(2-) or SO3(2-). Incorporation of 32P from [gamma-32P]ATP into the 100-kDa intermediate of this Ca2+ATPase was also higher in the presence of Cl-, Br-, NO3- or SCN- as compared to cyclamate-, SO4(2-) or SO3(2-). When the membrane permeability barrier to anions was abolished by breaking vesicle membrane with the detergent Triton X-100 (0.015%) (Ca2+ + K+)-Mg2+ATPase activity in the presence of weakly permeant anions, such as SO4(2-) and cyclamate-, increased to the level obtained with Cl-. However, 32P incorporation into 100-kDa protein was still higher in the presence of Cl- as compared to cyclamate-, indicating a direct effect of Cl- on the Ca2+ATPase molecule. The anion transport blocker 4,4-diisothiocyanostilbene-2,2-disulfonate (DIDS) inhibited (Ca2+ + K+)-Mg2+ATPase activity to about 10% of the Cl- stimulation level, irrespective of the sort of anions present in both intact and broken vesicles. This indicates a direct effect of DIDS on (Ca2+ + K+)-Mg2+ATPase. K+ ionophore valinomycin influenced (Ca2+ + K+)-Mg2+ATPase activity according to the actual K+ gradient: Ko+ greater than Ki+ caused inhibition, Ko+ less than Ki+ caused stimulation. From these results we conclude that Ca2+ transport into endoplasmic reticulum is coupled to ion movements which must occur to maintain electroneutrality.
利用腺苷5'-[γ-32P]三磷酸（[γ-32P]ATP），对大鼠胰腺腺泡细胞内质网“完整”和“破碎”囊泡中（Ca2+ + K+）刺激的Mg2+依赖性转运ATP酶及其磷酸化中间体的阴离子依赖性进行了表征。在完整囊泡中，与NO3-、SCN-、环己基氨基磺酸盐-、SO4(2-)或SO3(2-)相比，在Cl-或Br-存在下，（Ca2+ + K+）-Mg2+-ATP酶活性更高。与环己基氨基磺酸盐-、SO4(2-)或SO3(2-)相比，在Cl-、Br-、NO3-或SCN-存在下，[γ-32P]ATP中的32P掺入该Ca2+ATP酶的100 kDa中间体中的量也更高。当用去污剂Triton X-100（0.015%）破坏囊泡膜消除膜对阴离子的通透性屏障时，在弱渗透性阴离子（如SO4(2-)和环己基氨基磺酸盐-）存在下，（Ca2+ + K+）-Mg2+ATP酶活性增加到与Cl-存在时相同的水平。然而，与环己基氨基磺酸盐-相比，在Cl-存在下，32P掺入100 kDa蛋白中的量仍然更高，这表明Cl-对Ca2+ATP酶分子有直接作用。阴离子转运阻滞剂4,4-二异硫氰基芪-2,2-二磺酸盐（DIDS）将（Ca2+ + K+）-Mg2+ATP酶活性抑制至Cl-刺激水平的约10%，无论完整和破碎囊泡中存在何种阴离子。这表明DIDS对（Ca2+ + K+）-Mg2+ATP酶有直接作用。K+离子载体缬氨霉素根据实际K+梯度影响（Ca2+ + K+）-Mg2+ATP酶活性：Ko+大于Ki+导致抑制，Ko+小于Ki+导致刺激。从这些结果我们得出结论，Ca2+向内质网的转运与为维持电中性而必须发生的离子运动相偶联。
