Gupta R C, Khandelwal R L, Sulakhe P V
Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Canada.
Mol Cell Biochem. 1990 Sep 3;97(1):53-66. doi: 10.1007/BF00231701.
The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn2+, was unaffected by Mg2+, Ca2+, and Ba2+, and was markedly inhibited by Ni2+, Fe2+, Zn2+ and Cu2+. When assayed in the presence of calmodulin, many divalent metals (Ni2+, Zn2+, Pb2+, Cd2+), besides Mn2+, increased modestly the phosphatase activity at low concentrations (10-100 microM) and inhibited it markedly at high concentrations. Ca2(+)-calmodulin stimulated phosphatase activity was antagonized by Ni2+, Zn2+, Fe2+, Cu2+, Pb2+, at low concentrations (50 microM), and by Ba2+, Cd2+ at slightly higher concentrations (greater than 100 microM); Mn2+ and Co2+ (50 microM to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn2+ (+/- calmodulin) and Ca2+ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn2+ led to a high activity conformation state of calcineurin that was 'long-lived' or 'pseudo-irreversible'. Such Mn2(+)-activated state of calcineurin exhibited no discernible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg2+ supported the intrinsic phosphatase activity, although to a lesser degree than Mn2+. The latter cation, compared to Mg2+ and Ni2+, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.
研究了二价金属、金属螯合剂（乙二胺四乙酸、乙二醇双（2-氨基乙基醚）四乙酸）和十二烷基硫酸钠对分离的牛脑钙调神经磷酸酶磷酸酶活性的影响，该活性在不存在钙调蛋白（称为内在活性）和存在钙调蛋白的情况下进行测定。内在磷酸酶活性在锰离子作用下增强，在镁离子、钙离子和钡离子作用下无变化，在镍离子、亚铁离子、锌离子和铜离子作用下受到显著抑制。在钙调蛋白存在的情况下进行测定时，除锰离子外，许多二价金属（镍离子、锌离子、铅离子、镉离子）在低浓度（10 - 100微摩尔）时适度增加磷酸酶活性，在高浓度时则显著抑制该活性。钙离子 - 钙调蛋白刺激的磷酸酶活性在低浓度（50微摩尔）时受到镍离子、锌离子、亚铁离子、铜离子、铅离子的拮抗，在稍高浓度（大于100微摩尔）时受到钡离子、镉离子的拮抗；而锰离子和钴离子（50微摩尔至1毫摩尔）实际上增强了该活性。乙二胺四乙酸和乙二醇双（2-氨基乙基醚）四乙酸以浓度和时间依赖的方式抑制内在磷酸酶活性，尤其是胰蛋白酶处理的钙调神经磷酸酶的活性。低浓度（0.005%）的十二烷基硫酸钠增强磷酸酶活性，高浓度时则抑制该活性。锰离子（无论有无钙调蛋白）和仅在有钙调蛋白存在时的钙离子，可增加用低浓度十二烷基硫酸钠测定的磷酸酶活性。在含有十二烷基硫酸钠的测定中，乙二胺四乙酸对内在磷酸酶的抑制作用几乎被消除。钙调神经磷酸酶预先暴露于锰离子会导致其处于高活性构象状态，这种状态“寿命长”或“假不可逆”。这种锰离子激活的钙调神经磷酸酶状态在对髓鞘碱性蛋白的亲和力或其受三氟拉嗪抑制方面没有明显变化。在碱性pH值下，镁离子支持内在磷酸酶活性，尽管程度低于锰离子。与镁离子和镍离子相比，锰离子也是以组蛋白（III - S）和肌球蛋白轻链为底物测定钙调神经磷酸酶活性的更强有力的刺激剂。
