Cuppoletti J, Segel I H
Biochemistry. 1975 Oct 21;14(21):4712-8. doi: 10.1021/bi00692a023.
The active transport of inorganic sulfate by an ATP sulfurylase-negative strain of Penicillium notatum is promoted by H+ ions and metal ions (divalent metal ions being more effective than monovalent metal ions). Initial velocity studies suggest that H+ and SO4(2-) add to the carrier in an ordered sequence (H+ before SO4(2-)), with H+ at equilibrium with free carrier and carrier-H+ complex. The linear reciprocal plots and replots suggest a 1:1 stoichiometry between H+ and SO4(2-). Ca2+ and other divalent metal ions stimulate sulfate transport markedly in buffered suspensions of low ionic strength. The kinetics of the Ca2+/SO4(2-) interaction suggest that Ca2+ (like H+) adds to the carrier before SO4(2-) and is at equilibrium with free carrier and carrier-Ca2+ complex. The linear reciprocal plots and replots indicate a 1:1 stoichiometry between Ca2+ and SO4(2-). Thus the fully loaded carrier-SO4(2-) -Ca2+ -H+ complex has a net positive charge relative to that of the free carrier, a fact consistent with the chemiosmotic hypothesis of membrane transport. The kinetics of the H+/Ca2+ interaction point to a random A-B (rapid equilibrium), ordered C sequence with A = H+, B = Ca2+, and C = SO4(2-). Selenate (an alternate substrate competitive with sulfate) is an uncompetitive inhibitor with respect to Ca2+, in agreement with the suggested mechanism. Internal charge balance is not accomplished by a stoichiometric coaccumulation of Ca2+ and SO4(2-). Sulfate transport does, however, promote 45Ca2+ uptake. A significant fraction of the added Ca2+ is bound by the mycelial surface. Binding is extremely rapid, but reversible.
一株产黄青霉ATP硫酸化酶阴性菌株对无机硫酸盐的主动转运受H⁺离子和金属离子（二价金属离子比一价金属离子更有效）的促进。初速度研究表明，H⁺和SO₄²⁻以有序序列（H⁺先于SO₄²⁻）添加到载体上，H⁺与游离载体及载体 - H⁺复合物处于平衡状态。线性双倒数图和重新绘制的图表明H⁺与SO₄²⁻之间的化学计量比为1:1。在低离子强度的缓冲悬浮液中，Ca²⁺和其他二价金属离子显著刺激硫酸盐转运。Ca²⁺/SO₄²⁻相互作用的动力学表明，Ca²⁺（如H⁺）在SO₄²⁻之前添加到载体上，并与游离载体及载体 - Ca²⁺复合物处于平衡状态。线性双倒数图和重新绘制的图表明Ca²⁺与SO₄²⁻之间的化学计量比为1:1。因此，相对于游离载体，完全负载的载体 - SO₄²⁻ - Ca²⁺ - H⁺复合物具有净正电荷，这一事实与膜转运的化学渗透假说一致。H⁺/Ca²⁺相互作用的动力学表明其为随机的A - B（快速平衡）、有序的C序列，其中A = H⁺，B = Ca²⁺，C = SO₄²⁻。硒酸盐（一种与硫酸盐竞争的替代底物）对Ca²⁺而言是反竞争性抑制剂，这与所提出的机制一致。内部电荷平衡并非通过Ca²⁺和SO₄²⁻的化学计量共积累来实现。然而，硫酸盐转运确实促进了⁴⁵Ca²⁺的摄取。添加的Ca²⁺有很大一部分被菌丝体表面结合。结合极其迅速，但可逆。
