梭子鱼II型小清蛋白与钙和铽离子的相互作用。

Interaction of parvalbumin of pike II with calcium and terbium ions.

作者信息

Eberspach I, Strassburger W, Glatter U, Gerday C, Wollmer A

机构信息

Abteilung Physiologische Chemie, Rheinisch-Westfälische Technische Hochschule Aachen, F.R.G.

出版信息

Biochim Biophys Acta. 1988 Jan 4;952(1):67-76. doi: 10.1016/0167-4838(88)90102-1.

DOI:10.1016/0167-4838(88)90102-1

PMID:3334854

Abstract

Fluorimetric titrations of parvalbumin II (pI 4.2) of pike (Pike II) with Ca2+ and Tb3+ show the CD and EF binding sites to be non-equivalent. The intrinsic binding constants of the strong and the weak sites obtained for Ca2+ are: KsCa = 1.6.10(8) M-1; KwCa = 6.6.10(5) M-1. Differences of the order of 100% were encountered between the Tb3+ binding constants obtained with four different versions of titration. Their average values are: KsTb = 1.9.10(11) M-1; KwTb = 1.0.10(7) M-1. The distances of the strong and the weak sites from the singular Tyr-48, rs = 9.5 A and r2 = 11.5 A, were derived from Förster-type energy transfer and proved compatible with the X-ray structure of parvalbumin III (pI 4.2) of carp (CarpIII). From the distances, it is suggested that CD is the strong and EF the weak metal-binding site of PikeII. Tb3+ was shown by CD spectroscopy to have the same structural effect on PikeII as Ca2+. Removal of the metal ions from PikeII results in a decrease of helix content as monitored by CD spectroscopy. This decrease is larger than that in CarpIII. A concomitant decrease of the fluorescence quantum yield at nearly constant decay time is indicative of mainly static quenching, probably by the non-coordinating carboxylate groups. The maximum helix content is almost completely reestablished upon binding of the first metal ion. However, small changes of the energy transfer in PikeII with one terbium ion bound to the strong site indicate fine structural rearrangements of the strong binding site when Ca2+ is bound to the weak one.

摘要

用钙离子（Ca²⁺）和铽离子（Tb³⁺）对梭子鱼的小清蛋白II（pI 4.2，即梭子鱼II）进行荧光滴定，结果表明钙调蛋白（CD）结合位点和肌钙蛋白（EF）结合位点是不等价的。对于Ca²⁺，强结合位点和弱结合位点的固有结合常数分别为：KsCa = 1.6×10⁸ M⁻¹；KwCa = 6.6×10⁵ M⁻¹。用四种不同的滴定方法得到的Tb³⁺结合常数之间相差约100%。它们的平均值为：KsTb = 1.9×10¹¹ M⁻¹；KwTb = 1.0×10⁷ M⁻¹。通过Förster型能量转移得出，强结合位点和弱结合位点与单一的酪氨酸-48之间的距离分别为rs = 9.5 Å和r₂ = 11.5 Å，并且证明与鲤鱼的小清蛋白III（pI 4.2，即鲤鱼III）的X射线结构相符。根据这些距离，推测梭子鱼II中CD是强金属结合位点，EF是弱金属结合位点。圆二色光谱显示，Tb³⁺对梭子鱼II的结构影响与Ca²⁺相同。通过圆二色光谱监测发现，从梭子鱼II中去除金属离子会导致螺旋含量降低。这种降低幅度比鲤鱼III中的更大。在几乎恒定的衰减时间下，荧光量子产率随之降低，这表明主要是静态猝灭，可能是由非配位羧酸基团引起的。结合第一个金属离子后，最大螺旋含量几乎完全恢复。然而，当一个铽离子结合到强结合位点时，梭子鱼II中的能量转移发生了微小变化，这表明当Ca²⁺结合到弱结合位点时，强结合位点发生了精细的结构重排。