Pozdnyakov N, Goraczniak R, Margulis A, Duda T, Sharma R K, Yoshida A, Sitaramayya A
Eye Research Institute, Oakland University, Rochester, Michigan 48309, USA.
Biochemistry. 1997 Nov 18;36(46):14159-66. doi: 10.1021/bi971792l.
Calcium-dependent guanylate cyclase activator protein (CD-GCAP) is a low-molecular-weight retinal calcium-binding protein which activates rod outer segment guanylate cyclase (ROS-GC) in a calcium-dependent manner. This investigation was undertaken to determine the protein's structure and identity. Partial amino acid sequencing (72% of the protein), mass spectral analysis, cloning, and immunological studies revealed that CD-GCAP is identical to S100beta, another low-molecular-weight calcium-binding protein whose structure was known. We had shown earlier that the latter protein, which is usually called S100b (S100betabeta or dimer of S100beta), also activates ROS-GC but that the Vmax of activated cyclase was about 50% lower than when stimulated by CD-GCAP. S100b also required about 15 times more calcium (3.2 x 10(-)5 vs 1.5 x 10(-)6 M) for half-maximal stimulation of cyclase. To investigate the possibility that CD-GCAP is a post-translationally modified form of S100b, both proteins were treated with 1 M hydroxylamine which is known to deacylate proteins. After the treatment, CD-GCAP did not activate cyclase while S100b activation remained unaffected suggesting that CD-GCAP could not be a modified form of S100b. Hydroxylamine also broke down CD-GCAP into smaller fragments while leaving S100b intact. It therefore appeared that in spite of identical primary structures, the conformations of the two proteins were different. We then investigated the possibility that the purification procedures of the two proteins, which were quite different, could have contributed to such conformational differences: CD-GCAP purification included a step of heating at 75 degrees C in 5 mM Ca, while S100b purification included zinc affinity chromatography. To test the influence of these treatments on the properties of the proteins, CD-GCAP was subjected to zinc affinity chromatography and purified as S100b (CD-GCAP-->S100b) and S100b was heated in Ca and purified as CD-GCAP (S100b-->CD-GCAP). Cyclase activation, calcium-sensitivity, and hydroxylamine-lability measurements revealed that CD-GCAP-->S100b is identical to S100b and that S100b-->CD-GCAP is identical to CD-GCAP. Taken together the results demonstrate that CD-GCAP and S100b are one and the same protein and that their functional differences are due to different interconvertible conformational states.
钙依赖性鸟苷酸环化酶激活蛋白（CD-GCAP）是一种低分子量的视网膜钙结合蛋白，它以钙依赖性方式激活视杆外段鸟苷酸环化酶（ROS-GC）。本研究旨在确定该蛋白的结构和身份。部分氨基酸测序（占该蛋白的72%）、质谱分析、克隆和免疫学研究表明，CD-GCAP与S100β相同，S100β是另一种结构已知的低分子量钙结合蛋白。我们之前已经表明，后一种蛋白，通常称为S100b（S100ββ或S100β的二聚体），也能激活ROS-GC，但激活的环化酶的Vmax比CD-GCAP刺激时低约50%。S100b在半最大激活环化酶时也需要大约15倍的钙（3.2×10⁻⁵对1.5×10⁻⁶M）。为了研究CD-GCAP是否是S100b的翻译后修饰形式，两种蛋白都用已知能使蛋白脱酰基的1M羟胺处理。处理后，CD-GCAP不能激活环化酶，而S100b的激活不受影响，这表明CD-GCAP不可能是S100b的修饰形式。羟胺还将CD-GCAP分解成更小片段，而S100b保持完整。因此，尽管一级结构相同，但这两种蛋白的构象似乎不同。然后我们研究了两种蛋白完全不同的纯化程序是否可能导致了这种构象差异：CD-GCAP的纯化包括在5mM钙中75℃加热步骤，而S100b的纯化包括锌亲和色谱。为了测试这些处理对蛋白性质的影响，将CD-GCAP进行锌亲和色谱并按S100b的方式纯化（CD-GCAP→S100b），将S100b在钙中加热并按CD-GCAP的方式纯化（S100b→CD-GCAP）。环化酶激活、钙敏感性和羟胺敏感性测量表明，CD-GCAP→S100b与S100b相同，S100b→CD-GCAP与CD-GCAP相同。综合这些结果表明，CD-GCAP和S100b是同一种蛋白且它们的功能差异是由于不同的可相互转化的构象状态。
