Pattison S E, Dunn M F
Biochemistry. 1975 Jun 17;14(12):2733-9. doi: 10.1021/bi00683a027.
The 7S nerve growth factor (7S NGF) is an oligomeric protein consisting of three distinct classes of subunits, alpha,beta, and gamma (A. P. Smith, S. Varon, and E. M. Shooter (1968), Biochemistry 7, 3259). The beta subunit contains the growth promoting activity while gamma is a potent esteropeptidase. The proteolytic activity of gamma is virtually completely inhibited in the 7S NGF aggregate (L. A. Greene, E. M. Shooter, and S. Varon (1969), Biochemistry 8, 3735). In this paper, we report that divalent metal ion chelating agents effect a seven- to tenfold increase in the esteropeptidase activity of 7S NGF at pH 7.40. Plots of esteropeptidase activity vs. chelator concentration give saturation curves which are either sigmoidal (EDTA) or hyperbolic (o-phenanthroline) depending on the chemical structure of the chelator. A survey of common divalent metal ions shows that only zinc ion (Ki = 8 times 10(7) M) and, to a lesser extent, cadmium ion are effective, reversible inhibitors of both 7S NGF and the gamma subunit esteropeptidase activities. We have found that during isolation of 7S NGF, Zn2+ is selectively associated with the oligomer in a ratio of approximately 1-2 g-atoms of zinc/mol of 7S NGF with an apparent affinity which is orders of magnitude tighter than is indicated by the Ki value for the gamma subunit. Dialysis to pH 4.0 where 7S NGF is known to undergo a reversible dissociation (A. P. Smith, S. Varon, and E. M. Shooter (1968), Biochemistry 7, 3259) brings about a tenfold reduction in the zinc ion content of the protein. This reduction is reversed on dialysis back to pH 7.4. In contrast, the isolated subunits contain only trace amounts of zinc ion at pH 7.4. Preliminary metal ion exchange experiments indicate that, of the common metal ions known to substitute for zinc in other zinc-metalloproteins, only cadmium ion is effective in substituting for zinc ion in 7S NGF. The fact that zinc ion is specifically bound to native 7S NGF, and that the zinc ion content of the system is critically dependent on the subunit aggregation state strongly suggests that zinc ion is an integral structural component of native 7S NGF.
7S神经生长因子(7S NGF)是一种寡聚蛋白,由α、β和γ三类不同的亚基组成(A.P.史密斯、S.瓦龙和E.M.舒特(1968年),《生物化学》7卷,3259页)。β亚基具有促进生长的活性,而γ是一种强效酯肽酶。在7S NGF聚集体中,γ的蛋白水解活性几乎完全受到抑制(L.A.格林、E.M.舒特和S.瓦龙(1969年),《生物化学》8卷,3735页)。在本文中,我们报告二价金属离子螯合剂在pH 7.40时可使7S NGF的酯肽酶活性提高7至10倍。酯肽酶活性与螯合剂浓度的关系图给出了饱和曲线,根据螯合剂的化学结构,这些曲线要么是S形的(乙二胺四乙酸),要么是双曲线形的(邻菲罗啉)。对常见二价金属离子的研究表明,只有锌离子(Ki = 8×10⁻⁷ M)以及在较小程度上镉离子是7S NGF和γ亚基酯肽酶活性的有效、可逆抑制剂。我们发现,在7S NGF的分离过程中,Zn²⁺以大约1 - 2克原子锌/摩尔7S NGF的比例选择性地与寡聚体结合,其表观亲和力比γ亚基的Ki值所表明的亲和力强几个数量级。透析至pH 4.0(已知7S NGF在此pH下会发生可逆解离(A.P.史密斯、S.瓦龙和E.M.舒特(1968年),《生物化学》7卷,3259页))会使蛋白质中的锌离子含量降低10倍。透析回pH 7.4时,这种降低会逆转。相比之下,分离出的亚基在pH 7.4时仅含有痕量的锌离子。初步的金属离子交换实验表明,在已知可在其他锌金属蛋白中替代锌的常见金属离子中,只有镉离子能有效替代7S NGF中的锌离子。锌离子特异性地结合到天然7S NGF上,且系统中的锌离子含量严重依赖于亚基聚集状态,这一事实强烈表明锌离子是天然7S NGF不可或缺的结构成分。