Yang Z W, Babitch J A
Chemistry of Behavior Program, Texas Christian University, Fort Worth 76129.
Biochemistry. 1988 Sep 6;27(18):7038-45. doi: 10.1021/bi00418a055.
Glial fibrillary acidic protein (GFAP) is soluble in low ionic strength solutions but shows a strong tendency toward assembly with increasing ionic strength as revealed by electron microscopy and turbidity measurements. Increasing K+, Na+, and Li+ concentrations cause an increase followed by a decrease in GFAP turbidity with a maximum at 200 mM, but their effects are much weaker than effects of divalent cations at the same ionic strength. Ca2+, Mg2+, Mn2+, and Ba2+ promote assembly at millimolar concentrations, and 10 microM Cu2+ causes rapid aggregation. The critical concentration for GFAP assembly was 0.08 +/- 0.04 mg/mL in 2 mM Tris-HCl, 60 mM KCl, and 1 mM CaCl2, pH 6.8. The Mr 38,000 rod domain of GFAP obtained by limited chymotryptic digestion is more soluble in 100 mM imidazole hydrochloride buffer, pH 6.8, than the intact molecule, and removal of the end pieces greatly reduces the ability of GFAP to form filaments. BNPS-skatole (2-[(2-nitrophenyl)sulfenyl]-3-methyl-3-bromoindolenine) treatment releases a Mr 30,000 N-terminus and a Mr 20,000 C-terminus. The Mr 30,000 polypeptide shows a higher affinity than the Mr 20,000 fragment for intact GFAP. Arginine and lysine at low concentrations slightly accelerate GFAP assembly, but above 100 mM both amino acids inhibit assembly. ATP, GTP, CTP, and UTP do not show significant effects on GFAP assembly. Dephosphorylation by alkaline phosphatase slightly reduces the assembly ability of GFAP, but phosphatase-treated GFAP still is assembly competent.
胶质纤维酸性蛋白（GFAP）可溶于低离子强度溶液，但随着离子强度增加，其显示出强烈的组装倾向，这通过电子显微镜和浊度测量得以揭示。随着K⁺、Na⁺和Li⁺浓度增加，GFAP浊度先升高后降低，在200 mM时达到最大值，但在相同离子强度下，它们的作用比二价阳离子弱得多。Ca²⁺、Mg²⁺、Mn²⁺和Ba²⁺在毫摩尔浓度下促进组装，10 μM Cu²⁺导致快速聚集。在2 mM Tris-HCl、60 mM KCl和1 mM CaCl₂，pH 6.8条件下，GFAP组装的临界浓度为0.08±0.04 mg/mL。通过有限胰凝乳蛋白酶消化获得的GFAP的38,000 Mr杆状结构域在pH 6.8的100 mM咪唑盐酸盐缓冲液中比完整分子更易溶，去除末端片段大大降低了GFAP形成细丝的能力。BNPS-粪臭素（2-[(2-硝基苯基)硫代]-3-甲基-3-溴吲哚啉）处理可释放出30,000 Mr的N末端和20,000 Mr的C末端。30,000 Mr的多肽对完整GFAP的亲和力高于20,000 Mr的片段。低浓度的精氨酸和赖氨酸略微加速GFAP组装，但在100 mM以上，这两种氨基酸均抑制组装。ATP、GTP、CTP和UTP对GFAP组装无显著影响。碱性磷酸酶去磷酸化略微降低了GFAP的组装能力，但经磷酸酶处理的GFAP仍具有组装能力。
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