Modulation of filament formation by glial fibrillary acidic protein and involvement of calcium

Abstract

A number of factors affect filament formation by glial fibrillary acidic protein (GFAP). GFAP is soluble in low ionic strength solution but shows a strong tendency toward assembly with increasing ionic strength. Increases in K$\sp+$, Na$\sp+$, or Li$\sp+$ concentrations cause a periodic change in GFAP turbidity with a maximum at 200 mM, but their effects are much weaker than effects of divalent cations, such as Ca$\sp{2+}$, Mg$\sp{2+}$, Mn$\sp{2+}$ or Ba$\sp{2+}$, at millimolar concentrations. Cu$\sp{2+}$ at 0.01 mM causes rapid aggregation. The 38,000 Mr rod domain of GFAP obtained by limited chymotryptic digestion is more soluble than the intact molecule, and removal of the endpieces reduces the ability of GFAP to form filaments. BNPS-skatole treatment releases a 30,000 Mr N-terminus and a 20,000 Mr C-terminus. The 30,000 Mr fragment shows a higher affinity than the 20,000 Mr C-terminus for intact GFAP. At high concentrations, above 100 mM, both arginine and lysine inhibit GFAP 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. The results of flow dialysis experiments suggest that in 0.5 mM MgSO$\sb4$ solution GFAP contains approximately two calcium-specific binding sites with a dissociation constant of 2.16 $\pm$ 0.25 x 10$\sp{-6}$M. GFAP fragments produced by enzymatic or chemical cleavages are examined for calcium-binding by $\sp{45}$calcium-autoradiography. From BrCN digest a 18,500 Mr fragment, covering 46-193 amino acid positions, is found to contain calcium-binding sites. A 23,000 Mr trypsin-resistant fragment also shows high affinity calcium-binding. These data suggest that the calcium-specific binding sites are located in Coil I. Postsynaptic density was predicted to be rich in basic proteins (Kelly & Cotman, 1978). Improved IEF-SDS two-dimensional gels extend the pH to 9.2 and, as a consequence, four new basic proteins--76,000 Mr (pI $>$ 9.2), 58,000 Mr (pI 8.1-8.8, heterogeneous), 40,000 Mr (pI 9.0), and 24,000 Mr (pI 8.9)--were detected on these gels. Nonequilibrium pH gradient-SDS two-dimensional gels further revealed six more basic proteins with pI values higher than 9.2: 76,000 Mr, 52,000 Mr, 47,000 Mr, 45,000 Mr, 36,000 Mr, and 24,000 Mr. Most of these basic proteins are distinguishable from those of brain mitochondria, the major contaminant. A number of PSD proteins including the basic proteins of 76,000 Mr and 58,000 Mr are phosphorylated by endogeneous protein kinase(s) in the presence of Ca$\sp{2+}$, Mg$\sp{2+}$, and ($\gamma$-$\sp{32}$P) ATP. No basic protein bind Ca$\sp{2+}$.