Characterization of the cation-binding properties of porcine neurofilaments

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Abstract
In the presence of physiological levels of Na+ (10 mM), K+ (150 mM), and Mg2+ (2 mM), dephosphorylated neurofilaments contained two Ca2+ specific binding sites with Kd = 11 .mu.M per unit consisting of eight low, three middle, and three high molecular subunits, as well as 46 sites with Kd = 620 .mu.M. Only one class of 126 sites with Kd = 740 .mu.M was detected per unit of untreated neurofilaments. A chymotryptic fraction enriched in the .alpha.-helical domains of neurofilament subunits contained one high-affinity Ca2+-binding site (Kd = 3.6 .mu.M) per domain fragment of .apprx. 32 kDa. This site may correspond to a region in coil 2b of the .alpha.-helical domain, which resembles the I-II Ca2+-binding site in intestinal Ca2+-binding protein. Homopolymeric filaments composed of the low or middle molecular weight subunits contained low-affinity Ca2+-binding sites with Kd = 37 .mu.M, respectively, while the Kd values for the low-affinity sites in heteropolymeric filaments were 8-10-fold higher. Competitive binding studies, using the chymotryptic fraction to assay the high-affinity Ca2+-binding sites and 22Na+ to monitor binding to the phosphate-containing low-affinity sites, yielded Kd values for Al3+ of 0.01 .mu.M and 4 .mu.M, respectively. This suggests that the accumulation of Al3+ in neurons may be due in part to its binding to neurofilaments.