Characterization of the cation-binding properties of porcine neurofilaments.
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 microM per unit consisting of eight low, three middle, and three high molecular subunits, as well as 46 sites with Kd = 620 microM. Only one class of 126 sites with Kd = 740 microM 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 microM) per domain fragment of approximately 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 microM and 24 microM, 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 microM and 4 microM, respectively. This suggests that the accumulation of Al3+ in neurons may be due in part to its binding to neurofilaments.
Lefebvre
S
S
Department of Biochemistry, McGill University, Montréal, Québec, Canada.
Mushynski
W E
WE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002412:Cations; D003599:Cytoskeleton / Q000378:metabolism*; D066298:In Vitro Techniques; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007382:Intermediate Filaments / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins; D012964:Sodium / Q000378:metabolism; D013552:Swine
D002412:Cations; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D012964:Sodium; D002118:Calcium