Thermodynamics of interaction of the subunits of 7 S nerve growth factor. The mechanism of activation of the esteropeptidase activity by chelators.

The effect of zinc ion on the affinity of association of the subunits in 7 S nerve growth factor (NGF) was measured using three different techniques including rapid chromatographic separation on carboxymethylcellulose of 7 S NGF and dissociated products, quantitative frontal analysis of profiles of elution of 7 S NGF from Bio-Gel P-200, and analysis, using LineweaverBurk plots, of the inhibition of the arginyl-esteropeptidase activity of y subunit by PNGF and by the cu$ complex. The results of all three techniques demonstrate that the equilibrium constant for dissociation of the y subunit from 7 S NGF is 100 nM, that artificial arginyl amide substrates of the y subunit competitively compete with PNGF and with the a$ complex for binding to y subunit, and that the presence of zinc ion greatly stabilizes the association of y subunit in the 7 S NGF complex. Analysis of the inhibition of the y subunit’s enzymatic activity by CI$ indicates that y subunit is enzymatically completely inactive when in the 7 S NGF complex, even in the absence of bound zinc and also demonstrates that the equilibrium constant for dissociation of y subunit from 7 S NGF decreases progressively with increasing zinc ion concentration to a minimum equilibrium constant of 1 PM at zinc ion concentrations of 1 pM or greater. The results are interpreted in terms of the hypothesis that y subunit functions to enzymatically convert pro+?NGF to PNGF by cleavage at an arginyl residue, and that the product., PNGF, subsequently remains associated with y subunit due to the interaction of the carboxyterminal arginyl residue of /?NGF with the enzymatic active site of y subunit.