Subunit interactions of 7 S nerve growth factor. Gamma-esterase activity, rates, and conformational changes during reassociation.
The gamma-esterase activity of 7 S nerve growth factor (NGF) is depressed relative to free subunit because of constraints within the oligomer. 4 M urea causes a reversible dissociation of 7 S NGF and concomitant increase in esterase activity. The gamma-esterase activity with tosyl-arginyl-methyl-ester increased between 1 and 4 M urea and was inhibited at higher concentrations. Upon dilution of 7 S NGF from 6 M urea, the esterase activity was initially identical with that of the similarly treated gamma-subunit but decayed rapidly (10 min) to approach that of the native 7 S NGF. In the presence of 100 microM EDTA the activity was higher, but still required the same length of time to reach a constant depressed value. Upon dilution of 7 S NGF from 2 M NaCl the rapid decay was not observed and the activity remained constant at about the same level as the equilibrium values for the urea-treated and renatured 7 S NGF. A red shift in the fluorescence maximum and a concomitant increase in quantum yield occurred for the 7 S in 8 M urea. Dilution into buffer resulted in a rapid decay and return to native 7 S fluorescence but not when EDTA was included. Both the decay in esterase activity and in fluorescence intensity upon dilution from urea were first order reactions with a rate constant of about 8 X 10(-3) s-1 suggesting that both methods were measuring a unimolecular renaturation process. Renaturation from NaCl or recombination of isolated subunits was much more rapid, indicating a simple combination of subunits in a native conformation. The circular dichroism spectra of urea and NaCl-treated 7 S NGF were different, and spectra of the renatured species with and without EDTA differed. Reassociation to a conformationally different 7 S NGF probably occurs in the presence of EDTA. The results emphasize the role of zinc in 7 S NGF formation and the influence of conformational changes in the effect of beta-NGF on the activity of the gamma-esterase in 7 S NGF.
D000818:Animals; D002942:Circular Dichroism; D004950:Esterases / Q000378:metabolism*; D005470:Fluorometry; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D009414:Nerve Growth Factors / Q000378:metabolism*; D011487:Protein Conformation; D013997:Time Factors; D014106:Tosylarginine Methyl Ester / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D046911:Macromolecular Substances; D009414:Nerve Growth Factors; D014508:Urea; D014106:Tosylarginine Methyl Ester; D004950:Esterases; D015032:Zinc