the linear amino acid sequence alone is not suf- 
ficient for neurotrophic activity but that secon- 
dary structural features, i.e., disulfide bonds, are 
important for both neurite extension and neuronal 
survival activities. In addition, the fact that both 
cysteine residues are necessary for biological activ- 
ity suggests an involvement of both residues in the 
disulfide linkage of the active species of the 
protein. An analysis of the secondary structural 
constraints on the protein for activity was begun 
by changing the relative position of the two cys- 
teines in the linear amino acid sequence. A mutant 
was prepared with Ser-62 changed to cysteine and 
Cys-68 changed to valine. The mutant still has 
two cysteine residues, but the cysteine that is nor- 
mally found at residue 68 has been moved to resi- 
due 62. This mutant stimulated neurite extension 
and enhanced cell survival in a manner similar 
to the unmodified VUSB-1. These data suggest that 
the structural requirements for neurotrophic ac- 
tivity are somewhat flexible; the relative position 
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of the two cysteine residues can be altered with- 
out significantly diminishing the neurotrophic ac- 
tivity. 
This flexibility in the relative positioning of the 
cysteine residues in the linear sequence has impor- 
tant pharmacological implications. The ability to 
design synthetic neurotrophic agents based on the 
SIOOP structure has the potential to provide a ra- 
tional approach to the development of reagents 
useful for nerve regeneration or selective mainte- 
nance of neuronal function. In addition, the data 
support the hypothesis that a disulfide-linked form 
of SlOOp may act as a neurotrophic factor in the 
CNS, provide a foundation for future studies into 
the effects of NEF in vivo during CNS development, 
and offer insights into the molecular regulation of 
neuronal development and maintenance. 
Dr. Van Eldik is Associate Professor of Pharmacol- 
ogy and Cell Biology at Vanderbilt University 
School of Medicine. 
Article 
Zimmer, D.B., and Van Eldik, L.J. 1989. Analysis of the calcium-modulated proteins, SlOO and calmodulin, 
and their target proteins during C6 glioma cell differentiation. / Cell Biol 108: 141-15 1. 
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