Biochemical characterization of netrin-synergizing activity.
The netrin-1 protein elicits spinal commissural axon outgrowth and turning in vitro and has been shown to be required for commissural axon guidance in vivo in the developing spinal cord. Biochemical observations made during the purification of netrin-1 suggest that this ligand and its receptor, DCC, may not function alone in directing commissural axon guidance. Recombinant netrin-1 protein is approximately 10 times more active in eliciting axon outgrowth from embryonic day (E) 13 rat dorsal spinal cord explants than from E11 rat dorsal spinal cord explants (Serafini, T., Kennedy, T. E., Galko, M. J., Mirzayan, C., Jessell, T. M., and Tessier-Lavigne, M. (1994) Cell 78, 409-424) even though the starting material for the netrin purification, a high salt extract of E10 chicken brain membranes, is equally active on E13 and E11 explants. We previously reported an activity termed netrin-synergizing activity (NSA) that can potentiate the outgrowth-promoting activity of netrin-1 on E11 explants (Serafini et al.). Here we report a biochemical characterization of NSA in netrin-depleted high salt extracts of E10 chicken brain membranes. We provide evidence that NSA is composed of a denaturation-resistant basic protein(s) in the 25-35-kDa size range. We also provide evidence that the activity may be heterogeneous, splitting into three species that may be distinct or related. The results reported here should facilitate purification of this activity from a more abundant source or identification of the activity based on similarity to known proteins that share its distinctive biochemical properties.
Galko
M J
MJ
Howard Hughes Medical Institute, Departments of Anatomy and of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0452, USA.
Tessier-Lavigne
M
M
D000818:Animals; D001369:Axons / Q000187:drug effects*; D001923:Brain Chemistry; D002450:Cell Communication; D002846:Chromatography, Affinity; D004357:Drug Synergism; D006493:Heparin; D066298:In Vitro Techniques; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D000075388:Netrin-1; D011485:Protein Binding; D011489:Protein Denaturation; D051381:Rats; D013116:Spinal Cord / Q000187:drug effects / Q000196:embryology*; D025521:Tumor Suppressor Proteins
D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; C000617354:Ntn1 protein, rat; D025521:Tumor Suppressor Proteins; D000075388:Netrin-1; D006493:Heparin