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Biochemical Characterization of Netrin-synergizing Activity

2000; Elsevier BV; Volume: 275; Issue: 11 Linguagem: Inglês

10.1074/jbc.275.11.7832

1083-351X

Michael J. Galko, Marc Tessier‐Lavigne,

Neurogenesis and neuroplasticity mechanisms

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 ∼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 (Serafiniet 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. 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 ∼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 (Serafiniet 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. embryonic day netrin-synergizing activity polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate N,N,N′,N′-tetramethylethylenediamine The faithful guidance of axons to their targets during development of the embryonic nervous system is believed to occur through the action of both positive and negative guidance factors (1.Goodman C.S. Annu. Rev. Neurosci. 1996; 19: 341-377Crossref PubMed Scopus (462) Google Scholar, 2.Mueller B.K. Annu. Rev. Neurosci. 1999; 22: 351-388Crossref PubMed Scopus (378) Google Scholar). These factors may act either locally or at a distance within the terrain of the developing embryo. A large body of experimental evidence has implicated the phylogenetically conserved netrin gene family in directing the long-range attraction of circumferentially migrating neurons inCaenorhabditis elegans, Drosophila, and vertebrates (reviewed in Ref. 1.Goodman C.S. Annu. Rev. Neurosci. 1996; 19: 341-377Crossref PubMed Scopus (462) Google Scholar). In vertebrates, the netrin-1 mRNA is expressed in the floor plate, the ventral midline intermediate target of migrating commissural neurons, before and during the migration of these neurons toward this structure (3.Kennedy T.E. Serafini T. de la Torre J.R. Tessier-Lavigne M. Cell. 1994; 78: 425-435Abstract Full Text PDF PubMed Scopus (1121) Google Scholar). The netrin-1 receptor on migrating commissural neurons is the netrin-binding DCC (deleted in colorectal cancer) protein, which is expressed on commissural axons as they migrate toward the floor plate (4.Keino-Masu K. Masu M. Hinck L. Leonardo E.D. Chan S.S. Culotti J.G. Tessier-Lavigne M. Cell. 1996; 87: 175-185Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar). Mutant mice deficient for either the netrin-1 gene product or DCC exhibit defects in commissural axon migration from their birthplace in the dorsal spinal cord to the floor plate, demonstrating that their proper functioning is necessary for commissural axon guidance (5.Fazeli A. Dickinson S.L. Hermiston M.L. Tighe R.V. Steen R.G. Small C.G. Stoeckli E.T. Keino-Masu K. Masu M. Rayburn H. Simons J. Bronson R.T. Gordon J.I. Tessier-Lavigne M. Weinberg R.A. Nature. 1997; 386: 796-804Crossref PubMed Scopus (667) Google Scholar, 6.Serafini T. Colamarino S.A. Leonardo E.D. Wang H. Beddington R. Skarnes W.C. Tessier-Lavigne M. Cell. 1996; 87: 1001-1114Abstract Full Text Full Text PDF PubMed Scopus (1049) Google Scholar).The vertebrate netrin-1 protein was originally purified from a high salt extract of embryonic day (E)1 10 chicken brain membranes on the basis of its ability to mimic the axon outgrowth-promoting effect of floor plate extracts from dorsal spinal cord explants cultured in vitro within three-dimensional collagen matrices (7.Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1149) Google Scholar). During this purification, an activity was discovered that collaborates with netrin-1 to promote outgrowth. Although recombinant netrin-1 protein is ∼10 times more active in eliciting axon outgrowth from E13 rat dorsal spinal cord explants than from E11 rat dorsal spinal cord explants, a high salt extract of E10 chicken brain membranes is equally active on E13 and E11 explants. We show that the difference is due to the presence in brain extracts of a distinct activity, netrin-synergizing activity (NSA), that is capable of potentiating the axon outgrowth-promoting effects of netrin-1 from E11 explants (7.Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1149) Google Scholar). Because of its dramatic potentiation of netrin-mediated axon outgrowth in vitro, it seems likely that this activity may play an important role in commissural axon guidance in vivo. As a first step toward identifying NSA and determining its role in commissural axon guidance in vivo, we report here the results of a biochemical characterization of NSA from a netrin-depleted high salt extract of E10 chicken brain membranes.DISCUSSIONAxon guidance cues can be classified into four categories: positive or negative cues that act either locally or at a distance to guide axons within the developing embryo (1.Goodman C.S. Annu. Rev. Neurosci. 1996; 19: 341-377Crossref PubMed Scopus (462) Google Scholar, 2.Mueller B.K. Annu. Rev. Neurosci. 1999; 22: 351-388Crossref PubMed Scopus (378) Google Scholar). The fact that NSA does not have any axon outgrowth-promoting activity on its own suggests that this activity may be acting in a mechanistically distinct manner from that of previously identified positive and negative axon guidance factors, through modulating the activity of a known axon guidance cue. It will be difficult to unravel the mechanism of action and in vivo function of this cue without its molecular identification. The results presented here should aid in that endeavor. Here we describe an improved assay for NSA and the use of this assay to define the biochemical properties of NSA. Fractionation of NSA reveals a number of important observations that should facilitate future molecular identification. First, despite its remarkable stability to a variety of denaturing conditions, this activity contains a necessary protein component for activity since it is abolished by protease treatment. This observation, coupled with the finding that NSA is a basic protein (indicated by the behavior of NSA on ion-exchange resins and on native acid/urea electrophoresis), indicates that NSA is not encoded by a glycosaminoglycan or proteoglycan moiety as was originally hypothesized (7.Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1149) Google Scholar) since brain-derived proteoglycans (but not NSA) bind to anion exchangers at 150 mm NaCl (15.Herndon M.E. Lander A.D. Neuron. 1990; 4: 949-961Abstract Full Text PDF PubMed Scopus (219) Google Scholar).It appears that the NSA present in our starting material, a netrin-depleted high salt extract of embryonic chick brain membranes, may be heterogeneous, as indicated by the existence of multiple activity peaks that elute from a C4 reverse-phase chromatography column. All of these peaks must be encoded by basic proteins (since they all elute from heparin at basic pH), and they are likely to all be in the same size range (25–35 kDa) since this is where all NSA-like activity is recovered in nonreducing SDS electrophoresis experiments. One could argue that some of these NSAs are stable to trifluoroacetic acid and not to SDS (a possibility our stability experiments do not address) and that we therefore really know the size of only one of the three NSAs. Although this is possible, we think it is unlikely since resistance to one denaturing condition generally implies a structure that imparts resistance to other denaturing conditions. Examples of this principle include the neuregulins (16.Peles E. Yarden Y. Bioessays. 1993; 15: 815-824Crossref PubMed Scopus (260) Google Scholar) and the tissue inhibitors of metalloproteases (17.Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (243) Google Scholar), both of which survive exposure to a number of denaturing conditions. The most likely interpretation of our nonreducing electrophoresis and reverse-phase chromatography experiments is that each peak observed on reverse-phase chromatography is in the 25–35-kDa size range we defined for NSA by nonreducing SDS-PAGE.Most of the difficulty in purifying this activity can be attributed to the facts that we have not been able to identify a high enrichment affinity chromatography step specific for this activity (since neither a panel of lectins nor netrin affinity resins bind NSA) and that the steps we have identified so far give only modest enrichment of NSA (Table III). Published biochemical purifications of other axon guidance molecules have all included affinity steps and may not have been possible without them (7.Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1149) Google Scholar, 18.Luo Y. Raible D. Raper J.A. Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (1005) Google Scholar, 19.Wang K.H. Brose K. Arnott D. Kidd T. Goodman C.S. Henzel W. Tessier-Lavigne M. Cell. 1999; 96: 771-784Abstract Full Text Full Text PDF PubMed Google Scholar). Through future fractionation experiments, we hope to determine whether the three activity peaks we observed with reverse-phase chromatography represent independent proteins, related but distinct proteins, or a single protein with several different modifications. Our suspicion, based on the results of nonreducing SDS-PAGE of NSA-containing fractions, is that NSA is encoded by minor protein species present in trace amounts in our fractions. Mass spectrometry and Edman degradation sequencing of the most abundant protein species that approximately cofractionate with our observed activity on reverse-phase chromatography (the ∼30-kDa bands shown in Fig. 5) bear this conclusion out; to date, the proteins we have sequenced from these fractions are all highly basic histone and ribosomal proteins. 2W. Henzel, C. Turck, M. Tessier-Lavigne, and M. J. Galko, unpublished observations. Although these proteins share biochemical properties with NSA, their intracellular nature makes it unlikely that they actually encode NSA. Due to low recoveries of NSA on reverse-phase chromatography, the lack of an affinity chromatography step, and the fact that NSA in embryonic chicken brain appears to be present in trace amounts, purification of NSA from embryonic chicken brain does not seem currently feasible. Identification of an affinity chromatography step or purification from a more abundant activity source (perhaps adult chicken or bovine brain) may surmount these obstacles to the molecular identification of NSAs. The faithful guidance of axons to their targets during development of the embryonic nervous system is believed to occur through the action of both positive and negative guidance factors (1.Goodman C.S. Annu. Rev. Neurosci. 1996; 19: 341-377Crossref PubMed Scopus (462) Google Scholar, 2.Mueller B.K. Annu. Rev. Neurosci. 1999; 22: 351-388Crossref PubMed Scopus (378) Google Scholar). These factors may act either locally or at a distance within the terrain of the developing embryo. A large body of experimental evidence has implicated the phylogenetically conserved netrin gene family in directing the long-range attraction of circumferentially migrating neurons inCaenorhabditis elegans, Drosophila, and vertebrates (reviewed in Ref. 1.Goodman C.S. Annu. Rev. Neurosci. 1996; 19: 341-377Crossref PubMed Scopus (462) Google Scholar). In vertebrates, the netrin-1 mRNA is expressed in the floor plate, the ventral midline intermediate target of migrating commissural neurons, before and during the migration of these neurons toward this structure (3.Kennedy T.E. Serafini T. de la Torre J.R. Tessier-Lavigne M. Cell. 1994; 78: 425-435Abstract Full Text PDF PubMed Scopus (1121) Google Scholar). The netrin-1 receptor on migrating commissural neurons is the netrin-binding DCC (deleted in colorectal cancer) protein, which is expressed on commissural axons as they migrate toward the floor plate (4.Keino-Masu K. Masu M. Hinck L. Leonardo E.D. Chan S.S. Culotti J.G. Tessier-Lavigne M. Cell. 1996; 87: 175-185Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar). Mutant mice deficient for either the netrin-1 gene product or DCC exhibit defects in commissural axon migration from their birthplace in the dorsal spinal cord to the floor plate, demonstrating that their proper functioning is necessary for commissural axon guidance (5.Fazeli A. Dickinson S.L. Hermiston M.L. Tighe R.V. Steen R.G. Small C.G. Stoeckli E.T. Keino-Masu K. Masu M. Rayburn H. Simons J. Bronson R.T. Gordon J.I. Tessier-Lavigne M. Weinberg R.A. Nature. 1997; 386: 796-804Crossref PubMed Scopus (667) Google Scholar, 6.Serafini T. Colamarino S.A. Leonardo E.D. Wang H. Beddington R. Skarnes W.C. Tessier-Lavigne M. Cell. 1996; 87: 1001-1114Abstract Full Text Full Text PDF PubMed Scopus (1049) Google Scholar). The vertebrate netrin-1 protein was originally purified from a high salt extract of embryonic day (E)1 10 chicken brain membranes on the basis of its ability to mimic the axon outgrowth-promoting effect of floor plate extracts from dorsal spinal cord explants cultured in vitro within three-dimensional collagen matrices (7.Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1149) Google Scholar). During this purification, an activity was discovered that collaborates with netrin-1 to promote outgrowth. Although recombinant netrin-1 protein is ∼10 times more active in eliciting axon outgrowth from E13 rat dorsal spinal cord explants than from E11 rat dorsal spinal cord explants, a high salt extract of E10 chicken brain membranes is equally active on E13 and E11 explants. We show that the difference is due to the presence in brain extracts of a distinct activity, netrin-synergizing activity (NSA), that is capable of potentiating the axon outgrowth-promoting effects of netrin-1 from E11 explants (7.Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1149) Google Scholar). Because of its dramatic potentiation of netrin-mediated axon outgrowth in vitro, it seems likely that this activity may play an important role in commissural axon guidance in vivo. As a first step toward identifying NSA and determining its role in commissural axon guidance in vivo, we report here the results of a biochemical characterization of NSA from a netrin-depleted high salt extract of E10 chicken brain membranes. DISCUSSIONAxon guidance cues can be classified into four categories: positive or negative cues that act either locally or at a distance to guide axons within the developing embryo (1.Goodman C.S. Annu. Rev. Neurosci. 1996; 19: 341-377Crossref PubMed Scopus (462) Google Scholar, 2.Mueller B.K. Annu. Rev. Neurosci. 1999; 22: 351-388Crossref PubMed Scopus (378) Google Scholar). The fact that NSA does not have any axon outgrowth-promoting activity on its own suggests that this activity may be acting in a mechanistically distinct manner from that of previously identified positive and negative axon guidance factors, through modulating the activity of a known axon guidance cue. It will be difficult to unravel the mechanism of action and in vivo function of this cue without its molecular identification. The results presented here should aid in that endeavor. Here we describe an improved assay for NSA and the use of this assay to define the biochemical properties of NSA. Fractionation of NSA reveals a number of important observations that should facilitate future molecular identification. First, despite its remarkable stability to a variety of denaturing conditions, this activity contains a necessary protein component for activity since it is abolished by protease treatment. This observation, coupled with the finding that NSA is a basic protein (indicated by the behavior of NSA on ion-exchange resins and on native acid/urea electrophoresis), indicates that NSA is not encoded by a glycosaminoglycan or proteoglycan moiety as was originally hypothesized (7.Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1149) Google Scholar) since brain-derived proteoglycans (but not NSA) bind to anion exchangers at 150 mm NaCl (15.Herndon M.E. Lander A.D. Neuron. 1990; 4: 949-961Abstract Full Text PDF PubMed Scopus (219) Google Scholar).It appears that the NSA present in our starting material, a netrin-depleted high salt extract of embryonic chick brain membranes, may be heterogeneous, as indicated by the existence of multiple activity peaks that elute from a C4 reverse-phase chromatography column. All of these peaks must be encoded by basic proteins (since they all elute from heparin at basic pH), and they are likely to all be in the same size range (25–35 kDa) since this is where all NSA-like activity is recovered in nonreducing SDS electrophoresis experiments. One could argue that some of these NSAs are stable to trifluoroacetic acid and not to SDS (a possibility our stability experiments do not address) and that we therefore really know the size of only one of the three NSAs. Although this is possible, we think it is unlikely since resistance to one denaturing condition generally implies a structure that imparts resistance to other denaturing conditions. Examples of this principle include the neuregulins (16.Peles E. Yarden Y. Bioessays. 1993; 15: 815-824Crossref PubMed Scopus (260) Google Scholar) and the tissue inhibitors of metalloproteases (17.Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (243) Google Scholar), both of which survive exposure to a number of denaturing conditions. The most likely interpretation of our nonreducing electrophoresis and reverse-phase chromatography experiments is that each peak observed on reverse-phase chromatography is in the 25–35-kDa size range we defined for NSA by nonreducing SDS-PAGE.Most of the difficulty in purifying this activity can be attributed to the facts that we have not been able to identify a high enrichment affinity chromatography step specific for this activity (since neither a panel of lectins nor netrin affinity resins bind NSA) and that the steps we have identified so far give only modest enrichment of NSA (Table III). Published biochemical purifications of other axon guidance molecules have all included affinity steps and may not have been possible without them (7.Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1149) Google Scholar, 18.Luo Y. Raible D. Raper J.A. Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (1005) Google Scholar, 19.Wang K.H. Brose K. Arnott D. Kidd T. Goodman C.S. Henzel W. Tessier-Lavigne M. Cell. 1999; 96: 771-784Abstract Full Text Full Text PDF PubMed Google Scholar). Through future fractionation experiments, we hope to determine whether the three activity peaks we observed with reverse-phase chromatography represent independent proteins, related but distinct proteins, or a single protein with several different modifications. Our suspicion, based on the results of nonreducing SDS-PAGE of NSA-containing fractions, is that NSA is encoded by minor protein species present in trace amounts in our fractions. Mass spectrometry and Edman degradation sequencing of the most abundant protein species that approximately cofractionate with our observed activity on reverse-phase chromatography (the ∼30-kDa bands shown in Fig. 5) bear this conclusion out; to date, the proteins we have sequenced from these fractions are all highly basic histone and ribosomal proteins. 2W. Henzel, C. Turck, M. Tessier-Lavigne, and M. J. Galko, unpublished observations. Although these proteins share biochemical properties with NSA, their intracellular nature makes it unlikely that they actually encode NSA. Due to low recoveries of NSA on reverse-phase chromatography, the lack of an affinity chromatography step, and the fact that NSA in embryonic chicken brain appears to be present in trace amounts, purification of NSA from embryonic chicken brain does not seem currently feasible. Identification of an affinity chromatography step or purification from a more abundant activity source (perhaps adult chicken or bovine brain) may surmount these obstacles to the molecular identification of NSAs. Axon guidance cues can be classified into four categories: positive or negative cues that act either locally or at a distance to guide axons within the developing embryo (1.Goodman C.S. Annu. Rev. Neurosci. 1996; 19: 341-377Crossref PubMed Scopus (462) Google Scholar, 2.Mueller B.K. Annu. Rev. Neurosci. 1999; 22: 351-388Crossref PubMed Scopus (378) Google Scholar). The fact that NSA does not have any axon outgrowth-promoting activity on its own suggests that this activity may be acting in a mechanistically distinct manner from that of previously identified positive and negative axon guidance factors, through modulating the activity of a known axon guidance cue. It will be difficult to unravel the mechanism of action and in vivo function of this cue without its molecular identification. The results presented here should aid in that endeavor. Here we describe an improved assay for NSA and the use of this assay to define the biochemical properties of NSA. Fractionation of NSA reveals a number of important observations that should facilitate future molecular identification. First, despite its remarkable stability to a variety of denaturing conditions, this activity contains a necessary protein component for activity since it is abolished by protease treatment. This observation, coupled with the finding that NSA is a basic protein (indicated by the behavior of NSA on ion-exchange resins and on native acid/urea electrophoresis), indicates that NSA is not encoded by a glycosaminoglycan or proteoglycan moiety as was originally hypothesized (7.Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1149) Google Scholar) since brain-derived proteoglycans (but not NSA) bind to anion exchangers at 150 mm NaCl (15.Herndon M.E. Lander A.D. Neuron. 1990; 4: 949-961Abstract Full Text PDF PubMed Scopus (219) Google Scholar). It appears that the NSA present in our starting material, a netrin-depleted high salt extract of embryonic chick brain membranes, may be heterogeneous, as indicated by the existence of multiple activity peaks that elute from a C4 reverse-phase chromatography column. All of these peaks must be encoded by basic proteins (since they all elute from heparin at basic pH), and they are likely to all be in the same size range (25–35 kDa) since this is where all NSA-like activity is recovered in nonreducing SDS electrophoresis experiments. One could argue that some of these NSAs are stable to trifluoroacetic acid and not to SDS (a possibility our stability experiments do not address) and that we therefore really know the size of only one of the three NSAs. Although this is possible, we think it is unlikely since resistance to one denaturing condition generally implies a structure that imparts resistance to other denaturing conditions. Examples of this principle include the neuregulins (16.Peles E. Yarden Y. Bioessays. 1993; 15: 815-824Crossref PubMed Scopus (260) Google Scholar) and the tissue inhibitors of metalloproteases (17.Murphy G. Willenbrock F. Methods Enzymol. 1995; 248: 496-510Crossref PubMed Scopus (243) Google Scholar), both of which survive exposure to a number of denaturing conditions. The most likely interpretation of our nonreducing electrophoresis and reverse-phase chromatography experiments is that each peak observed on reverse-phase chromatography is in the 25–35-kDa size range we defined for NSA by nonreducing SDS-PAGE. Most of the difficulty in purifying this activity can be attributed to the facts that we have not been able to identify a high enrichment affinity chromatography step specific for this activity (since neither a panel of lectins nor netrin affinity resins bind NSA) and that the steps we have identified so far give only modest enrichment of NSA (Table III). Published biochemical purifications of other axon guidance molecules have all included affinity steps and may not have been possible without them (7.Serafini T. Kennedy T.E. Galko M.J. Mirzayan C. Jessell T.M. Tessier-Lavigne M. Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1149) Google Scholar, 18.Luo Y. Raible D. Raper J.A. Cell. 1993; 75: 217-227Abstract Full Text PDF PubMed Scopus (1005) Google Scholar, 19.Wang K.H. Brose K. Arnott D. Kidd T. Goodman C.S. Henzel W. Tessier-Lavigne M. Cell. 1999; 96: 771-784Abstract Full Text Full Text PDF PubMed Google Scholar). Through future fractionation experiments, we hope to determine whether the three activity peaks we observed with reverse-phase chromatography represent independent proteins, related but distinct proteins, or a single protein with several different modifications. Our suspicion, based on the results of nonreducing SDS-PAGE of NSA-containing fractions, is that NSA is encoded by minor protein species present in trace amounts in our fractions. Mass spectrometry and Edman degradation sequencing of the most abundant protein species that approximately cofractionate with our observed activity on reverse-phase chromatography (the ∼30-kDa bands shown in Fig. 5) bear this conclusion out; to date, the proteins we have sequenced from these fractions are all highly basic histone and ribosomal proteins. 2W. Henzel, C. Turck, M. Tessier-Lavigne, and M. J. Galko, unpublished observations. Although these proteins share biochemical properties with NSA, their intracellular nature makes it unlikely that they actually encode NSA. Due to low recoveries of NSA on reverse-phase chromatography, the lack of an affinity chromatography step, and the fact that NSA in embryonic chicken brain appears to be present in trace amounts, purification of NSA from embryonic chicken brain does not seem currently feasible. Identification of an affinity chromatography step or purification from a more abundant activity source (perhaps adult chicken or bovine brain) may surmount these obstacles to the molecular identification of NSAs. We thank S. Faynboym for providing high salt extracts of stably transfected netrin-producing cells, W. Henzel and C. Turck for the use of their reverse-phase chromatography equipment and their efforts to identify NSA through mass spectrometry and Edman degradation of cofractionating bands, and D. Galko for his assistance in quantifying the axon outgrowth in Fig. 1. In addition, we thank the members of the Tessier-Lavigne and neighboring laboratories and friends for assistance in dissecting the ∼40,000 embryonic chicken brains required for this study.