Effects of 2,5-hexanedione on calpain-mediated degradation of human neurofilaments in vitro

2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, can structurally modify neurofilaments (NF) by pyrrole adduct formation and subsequent covalent cross-linking. 2,5-HD also induces accumulations of NF within the pre-terminal axon. We examined whether exposure of NF to 2,5-HD affected NF degradation. Two different models were used: (1) NF-enriched cytoskeletons isolated from human sciatic nerve were incubated with 2,5-HD in vitro and (2) differentiated human neuroblastoma cells (SK-N-SH) were exposed to 2, 5-HD in culture prior to isolation of cytoskeletal proteins. The cytoskeletal preparations were subsequently incubated with calpain II. The amount of NF-H and NF-L remaining after proteolysis was determined by SDS-PAGE and quantitative immunoblotting. NF-M proteolysis could not be quantified. Incubation of sciatic nerve cytoskeletal preparations with 2,5-HD resulted in cross-linking of all three NF proteins into high molecular weight (HMW) material with a range of molecular weights. Proteolysis of the NF-H and NF-L polypeptides was not affected by 2,5-HD-exposure. Degradation of the HMW material containing NF-H or NF-L was retarded when comparing with degradation of the NF-H and NF-L polypeptides, respectively, from control samples, but not as compared to the corresponding NF polypeptides from 2,5-HD-treated samples. Exposure of SK-N-SH cells to 2,5-HD also resulted in considerable cross-linking of NF. No differences were found between the proteolytic rates of NF-L and NF-H from exposed cells as compared with those subunits from control cells. Moreover, degradation of cross-linked NF-H was not different from monomeric NF-H. In conclusion, whether 2,5-HD affects calpain-mediated degradation of cross-linked NF proteins will depend on which model better reflects NF cross-linking as occurring in 2, 5-HD-induced axonopathy. However, with both models it was demonstrated that exposure of NF proteins to 2,5-HD without subsequent cross-linking is not adequate to inhibit NF proteolysis in vitro by added calpain.     

Characterization of mammalian neurofilament triplet proteins. Subunit stoichiometry and morphology of native and reconstituted filaments

The three major proteins of mammalian neurofilaments of molecular weights 179,000 (NF1), 129,000 (NF2), and 66,500 (NF3) have been purified to homogeneity by multiple anion-exchange and hydroxylapatite absorption chromatography in 8 M urea. Silver staining of polyacrylamide gels of the purified proteins show single bands. In order to gain further insight into the molecular organization of the neurofilament triplet proteins, the molar stoichiometries and morphologies of native and reconstituted filaments and those isolated from developing brain were studied. Denaturing polyacrylamide gel electrophoresis followed by quantitative dye-binding analysis shows that the molar ratio of the three components in neurofilaments isolated from bovine spinal cord myelinated nerve is 4:2:1 (NF3:NF2:NF1). Comparison of the molar ratios of each component in neurofilaments isolated from rat, bovine, and human brain shows a variation in the ratio of each of these polypeptides and raises questions about the physiological uniqueness of the molar composition of the neurofilament triplet. Reconstitution of the three bovine polypeptides into 10-nm filaments was accomplished under conditions in which the NF3 protein was limiting. Reassembly of 10-nm filaments with varying amounts of NF2 and NF1 indicate that the NF3 homopolymer has a limiting capacity to bind NF2 and NF1 and is saturated at a molar ratio of 2:2:1 (NF3:NF2:NF1). Isolation of the neurofilament complex at various stages of rat brain maturation indicates that NF3 and NF2 are integrated into the neurofilament complex as early as embryonic day 17, while NF1 copurifies with these proteins at postnatal day 16, eventually reaching a molar stoichiometry of 2:2:1 in the adult rat. The molecular stoichiometry of the neurofilament proteins, the differential integration of these proteins during brain development, and the variation of the molar composition between mammalian species suggest accessory roles for the NF2 and NF1 proteins in the neurofilament complex.     

Cross-Linking of Neurofilament Proteins of Rat Spinal Cord In Vivo After Administration of 2,5-Hexanedione

The aliphatic hexacarbons n-hexane, methyl-n-butyl ketone, and 2,5-hexanedione are known to produce a peripheral neuropathy that involves an accumulation of 10-nm neurofilaments above the nodes of Ranvier in the spinal cord and peripheral nerve. In this study, rats were treated with 0.5% 2,5-hexanedione in drinking water for 180 days, and their spinal cord neurofilaments were isolated after development of the neuropathy. Visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a significant reduction in content of the neurofilament triplet proteins in treated animals and the presence of bands migrating at 138K and 260K that were not present in control animals. Analysis of the lanes using immunoblotting procedures and anti-70K, anti-160K, and anti-210K neurofilament antibodies revealed many cross-linked peptides. The 138K band cross-reacted with the anti-160K neurofilament antibody. This suggests that the 138K band is an intramolecular cross-link of the 160K neurofilament subunit. In addition to this peptide, there were numerous high-molecular-weight peptides immunoreactive with all three neurofilament protein antibodies. In addition to cross-linking, there was also a diminished amount of immunoreactive breakdown product of all three neurofilament proteins. This report demonstrates direct evidence of 2,5-hexanedione-induced cross-linking of neurofilament proteins in vivo, which maybe responsible for the accumulation of neurofilament proteins pathognomic of this neuropathy.     

Appearance and phosphorylation of the 210 kDalton neurofilament protein in newborn rat brain,spinal cord,and sciatic nerve

The appearance and in vivo phosphorylation of the 210 kDalton (kD) neurofilament protein (NF210K) in newborn rat brain, spinal cord, and sciatic nerve were invetigated. Electron microscopic examination of neurofilaments isolated from newborn rat brain and spinal cord demonstrated morphologically distinct filaments which contained cross-bridging side arms. Neurofilament proteins, phosphorylated in vivo, were separated by sodium dodecyl sulfate slab gel electrophoresis and were transferred from acrylamide gels to nitrocellulose sheets. The nitrocellulose sheets were treated with antiserum to the 70 kD, 145 kD and 210 kD neurofilament proteins by the immunoblot technique. The three neurofilament proteins were found to be present in newborn brain, spinal cord and sciatic nerve. The presence of NF210K in newborn rat brain was further confirmed by 2-dimensional gel electrophoresis followed by indentification of this protein by the immunoblot technique. Exposure of the immunostained nitrocellulose sheets to x-ray film revealed that the NF210K, NF145K, and NF70K proteins were phosphorylated in filaments prepared from newborn rat central and peripheral nervous systems. These results suggest that the synthesis and posttranslational modification of the neurofilament proteins may be synchronized or developmentally regulated. It is feasible that phosphorylation of the NF210K subunit may be a prerequisite for the formation of neurofilament cross-bridging elements which are necessary for radial growth of axons.     

Immunoblot Analyses of the Relative Contributions of Cysteine and Aspartic Proteases to Neurofilament Breakdown Products Following Experimental Brain Injury in Rats

Analyses using either one or two-dimensional gel electrophoresis were performed to identify the contribution of several proteases to lower molecular weight (MW) neurofilament 68 (NF68) break down products (BDPs) detected in cortical homogenates following unilateral cortical impact injury in rats. One dimensional immunoblot of BDPs obtained from in vitro cleavage of enriched neurofilaments (NF) by purified -calpain, m-calpain, cathepsin, B, cathepsin D, and CPP32 (caspase-3) were compared to in vivo samples from rats following traumatic brain injury (TBI). Comparison of these blots provided information on the relative contribution of different cysteine or aspartic proteases to NF loss following brain injury. As early as 3 hrs post-injury, cortical impact resulted in the presence of several lower MW NF68 immunopositive bands having patterns similar to those previously reported to be produced by calpain mediated proteolysis of neurofilaments. Only -calpain and m-calpain in vitro digestion of enriched neurofilaments contributed to the presence of the low MW 57 kD NF68 break down product (BDP) detected in post-TBI samples. Cathepsin B, cathepsin D, and caspase-3 failed to produce either the 53 kD or 57 kD NF BDPs. Further, 1 and 2 dimensional peptide maps containing a 1:1 ratio of in vivo and in vitro tissue samples showed complete comigration of lower MW immunopositive spots produced by TBI or in vitro incubation with m-calpain, thus providing additional evidence for the potential role of calpain activation to the production of NF68 BDPs following TBI. More importantly, 2-dimensional gel electrophoresis detected that immunopositive NF68 spots shifted to the basic pole (+) suggesting that dephosphorylation of the NF68 subunit pool may be associated with NF protein loss following TBI, an observation not previously noted in any model of experimental brain injury.     

Microtubule-associated proteins bind specifically to the 70-kDa neurofilament protein

Morphological and biochemical evidence have suggested that the components of the neuronal cytoskeleton, microtubules and neurofilaments (NF), interact with each other. Microtubule-associated proteins (MAPs) are plausible candidates for mediating some of these interactions and have been shown to bind to neurofilaments, as well as induce the formation of a viscous complex between neurofilaments and microtubules. By binding 32P-labeled MAPs to neurofilament proteins, which were transferred electrophoretically to nitrocellulose, we determined that, of the three neurofilament subunits, only the core NF70 subunit bound MAPs. The binding to electrophoretically transferred NF70 was specific, saturable, and reversible. Binding parameters were estimated by binding 32P-labeled MAPs to purified NF70 immobilized on nitrocellulose. Approximately 1 mol of MAPs bound per 45 +/- 15 mol of NF70 with an approximate Kd approximately 2.0 +/- 0.9 X 10(-7) M (n = 8). Reassembled filaments in suspension were used to confirm the specific binding. Tubulin and NF70 apparently bind to different sites on MAPs.     

Neurofilament phosphorylation and [125I]Calmodulin binding by Ca2+/calmodulin-dependent protein kinase in the brain subcellular fractions of diisopropyl phosphorofluoridate (DFP)-treated hen

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in humans and sensitive animal species, e.g., adult chicken. The chickens were sacrificed 18 days after a single dose of DFP (1.7 mg/kg, sc.), which produced severe ataxia or paralysis in 10–14 days. We studied Ca²⁺/calmodulin-dependent in vitro neurofilament phosphorylation by the brain subcellular fractions of control and DFP-treated hens. There was enhanced phosphorylation of all three NF subunits by the brain supernatant of treated hens. This was accompanied by enhanced autophosphorylation of both Ca²⁺/CaM-dependent protein kinase II (CaM-kinase II) subunits and increased calmodulin binding using either¹²⁵I-CaM or biotinylated calmodulin to only subunit without concomitant increase in the amount of this enzyme. This enhanced phosphorylation of neurofilament subunits was completely and partially inhibited by mastoparan and KN-62, respectively. There was no alteration in the distribution of CaM-kinase II activity in treated hens and the activity was not related to its concentration in different subcellular fractions. The difference in¹²⁵I-CaM binding to CaM-kinase II subunit in the brain supernatants of control and DFP-treated hens was not altered by its phosphorylation or dephosphorylation. The increased CaM-kinase II activity in the soluble fraction of DFP-treated hen brain may be involved in the aberrant phosphorylation of axonal neurofilaments, and thus play a role in OPIDN.Abbreviations CaM calmodulin - CaM-kinase II Ca²⁺/calmodulin-dependent protein kinase II - DFP diisopropyl phosphorofluoridate - ECL enhanced chemiluminescence - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid - MAP-2 microtubule-associated protein-2 - MBP myelin basic protein - OPIDN organophosphorus ester-induced delayed neurotoxicity - PIPES 1,4-piperazinediethanesulfonic acid - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Acrylamide alters neurofilament protein gene expression in rat brain

Acrylamide, a prototype neurotoxin, alters neurofilament protein (NF) gene expression in rat brain. Levels of mRNA coding for neurofilament protein subunits NF-L, NF-M, and NF-H have been determined by Northern blot analysis using³²P-labeled cDNA probes. Acrylamide given acutely (100 mg/kg, single intraperitoneal injection) causes a selective increase in NF-M mRNA (approximately 50%) compared to controls. The expression of NF-L or NF-H mRNA is not affected by acrylamide. In contrast, chronic treatment with acrylamide [0.03% (w/v) in drinking water for 4 weeks] induces a modest but significant increase (approximately 22%) in NF-L mRNA compared to controls. Levels of NF-M, and NF-H mRNA are not altered by acrylamide treatment. The expression of -actin mRNA, an ubiquitous protein, is not affected by either treatment regimen of acrylamide. The results of this study show that acrylamide increases the expression of mRNA for NF protein subunits in rat brain. The increase of specific mRNA for NF subunits depends on the dose, duration and route of acrylamide administration.     

2,5-Hexanedione Induced Decrease in Cytoskeletal Proteins of Rat Sciatic–tibial Nerve

Exposure chronically to n-hexane produces peripheral–central axonopathy mediated by 2,5-hexanedione (HD). Previous studies have demonstrated decreases in neurofilament (NF) contents of peripheral and central nervous regions from rats intoxicated with HD, and recent analysis has demonstrated that axonal atrophy, instead of NF-filled swellings, is a specific component of morphologic alterations. To deeply investigate the alterations of cytoskeletal proteins in HD peripheral neuropathy, the relative levels of NF-L, NF-M, NF-H, -tubulin, -tubulin and -actin of rat sciatic–tibial nerves were determined by SDS-PAGE and immunoblotting. HD was administrated to Wistar rats by intraperitoneal injection at dosage of 200 or 400 mg/kg/day (five-times per week). Rats were sacrificed after 6 weeks of treatment, and sciatic–tibial nerves were dissected, homogenized, and used for the determination of cytoskeletal proteins. Except for supernatant NF-L that could not be assayed, the results showed HD intoxication was associated with significant decreases in NF subunits in both of the supernatant and the pellet fractions of sciatic–tibial nerve homogenates (P<0.01), and obvious reductions in -tubulin, -tubulin and -actin only in the supernatant (P<0.05 or P<0.01). Among these alterations, the falls in the levels of NF subunits tended to be greater compared to those of the other cytoskeletal proteins in all HD-exposed groups, and the trend for decrements in NF-M was greater than those in the other NF subunits. Thus, HD intoxication was associated with significant declines in cytoskeletal protein contents in rat sciatic–tibial nerves, and the decreases might be related to the involvement of the peripheral axonopathy induced by HD.     

[32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo

Newly synthesized neurofilament proteins become highly phosphorylated within axons. Within 2 days after intravitreously injecting normal adult mice with [³²P]orthophosphate, we observed that neurofilaments along the entire length of optic axons were radiolabeled by a soluble³²P-carrier that was axonally transported faster than neurofilaments.³²P-incorporation into neurofilament proteins synthesized at the time of injection was comparatively low and minimally influenced the labeling pattern along axons.³²P-incorporation into axonal neurofilaments was considerably higher in the middle region of the optic axons. This characteristic non-uniform distribution of radiolabel remained nearly unchanged for at least 22 days. During this interval, less than 10% of the total³²P-labeled neurofilaments redistributed from the optic nerve to the optic tract. By contrast, newly synthesized neurofilaments were selectively pulse-labeled in ganglion cell bodies by intravitreous injection of [³⁵S]methionine and about 60% of this pool translocated by slow axoplasmic transport to the optic tract during the same time interval. These findings indicate that the steady-state or resident pool of neurofilaments in axons is not identical to the newly synthesized neurofilament pool, the major portion of which moves at the slowest rate of axoplasmic transport. Taken together with earlier studies, these results support the idea that, depending in part on their phosphorylation state, transported neurofilaments can interact for short or very long periods with a stationary but dynamic neurofilament lattice in axons.Special issue dedicated to Dr. Sidney Ochs.     

The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate

The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681-693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail-deleted (NF-MtailDelta) mutant mice using an embryonic stem cell-mediated "gene knockin" approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailDelta mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail-mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.     

Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis

Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric ¹⁴N/¹⁵N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning.     

Neurofilament proteins of rat peripheral nerve and spinal cord

Intact neurofilaments were isolated in parallel from rat peripheral nerve and spinal cord by osmotic shock into hypotonic media containing divalent cation chelators. Isolated neurofilaments were washed and separated by multiple centrifugations in 0.1 M NaCl. Abundant intact neurofilaments were identified in the washed pellets by negative staining techniques. Their origin from neurofilaments was confirmed by immune electron microscopy. Washed neurofilaments were extracted from lipid and membranous components with 8 M urea. Analyses of neurofilament isolates on sodium dodecyl sulfate gels showed that proteins of 200,000, 150,000, and 69,000 mol wt were the major components of intact neurofilaments derived from rat peripheral and central nervous systems. These same proteins were identified in whole tissue homogenates of both sources and became enriched during the isolation of intact neurofilaments. A minor component of 64,000 mol wt arose during isolation. Other proteins were identified as contaminants. Small amounts of proteins with electrophoretic migration of tubulin and actin remain in neurofilament isolates.     

The imbalance between dynamic and stable microtubules underlies neurodegeneration induced by 2,5-hexanedione

Exposure to environmental toxins, including hydrocarbon solvents, increases the risk of developing Parkinson's disease. An emergent hypothesis considers microtubule dysfunction as one of the crucial events in triggering neuronal degeneration in Parkinson's disease. Here, we used 2,5-hexanedione (2,5-HD), the toxic metabolite of n-hexane, to analyse the early effects of toxin-induced neurodegeneration on the cytoskeleton in multiple model systems. In PC12 cells differentiated with nerve growth factor for 5 days, we found that 2,5-HD treatment affected all the cytoskeletal components. Moreover, we observed alterations in microtubule distribution and stability, in addition to the imbalance of post-translational modifications of α-tubulin. Similar defects were also found in vivo in 2,5-HD-intoxicated mice. Interestingly, we also found that 2,5-HD exposure induced significant changes in microtubule stability in human skin fibroblasts obtained from Parkinson's disease patients harbouring mutations in PRKN gene, whereas it was ineffective in healthy donor fibroblasts, suggesting that the genetic background may really make the difference in microtubule susceptibility to this environmental Parkinson's disease-related toxin. In conclusion, by showing the imbalance between dynamic and stable microtubules in hydrocarbon-induced parkinsonism, our data support the crucial role of microtubule defects in triggering neurodegeneration.     

Differential phosphorylation of microtubule proteins by ATP and GTP

Purified brain microtubule protein is phosphorylated by endogenous protein kinase activities in the presence of [-³²P] ATP or [-³²P] GTP. Here we show that certain microtubule-associated proteins are phosphorylated differently by GTP or ATP as direct phosphoryl donors, suggesting the presence of distinct kinase activities, with different specificities, associated with microtubule protein.     

Importance of Non-Selective Cation Channel TRPV4 Interaction with Cytoskeleton and Their Reciprocal Regulations in Cultured Cells

Background

TRPV4 and the cellular cytoskeleton have each been reported to influence cellular mechanosensitive processes as well as the development of mechanical hyperalgesia. If and how TRPV4 interacts with the microtubule and actin cytoskeleton at a molecular and functional level is not known.

Methodology and Principal Findings

We investigated the interaction of TRPV4 with cytoskeletal components biochemically, cell biologically by observing morphological changes of DRG-neurons and DRG-neuron-derived F-11 cells, as well as functionally with calcium imaging. We find that TRPV4 physically interacts with tubulin, actin and neurofilament proteins as well as the nociceptive molecules PKCε and CamKII. The C-terminus of TRPV4 is sufficient for the direct interaction with tubulin and actin, both with their soluble and their polymeric forms. Actin and tubulin compete for binding. The interaction with TRPV4 stabilizes microtubules even under depolymerizing conditions in vitro. Accordingly, in cellular systems TRPV4 colocalizes with actin and microtubules enriched structures at submembranous regions. Both expression and activation of TRPV4 induces striking morphological changes affecting lamellipodial, filopodial, growth cone, and neurite structures in non-neuronal cells, in DRG-neuron derived F11 cells, and also in IB4-positive DRG neurons. The functional interaction of TRPV4 and the cytoskeleton is mutual as Taxol, a microtubule stabilizer, reduces the Ca²⁺-influx via TRPV4.

Conclusions and Significance

TRPV4 acts as a regulator for both, the microtubule and the actin. In turn, we describe that microtubule dynamics are an important regulator of TRPV4 activity. TRPV4 forms a supra-molecular complex containing cytoskeletal proteins and regulatory kinases. Thereby it can integrate signaling of various intracellular second messengers and signaling cascades, as well as cytoskeletal dynamics. This study points out the existence of cross-talks between non-selective cation channels and cytoskeleton at multiple levels. These cross talks may help us to understand the molecular basis of the Taxol-induced neuropathic pain development commonly observed in cancer patients.     

Characteristics of the protein kinase activity associated with rat neurofilament preparations

Some properties of the protein kinase activity associated with neurofilaments isolated from the brain stem and spinal cord of rats have been investigated. The activity had an apparent Km for ATP of 20 microM, a pH optimum of 8.0 and phosphorylated both serine and threonine residues in neurofilament proteins. Cyclic AMP had no effect on the in vitro reaction and casein was a preferred exogenous substrate in comparison to histone. Phosphopeptide mapping of the 145 kDa subunit from neurofilaments phosphorylated in the presence and absence of microtubule proteins indicated that the neurofilament-associated activity was distinct from the microtubule-associated protein kinase. Limited proteolysis of neurofilaments with chymotrypsin indicated that the enzyme activity was not associated with a domain of the 200 kDa subunit which may form the side-arm projections on neurofilaments.     

Bulk Preparation of CNS Cytoskeleton and the Separation of Individual Neurofilament Proteins by Gel Filtration: Dye-Binding Characteristics and Amino Acid Compositions

Abstract: The three major proteins of mammalian neurofilaments, of molecular weight 70,000, 160,000, and 210,000, have been resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel eJectrophoresis, and more recently, by ion-exchange chromatography in urea solution. We describe here a method to separate the neurofilament proteins by gel filtration without the use of SDS. A bulk preparation of cytoskeleton from rat spinal cord was first characterized. This preparation was then solubilized in a buffer containing 8 M urea and subjected to gel filtration. Individual neurofilament proteins, in milligram quantities, were harvested following the pooling of appropriate fractions. Gel electrophoresis showed a high degree of homogeneity in each of the three pooled fractions. Dye binding studies demonstrated that the protein of molecular weight 210,000 was relatively underrepresented when stained with Coomassie Blue, while all three neurofilament proteins showed similar dye binding properties with Fast Green. Amino acid analysis indicated that (1) all three neurofilament proteins contained a high content of acidic residues; (2) the molecular weight 210.000 protein contained >8 mol% proline; and (3) no simple oligomeric relationship existed among the neurofilament triplets.