Brain Research, 133 (1977) 341-349
341
© Elsevier/North-Holland Biomedical Press
Short Communications
Anomalous electrophoretic properties of brain filament protein subunits
HOWARD FEIT, URSULA NEUDECK and JERRY SHAY Departments of Neurology and Cell Biology, University of Texas Health Science Center at Dallas, Dallas, Texas' 75235 (U.S.A.)
(Accepted April 27th, 1977)
Introduction
Filaments 10 nm in diameter are prominent constituents of the cytoskeleton of neuronal and glial cells. In nerve cells, the 10 nm filaments are called neurofilaments*, although similar filaments are found in a variety of other cell types and are generally referred to as tonofilaments or intermediate sized filaments so as to distinguish them from the filamentous forms of actin and myosin. Recently, 10 nm filaments have been isolated from myelinated bovine axonsZ, 21 and chicken smooth muscle 1,1z and from both of these sources, a polypeptide with a mol. wt. of 50,000 has been identified as the major constituent. An alternate approach to the identification of the protein subunits of the neurofilament was recently reported by Hoffman and Lasek 9. These workers took advantage of the electron microscope autoradiographic evidence that the slow component of axoplasmic flow contains mainly neurofilaments and neurotubules 3,4. Following injection of a radioactive amino acid precursor into the ventral horn of the spinal cord, a peak of radioactive protein corresponding to the slow component of axoplasmic flow was isolated from the ventral roots and analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The radioactivity was associated with tubulin and three other polypeptides: a major component at 68,000 and two minor components at 160,000 and 212,000. It was shown that these three polypeptides migrated down the axon at a velocity slightly slower than tubulin and were tentatively identified as components of a single structure, the 10 nm neurofilament. Thus, different methodologies have identified different polypeptides as constituents of the neurofilament. Direct isolation of brain filaments resulted in a single 50,000 mol. wt. subunit2,21; in contrast, analysis of axoplasmic flow identified three components with tool. wt. of 68,000, 160,000 and 212,0009. In this report, we show that the major component of a fraction enriched in brain filaments has an apparent tool. * In this report, the term neurofilament is used for 10 nm filaments of neuronal origin, and the term brain filament is used when it is uncertain if the 10 nm filaments are of neuronal or glial origin.
342 TABLEI
Gels Stacking gel Acrylamide Bisacrylamide Tris pH SDS Separating gel Acrylamide Bisacrylamide Tris Urea pH SDS Running buffer Upper reservoir Tris Boric acid Glycine pH SDS Lower reservoir Tris Glycine pH SDS
System A G
System B 1°
System C 9'14
3% 0.08 % 50 mM 6.8 0.1%
5.1% 0.14 % 63 mM 6.8 0.1%
3% 0.08 % 50 mM 6.1 0.1%
7.5 % 0.2 % 200 mM 8M 8.8 0.1%
7.5 % 0.2 % 400 mM 8.8 0.1 ~
7.5 0.2 % 400 mM ~I 9.2 0.1%
40 mM 200 mM 8.8 0.1%
40 mM 200 mM 8.8 0.1%
40 mM 8.6 0.1%
4C'mM 200 mM 8.8 0.1%
40 mM 200 mM 8.8 0.1%
60 mM 6.7 0.1%
wt. o f either 50,000, 68,000 or 90,000 depending on the polyacrylamide gel electrophoresis system. In addition, the filament fraction also contains higher molecular weight associated proteins as previously suggested by H o f f m a n and Lasek 9. The anomalous electrophoretic behavior of the major c o m p o n e n t o f the brain filament explains the apparent discrepancy in the reported molecular weight for this protein by different laboratories.
Materials and methods Materials. Phenylmethylsulfonyl fluoride (PMSF) was obtained f r o m Sigma Chemical C o m p a n y . Reagents used in electrophoresis were purchased f r o m the BioR a d Corporation. The urea was ultra pure grade obtained from Schwarz/Mann. All other chemicals were of reagent grade. Neurofilament preparation. Bovine brains were obtained f r o m a local slaughter house approximately 30 m i n after the animal's death. The isolation o f filaments was basically performed according to the procedure o f Yen et al. 21 except for minor modifications: all solutions were 1 m M P M S F , and the initial homogenate volume was one liter. The crude axoplasmic pellet was layered onto the 1.0-1.5-2.0 M sucrose gradient to obtain the enriched neurofilament layer on the 1.5-2.0 M interface. Mitoehondrial preparation. The mitochondrial preparation was performed according to the procedure of G r a y and Whittaker s.
Fig. 1. Transmission electron micrographs of subcellular fractions: (a) filament fraction (10,000 x); (b) mitechondria (10,000 x ); (c) filaments (35,000 >,") and (d) filaments with entrapped mitochondria (35,000 x).
344
Fig. 2. SDS-polyacrylamide gel electrophoresis (System A). A: crude axoplasm; B: filament fraction; C: mitochondria; D: mixture of equal protein concentrations of b and c (see legend to Fig. 1).
Tubulin preparation. Tubulin was isolated from adult mouse brain by the polymerization procedure described by Shelanski et al. 16.
Protein solubilization and polyacrylamide gel electrophoresis. Proteins were solubilized in sample buffer containing 8 M urea, 0.1 ~ SDS, 0.12 M mercaptoethanol, 0.12 M Tris and 0.001 ~ Bromophenol blue. The composition of the three discontinuous polyacrylamide gel systems used is given in Table I. Following electrophoresis,
345
A
B
C
Fig. 3. SDS-polyacrylamide gel electrophoresis (System A). A: filaments; B: tubulin; C: mixture of equal protein concentrations of a and b (see legend to Fig. 1). the gels were stained with Coomassm blue or with periodic acid-Schiff reagent 5. The gels were scanned on a Gilford spectrophotometer. The electrophoretic mobilities were calculated according to the procedure of Weber and Osborn 2°. Elution procedure. After the gels had destained, the appropriate bands were cut out and eluted electrophoretically by the procedure of Stephens is. The elutant was lyophilized and taken up in sample buffer.
346
e
f~ g
%
h
~, 9F T
Fig. 4. Comparison of the electrophoretic mobility of the major component of the filament under various conditions for SDS-polyacrylamidegel electrophoresis (see Table I). Gels labeled a and b were System A; gels c and d were System C; gels e and f were System B; gels g and h were System C.The reference proteins were myophosphorylase (94,000) P; tubulin (56,000 and 53,000) T; and albumin (68,000) A. The major component of the filament fraction is indicated by F. Each pair of gels shows the filament fraction alone on the right and mixture of filament protein and reference proteins on the left. Results
The p r o c e d u r e we used for isolating filaments f r o m bovine m y e l i n a t e d axons was modified f r o m the p r o c e d u r e o f Y e n et al. 21 b y the inclusion o f P M S F t h r o u g h o u t the isolation p r o c e d u r e a n d m i n o r changes in centrifugation a n d h o m o g e n i z a t i o n . The final fraction was enriched in bundles o f filaments b u t also c o n t a i n e d occasional m e m b r a n e a n d myelin f r a g m e n t s a n d m i t o c h o n d r i a t r a p p e d within filament b u n d l e s
347 (Fig. 1). Polyacrylamide gel electrophoresis (system A) revealed a major component with mol. wt. 90,000 which accounted for 75 ~ of the protein in this fraction (Fig. 2B). There was also a higher molecular weight doublet which represented 15 ~ of the total protein (Fig. 2A, arrow). In the absence of PMSF, the component at 90,000 separated into a series of bands with decreasing intensity suggesting terminal degradation of the polypeptide. Although polyacrylamide gel electrophoresis revealed a major component in the filament fraction, the possibility that other polypeptides were associated with the filament was also considered. Since the most abundant contaminants in the filament fraction were trapped mitochondria, we compared the filament fraction and a mitochondrial fraction by polyacrylamide gel electrophoresis. The filament fraction (Fig. 2B) contained a higher molecular weight doublet which was absent in the mitochondrial fraction (Fig. 2C). The high molecular weight doublet in the filament fraction (Fig. 3A, arrow) could be readily distinguished from the high molecular weight microtubule-associated proteins (Fig. 3B). The filament-associated proteins had mol. wt. of 210,000 and 180,000 as estimated by relative mobility on 5 ~ polyacrylamide gels, system A. These results suggest that, in analogy to microtubules, filaments also contain high molecular weight-associated proteins, in agreement with the axoplasmic flow data of Hoffman and Lasek 9. The molecular weight (90,000) of the major component of the filament fraction that we obtained differed substantially from 50,000 or 68,000 as reported by other investigators. Although SDS polyacrylamide gel electrophoresis had been used by each group, the exact methodology varied. Therefore, we compared the electrophoretic mobility of the major component of the filament fraction with the mobility of tubulin (53,000 and 56,000), albumin (68,000) and myophosphorylase (94,000) under a variety of electrophoretic conditions. Depending upon the formulation of the running gel, the major component of the filament had a mobility corresponding to either 90,000 (Fig. 4a and b), 68,000 (Fig. 4c and d or 4g and h) or 50,000 (Fig. 4e and f). The higher molecular weight was favored by the presence of high concentrations of urea and low concentrations of Tris. The composition of the spacer gel did not affect the mobility of the filament protein. Since it has been reported that some proteins such as the erythrocyte membrane protein glycophoran can form stable dimers in the presence of SDS at room temperature13,17, we determined whether heating in the presence of 1 SDS and 5 ~ mercaptoethanol prior to electrophoresis would cause the 90,000 dalton fraction to become smaller. This treatment did not alter the mobility of this polypeptide. Thus, the observed mobility seemed to be determined by the composition of the running gel. This was confirmed when it was observed that the 50,000 tool. wt. polypeptide could be eluted from one gel and run on a second gel under conditions that resulted in a mobility corresponding to 90,000. Similar anomalous behavior upon SDS gel electrophoresis has been reported for glycoproteins 19. However, the major component of the filament fraction did not stain with the periodic acid-Schiff reagent. Discussion
On the basis of the observations reported here and the results in previous in-
348 vestigations z,9,1z,21, there is considerable evidence that mammalian brain filaments are composed of a major component that exhibits anomalous behavior upon SDS polyacrylamide gel electrophoresis. These results explain why several laboratories using different approaches have obtained different molecular weights for this major component. The physical basis for the anomalous electrophoresis behavior of this subunit is unclear at present. One possible explanation would be to assume that the observed bands are different oligomers of a smaller subunit with a size in the range of 18,000-20,000. Then the series of molecular weights 50,000, 68,000 and 90,000 could be explained as oligomers of 3, 4 or 5 subunits respectively. However, at present, there is no independent evidence for the existence of this hypothetical 18,000-20,000 mol. wt. subunit. Furthermore, the particular electrophoretic mobilities may be the result of the particular experimental conditions selected and other conditions might yield other electrophoretic mobilities. Further studies of the effects of various forms of protein modification such as carboxymethylation, dansylation etc. should provide additional information about the physical basis for the unusual electrophoretic behavior of this protein. Neurofilaments have also been isolated from the invertebrate Myxicola7,11 but the protein obtained differs from the mammalian brain filament. The major polypeptides in the invertebrate neurofilament have mol. wt. near 152,000 and 160,000 v,11. The neurofilaments from Myxicola are soluble at an ionic strength greater than 0.3, whereas the mammalian brain filament is soluble only under strong denaturing conditions. There is also present in the axoplasm of Myxicola an endogenous calciumactivated protease which readily cleaves the neurofilament protein at thiol sites 7. We cannot exclude the possibility that the calf brain filaments were acted upon by a PSMF-resistant protease during isolation. Nevertheless, the variability reported for the size of the major polypeptide in mammalian brain filaments results not from proteolysis but rather from its unusual electrophoretic properties. In addition to a major component, evidence is accumulating that there are specific higher molecular weight proteins which are also associated with the neurofilamerit, just as there are specific higher molecular weight microtubule-associated proteins. Hoffman and Lasek 9 showed that a protein triplet migrated down the axon with the slow component of axoplasmic flow at a velocity that was unique from tubulin. The two larger proteins in that triplet had mol. wt. of 160,000 and 212,000, which is in reasonable agreement with the 180,000 and 210,000 components that were present in our filament fractions. These two components were absent in mitochondria, which were the most abundant contaminant in the filament fraction. Although we cannot exclude the possibility that these proteins are merely absorbed on to the filaments, their possible role as integral components of the neurofilament should be considered. These particular molecular weights are intriguing because the heavy chain of myosin in mammals is 210,000 daltons and in certain invertebrates is 160,000 daltons 15. In addition, Lazarides has recently reported the isolation of 10 nm filaments from smooth muscle, and this preparation also clearly contains material that co-migrates with myosin heavy chain in addition to a more prominent 50,000 tool. wt. componentlL However, other evidence, such as repolymerization studies in vitro or immunocytochemistry, will be
349 needed before it can be u n a m b i g u o u s l y concluded that 10 n m filaments c o n t a i n higher molecular weight proteins or proteins related to myosin. This work was supported by G r a n t 5-RO1-NS-12743-03 from the U.S.P.H.S. a n d by a g r a n t from the M u s c u l a r Dystrophy Association, Inc., to J.S.
1 Cooke, P., Filamentous cytoskeleton in veItebrate smooth muscle fibers, J. Cell Biol., (1976) 539-556. 2 Davison, P. F. and Boyd, W., The protein subunit of calf brain neurofilament, J. Neurobiol., 5 (1974) 119-133. 3 Droz, B. and Koenig, H. L., Localization of protein metabolism in neurons. In A. Lajtha (Ed.), Protein Metabolism of the Nervous System, Plenum Publ. Corp., New York, 1970, pp. 93-108. 4 Droz, B., Koenig, H. L. and DiGiamberardino, L., Axonal migration of protein and glycoprotein to nerve endings. I. Radioautographic analysis of the renewal of proteins in nerve endings of chicken ciliary ganglion after intracerebral injection of [3H]lysine, Brain Research, 60 (1973) 93127. 5 Fairbanks, G., Steck, T. L. and Wallach, D. F., Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane, Biochemistry, 10 (1971) 2606--2617. 6 Feit, H., Slusarek, L. and Shelanski, M. L., Heterogeneity of tubulin subunits, Proc. Nat. Acad. Sci. (Wash.), 68 (197l) 2028-2031. 7 Gilbert, D. S., Newby, B. J. and Anderton, B. H., Neurofilament disguise, destruction and discipline, Nature (Lond.), 256, 5518, (1975) 586-589. 8 Gray, E. G. and Whittaker, V. P., The isolation of nerve endings from brain: an electron microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat. (Lond.), 96 (1962) 79 87. 9 Hoffman, P. N. and Lasek, F. J., The slow component of axonal transport, J. Cell Biol., 66 (1975) 351-366. 10 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond.), 227 (1970) 680-685. 11 Lasek, R. J., Krishnan, N. and Kaiserman-Abramof, I., Comparative studies of 10 nm neurofilaments isolated from molluscan and annelid giant axons, J. Cell Biol., 67 (1975) 234a. 12 Lazarides, E, and Hubbard, B. D., Immunological characterization of the subunit of the 100 filaments from muscle cells, Proc. Nat. Acad. Sci. (Wash.), 73 (1976) 4344~348. 13 Marton, L. S. G. and Garvin J. E., Subunit structure of the major human erythrocyte glycoprotein: depolymerization by heating ghosts with sodium dodecyl sulfate, Biochem. biophys. Res. Commun., 52 (1973) 1435-1462. 14 Neville, D. M., Jr., Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system, J. biol. Chem., 246 (1971) 6328-6334. 15 Pollard, T. D., The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba, J. Cell Biol., 68 (1976) 579-601. 16 Shelanski, M. L., Haskin, F. and Cantor, C. R., Microtubule assembly in the absence of added nucleotides, Proc. nat. Aead. Sci. (Wash.), 70 (1973) 765-768. 17 Slutzky, G. M. and Ji, T. H., The dissimilar nature of two forms of the major hyman erythrocyte membrane glycoprotein, Biochim. biophys. Acta (Amst.), 373 (1974) 337-346. 18 Stephens, R. E., High-resolution preparation SDS-polyacrylamide gel electrophoresis: fluorescent visualization and electrophoretic elution-concentration of protein bands, Analyt. Biochem., 65 (1975) 369-379. 19 Weber, K. and Osborn, H., Proteins and sodium dodec~¢lsulfate: molecular weight determinations on polyacrylamide gels and related procedures. In Robert L. Hill (Ed.), The Proteins, VoL I, 1975, pp. 179-223. 20 Weber, K. and Osborn, M., The reliability of molecular weight determination by dodecyl sulfatepolyacrylamide gel electrophoresis, J. biol. Chem., 244 (196) 4406~412. 21 Yen, S., Dahl, D., Schachner, M. and Shelanski, M. L., Biochemistry of the filaments of brain, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 529-533.
341
© Elsevier/North-Holland Biomedical Press
Short Communications
Anomalous electrophoretic properties of brain filament protein subunits
HOWARD FEIT, URSULA NEUDECK and JERRY SHAY Departments of Neurology and Cell Biology, University of Texas Health Science Center at Dallas, Dallas, Texas' 75235 (U.S.A.)
(Accepted April 27th, 1977)
Introduction
Filaments 10 nm in diameter are prominent constituents of the cytoskeleton of neuronal and glial cells. In nerve cells, the 10 nm filaments are called neurofilaments*, although similar filaments are found in a variety of other cell types and are generally referred to as tonofilaments or intermediate sized filaments so as to distinguish them from the filamentous forms of actin and myosin. Recently, 10 nm filaments have been isolated from myelinated bovine axonsZ, 21 and chicken smooth muscle 1,1z and from both of these sources, a polypeptide with a mol. wt. of 50,000 has been identified as the major constituent. An alternate approach to the identification of the protein subunits of the neurofilament was recently reported by Hoffman and Lasek 9. These workers took advantage of the electron microscope autoradiographic evidence that the slow component of axoplasmic flow contains mainly neurofilaments and neurotubules 3,4. Following injection of a radioactive amino acid precursor into the ventral horn of the spinal cord, a peak of radioactive protein corresponding to the slow component of axoplasmic flow was isolated from the ventral roots and analyzed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The radioactivity was associated with tubulin and three other polypeptides: a major component at 68,000 and two minor components at 160,000 and 212,000. It was shown that these three polypeptides migrated down the axon at a velocity slightly slower than tubulin and were tentatively identified as components of a single structure, the 10 nm neurofilament. Thus, different methodologies have identified different polypeptides as constituents of the neurofilament. Direct isolation of brain filaments resulted in a single 50,000 mol. wt. subunit2,21; in contrast, analysis of axoplasmic flow identified three components with tool. wt. of 68,000, 160,000 and 212,0009. In this report, we show that the major component of a fraction enriched in brain filaments has an apparent tool. * In this report, the term neurofilament is used for 10 nm filaments of neuronal origin, and the term brain filament is used when it is uncertain if the 10 nm filaments are of neuronal or glial origin.
342 TABLEI
Gels Stacking gel Acrylamide Bisacrylamide Tris pH SDS Separating gel Acrylamide Bisacrylamide Tris Urea pH SDS Running buffer Upper reservoir Tris Boric acid Glycine pH SDS Lower reservoir Tris Glycine pH SDS
System A G
System B 1°
System C 9'14
3% 0.08 % 50 mM 6.8 0.1%
5.1% 0.14 % 63 mM 6.8 0.1%
3% 0.08 % 50 mM 6.1 0.1%
7.5 % 0.2 % 200 mM 8M 8.8 0.1%
7.5 % 0.2 % 400 mM 8.8 0.1 ~
7.5 0.2 % 400 mM ~I 9.2 0.1%
40 mM 200 mM 8.8 0.1%
40 mM 200 mM 8.8 0.1%
40 mM 8.6 0.1%
4C'mM 200 mM 8.8 0.1%
40 mM 200 mM 8.8 0.1%
60 mM 6.7 0.1%
wt. o f either 50,000, 68,000 or 90,000 depending on the polyacrylamide gel electrophoresis system. In addition, the filament fraction also contains higher molecular weight associated proteins as previously suggested by H o f f m a n and Lasek 9. The anomalous electrophoretic behavior of the major c o m p o n e n t o f the brain filament explains the apparent discrepancy in the reported molecular weight for this protein by different laboratories.
Materials and methods Materials. Phenylmethylsulfonyl fluoride (PMSF) was obtained f r o m Sigma Chemical C o m p a n y . Reagents used in electrophoresis were purchased f r o m the BioR a d Corporation. The urea was ultra pure grade obtained from Schwarz/Mann. All other chemicals were of reagent grade. Neurofilament preparation. Bovine brains were obtained f r o m a local slaughter house approximately 30 m i n after the animal's death. The isolation o f filaments was basically performed according to the procedure o f Yen et al. 21 except for minor modifications: all solutions were 1 m M P M S F , and the initial homogenate volume was one liter. The crude axoplasmic pellet was layered onto the 1.0-1.5-2.0 M sucrose gradient to obtain the enriched neurofilament layer on the 1.5-2.0 M interface. Mitoehondrial preparation. The mitochondrial preparation was performed according to the procedure of G r a y and Whittaker s.
Fig. 1. Transmission electron micrographs of subcellular fractions: (a) filament fraction (10,000 x); (b) mitechondria (10,000 x ); (c) filaments (35,000 >,") and (d) filaments with entrapped mitochondria (35,000 x).
344
Fig. 2. SDS-polyacrylamide gel electrophoresis (System A). A: crude axoplasm; B: filament fraction; C: mitochondria; D: mixture of equal protein concentrations of b and c (see legend to Fig. 1).
Tubulin preparation. Tubulin was isolated from adult mouse brain by the polymerization procedure described by Shelanski et al. 16.
Protein solubilization and polyacrylamide gel electrophoresis. Proteins were solubilized in sample buffer containing 8 M urea, 0.1 ~ SDS, 0.12 M mercaptoethanol, 0.12 M Tris and 0.001 ~ Bromophenol blue. The composition of the three discontinuous polyacrylamide gel systems used is given in Table I. Following electrophoresis,
345
A
B
C
Fig. 3. SDS-polyacrylamide gel electrophoresis (System A). A: filaments; B: tubulin; C: mixture of equal protein concentrations of a and b (see legend to Fig. 1). the gels were stained with Coomassm blue or with periodic acid-Schiff reagent 5. The gels were scanned on a Gilford spectrophotometer. The electrophoretic mobilities were calculated according to the procedure of Weber and Osborn 2°. Elution procedure. After the gels had destained, the appropriate bands were cut out and eluted electrophoretically by the procedure of Stephens is. The elutant was lyophilized and taken up in sample buffer.
346
e
f~ g
%
h
~, 9F T
Fig. 4. Comparison of the electrophoretic mobility of the major component of the filament under various conditions for SDS-polyacrylamidegel electrophoresis (see Table I). Gels labeled a and b were System A; gels c and d were System C; gels e and f were System B; gels g and h were System C.The reference proteins were myophosphorylase (94,000) P; tubulin (56,000 and 53,000) T; and albumin (68,000) A. The major component of the filament fraction is indicated by F. Each pair of gels shows the filament fraction alone on the right and mixture of filament protein and reference proteins on the left. Results
The p r o c e d u r e we used for isolating filaments f r o m bovine m y e l i n a t e d axons was modified f r o m the p r o c e d u r e o f Y e n et al. 21 b y the inclusion o f P M S F t h r o u g h o u t the isolation p r o c e d u r e a n d m i n o r changes in centrifugation a n d h o m o g e n i z a t i o n . The final fraction was enriched in bundles o f filaments b u t also c o n t a i n e d occasional m e m b r a n e a n d myelin f r a g m e n t s a n d m i t o c h o n d r i a t r a p p e d within filament b u n d l e s
347 (Fig. 1). Polyacrylamide gel electrophoresis (system A) revealed a major component with mol. wt. 90,000 which accounted for 75 ~ of the protein in this fraction (Fig. 2B). There was also a higher molecular weight doublet which represented 15 ~ of the total protein (Fig. 2A, arrow). In the absence of PMSF, the component at 90,000 separated into a series of bands with decreasing intensity suggesting terminal degradation of the polypeptide. Although polyacrylamide gel electrophoresis revealed a major component in the filament fraction, the possibility that other polypeptides were associated with the filament was also considered. Since the most abundant contaminants in the filament fraction were trapped mitochondria, we compared the filament fraction and a mitochondrial fraction by polyacrylamide gel electrophoresis. The filament fraction (Fig. 2B) contained a higher molecular weight doublet which was absent in the mitochondrial fraction (Fig. 2C). The high molecular weight doublet in the filament fraction (Fig. 3A, arrow) could be readily distinguished from the high molecular weight microtubule-associated proteins (Fig. 3B). The filament-associated proteins had mol. wt. of 210,000 and 180,000 as estimated by relative mobility on 5 ~ polyacrylamide gels, system A. These results suggest that, in analogy to microtubules, filaments also contain high molecular weight-associated proteins, in agreement with the axoplasmic flow data of Hoffman and Lasek 9. The molecular weight (90,000) of the major component of the filament fraction that we obtained differed substantially from 50,000 or 68,000 as reported by other investigators. Although SDS polyacrylamide gel electrophoresis had been used by each group, the exact methodology varied. Therefore, we compared the electrophoretic mobility of the major component of the filament fraction with the mobility of tubulin (53,000 and 56,000), albumin (68,000) and myophosphorylase (94,000) under a variety of electrophoretic conditions. Depending upon the formulation of the running gel, the major component of the filament had a mobility corresponding to either 90,000 (Fig. 4a and b), 68,000 (Fig. 4c and d or 4g and h) or 50,000 (Fig. 4e and f). The higher molecular weight was favored by the presence of high concentrations of urea and low concentrations of Tris. The composition of the spacer gel did not affect the mobility of the filament protein. Since it has been reported that some proteins such as the erythrocyte membrane protein glycophoran can form stable dimers in the presence of SDS at room temperature13,17, we determined whether heating in the presence of 1 SDS and 5 ~ mercaptoethanol prior to electrophoresis would cause the 90,000 dalton fraction to become smaller. This treatment did not alter the mobility of this polypeptide. Thus, the observed mobility seemed to be determined by the composition of the running gel. This was confirmed when it was observed that the 50,000 tool. wt. polypeptide could be eluted from one gel and run on a second gel under conditions that resulted in a mobility corresponding to 90,000. Similar anomalous behavior upon SDS gel electrophoresis has been reported for glycoproteins 19. However, the major component of the filament fraction did not stain with the periodic acid-Schiff reagent. Discussion
On the basis of the observations reported here and the results in previous in-
348 vestigations z,9,1z,21, there is considerable evidence that mammalian brain filaments are composed of a major component that exhibits anomalous behavior upon SDS polyacrylamide gel electrophoresis. These results explain why several laboratories using different approaches have obtained different molecular weights for this major component. The physical basis for the anomalous electrophoresis behavior of this subunit is unclear at present. One possible explanation would be to assume that the observed bands are different oligomers of a smaller subunit with a size in the range of 18,000-20,000. Then the series of molecular weights 50,000, 68,000 and 90,000 could be explained as oligomers of 3, 4 or 5 subunits respectively. However, at present, there is no independent evidence for the existence of this hypothetical 18,000-20,000 mol. wt. subunit. Furthermore, the particular electrophoretic mobilities may be the result of the particular experimental conditions selected and other conditions might yield other electrophoretic mobilities. Further studies of the effects of various forms of protein modification such as carboxymethylation, dansylation etc. should provide additional information about the physical basis for the unusual electrophoretic behavior of this protein. Neurofilaments have also been isolated from the invertebrate Myxicola7,11 but the protein obtained differs from the mammalian brain filament. The major polypeptides in the invertebrate neurofilament have mol. wt. near 152,000 and 160,000 v,11. The neurofilaments from Myxicola are soluble at an ionic strength greater than 0.3, whereas the mammalian brain filament is soluble only under strong denaturing conditions. There is also present in the axoplasm of Myxicola an endogenous calciumactivated protease which readily cleaves the neurofilament protein at thiol sites 7. We cannot exclude the possibility that the calf brain filaments were acted upon by a PSMF-resistant protease during isolation. Nevertheless, the variability reported for the size of the major polypeptide in mammalian brain filaments results not from proteolysis but rather from its unusual electrophoretic properties. In addition to a major component, evidence is accumulating that there are specific higher molecular weight proteins which are also associated with the neurofilamerit, just as there are specific higher molecular weight microtubule-associated proteins. Hoffman and Lasek 9 showed that a protein triplet migrated down the axon with the slow component of axoplasmic flow at a velocity that was unique from tubulin. The two larger proteins in that triplet had mol. wt. of 160,000 and 212,000, which is in reasonable agreement with the 180,000 and 210,000 components that were present in our filament fractions. These two components were absent in mitochondria, which were the most abundant contaminant in the filament fraction. Although we cannot exclude the possibility that these proteins are merely absorbed on to the filaments, their possible role as integral components of the neurofilament should be considered. These particular molecular weights are intriguing because the heavy chain of myosin in mammals is 210,000 daltons and in certain invertebrates is 160,000 daltons 15. In addition, Lazarides has recently reported the isolation of 10 nm filaments from smooth muscle, and this preparation also clearly contains material that co-migrates with myosin heavy chain in addition to a more prominent 50,000 tool. wt. componentlL However, other evidence, such as repolymerization studies in vitro or immunocytochemistry, will be
349 needed before it can be u n a m b i g u o u s l y concluded that 10 n m filaments c o n t a i n higher molecular weight proteins or proteins related to myosin. This work was supported by G r a n t 5-RO1-NS-12743-03 from the U.S.P.H.S. a n d by a g r a n t from the M u s c u l a r Dystrophy Association, Inc., to J.S.
1 Cooke, P., Filamentous cytoskeleton in veItebrate smooth muscle fibers, J. Cell Biol., (1976) 539-556. 2 Davison, P. F. and Boyd, W., The protein subunit of calf brain neurofilament, J. Neurobiol., 5 (1974) 119-133. 3 Droz, B. and Koenig, H. L., Localization of protein metabolism in neurons. In A. Lajtha (Ed.), Protein Metabolism of the Nervous System, Plenum Publ. Corp., New York, 1970, pp. 93-108. 4 Droz, B., Koenig, H. L. and DiGiamberardino, L., Axonal migration of protein and glycoprotein to nerve endings. I. Radioautographic analysis of the renewal of proteins in nerve endings of chicken ciliary ganglion after intracerebral injection of [3H]lysine, Brain Research, 60 (1973) 93127. 5 Fairbanks, G., Steck, T. L. and Wallach, D. F., Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane, Biochemistry, 10 (1971) 2606--2617. 6 Feit, H., Slusarek, L. and Shelanski, M. L., Heterogeneity of tubulin subunits, Proc. Nat. Acad. Sci. (Wash.), 68 (197l) 2028-2031. 7 Gilbert, D. S., Newby, B. J. and Anderton, B. H., Neurofilament disguise, destruction and discipline, Nature (Lond.), 256, 5518, (1975) 586-589. 8 Gray, E. G. and Whittaker, V. P., The isolation of nerve endings from brain: an electron microscopic study of cell fragments derived by homogenization and centrifugation, J. Anat. (Lond.), 96 (1962) 79 87. 9 Hoffman, P. N. and Lasek, F. J., The slow component of axonal transport, J. Cell Biol., 66 (1975) 351-366. 10 Laemmli, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond.), 227 (1970) 680-685. 11 Lasek, R. J., Krishnan, N. and Kaiserman-Abramof, I., Comparative studies of 10 nm neurofilaments isolated from molluscan and annelid giant axons, J. Cell Biol., 67 (1975) 234a. 12 Lazarides, E, and Hubbard, B. D., Immunological characterization of the subunit of the 100 filaments from muscle cells, Proc. Nat. Acad. Sci. (Wash.), 73 (1976) 4344~348. 13 Marton, L. S. G. and Garvin J. E., Subunit structure of the major human erythrocyte glycoprotein: depolymerization by heating ghosts with sodium dodecyl sulfate, Biochem. biophys. Res. Commun., 52 (1973) 1435-1462. 14 Neville, D. M., Jr., Molecular weight determination of protein-dodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system, J. biol. Chem., 246 (1971) 6328-6334. 15 Pollard, T. D., The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba, J. Cell Biol., 68 (1976) 579-601. 16 Shelanski, M. L., Haskin, F. and Cantor, C. R., Microtubule assembly in the absence of added nucleotides, Proc. nat. Aead. Sci. (Wash.), 70 (1973) 765-768. 17 Slutzky, G. M. and Ji, T. H., The dissimilar nature of two forms of the major hyman erythrocyte membrane glycoprotein, Biochim. biophys. Acta (Amst.), 373 (1974) 337-346. 18 Stephens, R. E., High-resolution preparation SDS-polyacrylamide gel electrophoresis: fluorescent visualization and electrophoretic elution-concentration of protein bands, Analyt. Biochem., 65 (1975) 369-379. 19 Weber, K. and Osborn, H., Proteins and sodium dodec~¢lsulfate: molecular weight determinations on polyacrylamide gels and related procedures. In Robert L. Hill (Ed.), The Proteins, VoL I, 1975, pp. 179-223. 20 Weber, K. and Osborn, M., The reliability of molecular weight determination by dodecyl sulfatepolyacrylamide gel electrophoresis, J. biol. Chem., 244 (196) 4406~412. 21 Yen, S., Dahl, D., Schachner, M. and Shelanski, M. L., Biochemistry of the filaments of brain, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 529-533.
