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Limitations to the usefulness of N-succinimidyl propionate for the study of retrograde axonal transport
N ~ Letters, 36 (1983) 203-209 Elsevier Scientific Pubfishers Ireland Ltd.
203
LIMITATIONS TO THE USEFULNESS OF N-SUCCINIMIDYL PRO-
M.J. LOGAN, W.G. McLEAN and K.F. MEIRI*
Department of Pharmacology and Therapeutics, University o f Liverpool, P.O. Box 147, Liverpool L69 3BX (U.K.J (Rec~ved January 19th, 1983; Accepted February 18th, 1983)
Key words: axonal transport - N-succinimidyl propionate - Bolton and Hunter reagent - rat - sciatic nerve
Retrograde axonal transport of radiolabelled proteins was studied in rat sciatic nerve, after direct application of [~HlN-succinimidyl propionate. Waves of radiolabelled proteins were observed but only two proteins were predominantly labelled, one of molecular weight 68 kilodaltons (K) and the other of 19K. There was no evidence to confirm the waves as representing retrograde axonal transport of identifiable proteins, and the tendency of the covalent label to bind selectively in vivo to a small number of prominent proteins limits its usefulness for the detection of retrogradely transported proteins in general.
In a number of studies, Fink and Gainer [5-7, 9] have demonstrated both anterograde and retrograde axonal transport of proteins radiolabelled covalently with the acylating agent [3H]N-succinimidyl propionate ([JHINSP). The obvious advantage of the use of [3H]NSP for measurement of retrograde axonal transport is the fact that proteins can be radiolabelled by application of the agent to the nerve trunk or axonal processes, without involvement of protein synthesis, it should therefore be possible by this method to study the retrograde axoual transport of putative trophic factors which may be introduced to the axon by uptake at nerve terminals; this is in contrast to previous methods of measuring retrograde axonal transport, all of which have, of necessity, concentrated on cell body-derived proteins [2, 8]. However, a peculiarity of the early work by Fink and Gainer on rat sciatic nerve using the [3H]NSP label [6] was that only a wave of slowly transported proteins (3-6 mm/day) could be adequately distinguished moving in the retrograde direction. The same authors have recently shown that the major slowly transported protein in that wave is serum albumin [9]. We have used a similar technique and demonstrated waves of retrogradely * Current address: Department of Anatomy and Neurobiology. Washington University School of Medicine. St. Louis. MO 63110. U.S.A. 0304-3940/83/0000-0000/5
03.00 .~ 1983 Elsevier Scientific Publishers Ireland Ltd.
204
transported |JH]NSP-labelled protein travelling at two distinct faster rates in rat sciatic nerve [I 1]. Here we report on further attempts to characterize these waves. Female Wistar albino rats were anaesthetized with pentobarbitone (50 mg/kg i.p.)
in paraffin film and placed around approximately 5 mm of the nerve trunk and the wound closed. In some experiments |3HINSP was replaced by |t'51]N-succinimidyl 3-(4-hydroxy 5-iodophenyl)propionate (Bolton and Hunter reagent, 2000 Ci/mmol; Amersham). With both reagents the solvent in which it was supplied was blown off under a gentle stream of nitrogen; the saline was added before all the solvent had evaporated. In preliminary studies involving attempts to improve the efficiency of labelling mobile proteins with [3H]NSP, we applied up to ! pM unlabelled NSP (prepared according to the general method of Anderson et al. lID to the nerve 30 rain prior to application of [JH]NSP. in a number of experiments, the sciatic nerve was ligated 20 mm proximal to the cuff with a pair of silk thread ligatures tied 5 mm apart. Various times later the animals were killed by decapitation, the nerves removed and cut into 2 mm pieces. For analysis of the general distribution of radiolabelled proteins in the nerve, the pieces were soaked in 10070 trichloroacetic acid (TCA) overnight at 6°C, washed once more with TCA and dissolved in Protosol (New England Nuclear) for liquid scintillation counting (L$C). For analysis of the composition of the radiolabelled material the nerve pieces were homogenized in 60/d 2% (w/v) sodium dodecyl sulphate ($DS), 10070 (v/v) glycerol in water. The homogenatcs were then boiled for 3 min, 2-mercaptoethanol added to a final conccntration of 5010 (v/v), and the samples centrifuged at 13,000 g for 4 min. The supernatants wcrc applied to an 8-19e/o polyacrylamide vertical slab gel with a 507~ stacking gel, and electrophoresed for 16 h at 5 mA per gel, with bromophenol bV.ue as marker dye. Gels were fixed in 10o/0 (w/v) TCA, 3070 (w/v) sulphosalicylic a~id and stained with Coomassie Brillant Blue and, where [~H]NSP was used, cut into ! m m pieces which were dissolved in 0.5 ml 5007o (w/v) hydrogexi peroxide for LSC. Where the t-"~l-labelled reagent was used, gels were dried and pk:ced in contact with pre-flashed Fuji RX X-ray film for up to 6 days at - 7 0 ° C . Analysis of the distribution of the [3H]NSP-labelled proteins at various times after labelling produced typical waves of radioactivity within the nerves. A representative profile of radioactivity 3 h after labelling is shown in Fig. I A. In all 4 of the 3 h profiles studied there was a fast wave of protein moving at a mean rate of 7.4 mm/h, with a more clearly defined wave travelling at 4 mm/h. In order to determine if the waves of radioactivity remained distinct within the nerve during transport away from the cuff zone, we analyzed the distribution of radioactivity at later time points. At 4 h after radiolabelling only one wave could be distinguished clearly within each individual nerve (Fig. IB), with rather more variability in the position of the wavefront, which had reached a mean distance of
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Fig. I. The distribution of radiolabclled, TCA-precipitable protein in representative sciatic nerves proximal to a cuff containing 100/~Ci [JHINSP applied A, J h; B. 4 It; C. 8 h; D, 12 h, and E. 18 h previously.
20 mm. At 8 h. however, only one nerve (Fig. IC) out of 4 examined showed any evidence of increased radioactivity in the region of 32 mm from the cuff where a 4 mm/h transported wave would be expected. Twelve hour~ after radiolabelling0 all 4 nerves examined showed a further wave of radioactive proteins with a front at 16-20 mm from the cuff, i.e. with an apparent rate of 1.5 mm/h (Fig. ID). Again, however, at a later time point (viz. 18 h) only one out of 4 nerves showed the wave continuing to be transported as a separate entity (Fig. IE). When all the profiles from any one time point were averaged the resulting composite gave indistinct waves due to the variations in wave position. Furthermore, in individual profiles, the waves became very diffuse with increasing distance from the cuff zone. We considered that the lack of definition of the waves may have been due to their being masked by a high level of diffusion of unbound [3HINSP from the cuff and subsequent incorporation into proteins. We used three separate methods in an attempt to reduce this possible diffusion. (a) By application of ,nlabelled NSP to the cuff zone 30 min prior to application of the [3N]NSP. The aim of this was to occupy all the NSP-binding sites, transported proteins being replaced at the cuff area by retrograde transport before the radiolabel was applied. (b) By application of I% Triton X-100 to the cuff zone for I min prior to application of [~HINSP. The aim here was to improve permeability of the nerve, particularly perineurial barriers, to the radiolabel.
206
(c) By application of 4% bovine serum aJbumin to the nerve over a distance of 20 mm from the cuff zone at the time of application of [3HINSP. The aim was to "mop up" excess diffusing radiolabei. None of these methods led to sharper prof'des. In fact, in case (b) above the waves even less distinct, In a further series o f experiments, 60 mm lengths of sciatic nerve trunk were removed from the rat, manually desheathed and incubated in a two-compartment chamber, as described by McLean et al. IlO]. Five mm of the nerve trunk, either at the proximal or distal end, were exposed to 50/LCi [JHINSP and the nerves incubated for a further 3.5 h to allow anterograde or retrograde axonal transport respectively of the [~HlNSP-radiolahelled proteins to take place. Waves of radiolabelled protein were subsequently detected within the nerves (results not shown), but there was a marked variability in the positions of the waves and there was no distinction between the distance moved by the radiolabelled proteins in the anterograde and retrograde directions. Addition of I0-4 M vinblastine sulphate to the compartment containing the unlabelled portion of the nerve had no significant effect on the position or size of the waves. Two major cri. eria for identification of axonal transport, viz. the relatively higher rate of anteroggade transport compared with retrograde [3J, and the sensitivity to vinblastine [4], were therefore not fulfilled in these in vitro experiments. It is known that each wave of radiolabelled proteins undergoing anterogradc axonal transport after administration of radioactive aminoacid precursor to nerve cell bodies represents separate groups of proteins [12]. link and Gainer [6] identified both anterograde and retrograde slowly transported proteins by SDS gel electrophoresis of the waves of [~H]NSP-labelled material in the rat sciatic nerve. They were unable, however, to obtain suffici~ :ly high radiolabelling of fast transported proteins, in either direction, for accurate analysis after electrophoretic separation. Wc therefore collected the proteins which were apparently being transported at the 4 mm/h rate and at the 7.4 mm/h rate by tying ligatures on the nerve 20 mm proximal to the cuff. at appropriate times after application of [~H]NSP. The accumulated proteit~s were subjected to SDS-polyacrylamide gel electrophofesis and the r~'sults are shown in Fig. 2. Also shown are the electrophoretic profile of radiolabelled proteins at the cuff zone, and the profile of proteins radiolabelled with [~H]NSP in a homogenate of rat sciatic nerve, in all cases the most heavily labelled bands were of proteins with approximate molecular weight 68,000 and 19,000 daltons. Radiolabell,:d proteins at the cuff site (Fig. 2A) also included a less prominent 3 ! ,000 dalton protein which was absent in the profile of proteins collected from the 4 mm/h wave (Fig. 2B) but was just detectable in the 7.4 m m / h wave (Fig. 2(;). The only other differences between the proteins in the waves and the proteins under the cuff was a tendency for the ratio of 19,000 to 68,000 to be higher in the 7.4 mm/h proteins. The apparent split in th,: 68,000 peak seen in Fig. 2B and less markedly in 2C was also seen on some gels of proteins under the cuff. Radiolabell-
207
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Fig. 2. Electrophoretic analysis of radiolabelled proteins in rat sciatic nerve afler application of a cuff containing 100 ~Ci I~HJNSP. Proteins were separated on a di.~ontinuous 8-19% polyacrylamide-SDS gel. stained with Coomassi¢ Brilli-mt Blue, and the gels then sliced into I mm pieces, solubili/ed, and radioactivity determined by LSC. Estimated molecular weishts are shown in kilodaltons. Three diasrams ,,how proteins in a 2 mm piece from one representative nerve, taken front under the cuff (A) distal to a double ligature placed 20 mm proximal to the cuff at time points ~ as to accumulate those proteins moving at 4 mm/h (B) and tho~ proteins moving at 7.4 m m / h (C). I) shov,'s the radiolabelling pattern ~ f a homogenate of 2 mm sciatic nerve exposed to ~/4('i IJHJNSP and E is a densitontetrk" scan o f (,~oma~sie Brilliant Bluc stained proleins from 2 mm of unlabelled sciatic nerve (arbitrary units).
ing with 13HINSPof a homogenate of rat sciatic nerve (Fig. 2D), on the other hand, produced a labelling pattern which was much closer to thal of a densitometric scan of a Coomassie blue-stained gel (Fig. 2E), in which the most prominent peak is at 31,000, and a number of other proteins are clearly visible in addition to the 68,000 and 19,000 bands. We therefore confirm link and Gainer's observations [6] thai [JH]NSP radiolabels in vivo only a small number of selected proteins which can be detected in slices of polyacrylamide gels, with a protein of 68,000 molecular weigh[ predominating. One explanation for these findings was that the method of measurement of [3H]NSP-radiolabelled proteins in the gels was not sufficiently sensitive to detect other proteins which may have accumulated from the 4 mm/h or 7.4 mm/h waves. We therefore applied the IZSl-labelled analogue of NSP, viz. Bolton and Hunter reagent, to the nerve so that the more sensitive technique of autoradiography of the
20S
Fig. 3. Autoradiograph o f t2:lAabeBed proteins. 100 itC ~, I'~llBolton and Hunter reagent was applied in a cuff to the sciatic nerve. After 4 h the nerve was sectiotted into 2 m m pieces which were homogenized and dectrophoresecl on an
8- t9¢~ gradiempolyaa34mide-SDSgel (orion at top}. The gelsweredried, and exposed to LKB Ultrafilm for 16 days at - ?O°C. The segtion showlt is a 2 m m nerve piece tO m m proximal to the cuff, with the m a j o r labelled bands o f molecular weight 68,000 and 19,000 daltons.
gels could be employed (Fig. 3). Again two bands were predominantly labelled, the 19,000 protein and especially the 68,000 molecular weight protein. Very little '..abel could be visualized, even at the cuff zone, apart from these two bands, suggesting that our original findings with [~H]NSP were not as a consequence of insensitivity of technique, but due to a selectivity of binding of the labe; to these proteins. These two bands predominated even in those parts of the nerve containing the 4 mm/h attd 7.4 mm/h waves (Fig. 3) and even beyond these points (not shown). There was no evidence at any point in the nerve for any selective increase in individual proteins despite very intense radiolabelling of the 68,000 band. The wave-like appearance of the profiles of radiolabelled proteins travelling in a retrograde direction at various time points differed from the exponential distribution of radiolabeUing that would be expected if we were merely measuring diffusion of [~H]NSP away from the site of application. However, we are forced to conclude that, in our hands, there was no evidence to confirm the waves as representing the retrograde axonal transport of identifiable proteins. The tendency of [~H]NSP to label selectively only a small number of prominent proteins makes it useful, as has been demonstrated, for the measurement of the slow retrograde axonal transport of serum albumin [9]. However, this tendency limits its usefulness for the detection of retrogradely transported proteins in general. We are grateful to Ann McKay for technical assistance. The work was supported by a University of Liverpool research grant and postgraduate studentship to M.J.L.
209 I Anderson, G.W., Zimmerman, J.E. and Callahan, F.M., The use of esters of N-succinimide in peptide synthesis, J. Amer. Chem. Sot., 86 (1964) 1839-1842. 2 Bisby, M.A., Reversal of axonal transport: similarity of proteins transported in anterograde and retrograde directions, J. Neurochem., 36 (1981) 741-745. 3 Bisby, M.A. and Buchan, D.H., Vdocity of labelled protein undergoing anterograde and retrograde 4 Chan~ SiY.' Worthi R: ~ Ochs, S., Block o f axoplasmic transport in vitro by Vinca alkaloids, J. Neurobiol., 11 (1980) 251-264. 5 link, D.J. and Gainer, H., The use of a labelled acylating probe for the study of fast axonal transport, in vivo, Brain Res., 177 (1979) 208-213. 6 link, D.J. and Gainer, H., Axonal transport of proteins. A new view using in vivo covalent labelling, J. Cell Biol., 8S (1980) 175-186. 7 Fink, D.J. and Gainer, H., Retrograde axonal transport of endogenous proteins in sciatic nerve demonstrated by covalent labelling in vivo, Science, 208 (1980) 303-305. 8 Frizell, M., and gj0strand, J., Retrograde axonal transport of rapidly migrating proteins in the vagus and hypoglossal nerves of the rabbit, J. Neurochem., 25 (1974) 651-657. 9 Gainer. H. and Fink, D.J.. Evidence for slow retrograde transport of serum albumin in r~lt sciatic nerve, Brain ges., 2J3 (1982) 404-408. 10 McLean, W.G., Frizell, M. and Sj0strand, J., Axonal transport of labelled proteins in sensory fibres of rabbit vagus nerve in vitro, J. Neurocbem., 25 (1975) 695-698. 11 McLean, W.G. and Meiri, K.F., Two rapid rates of retrograde axonal transport of proteins radiolabelled with N-succinimidyl propiona~e in rat sciatic nerve, J. Physiol. (Lond.), 317 (198 I) 82P. 12 Tytell, M., Black, M.M., Garner, J. and Lasek, R.J., Axonal transport: Each major rate component reflects the movement of distinct macromolecular complexes, Science, 214 ( 198 I) 179-18 !.
203
LIMITATIONS TO THE USEFULNESS OF N-SUCCINIMIDYL PRO-
M.J. LOGAN, W.G. McLEAN and K.F. MEIRI*
Department of Pharmacology and Therapeutics, University o f Liverpool, P.O. Box 147, Liverpool L69 3BX (U.K.J (Rec~ved January 19th, 1983; Accepted February 18th, 1983)
Key words: axonal transport - N-succinimidyl propionate - Bolton and Hunter reagent - rat - sciatic nerve
Retrograde axonal transport of radiolabelled proteins was studied in rat sciatic nerve, after direct application of [~HlN-succinimidyl propionate. Waves of radiolabelled proteins were observed but only two proteins were predominantly labelled, one of molecular weight 68 kilodaltons (K) and the other of 19K. There was no evidence to confirm the waves as representing retrograde axonal transport of identifiable proteins, and the tendency of the covalent label to bind selectively in vivo to a small number of prominent proteins limits its usefulness for the detection of retrogradely transported proteins in general.
In a number of studies, Fink and Gainer [5-7, 9] have demonstrated both anterograde and retrograde axonal transport of proteins radiolabelled covalently with the acylating agent [3H]N-succinimidyl propionate ([JHINSP). The obvious advantage of the use of [3H]NSP for measurement of retrograde axonal transport is the fact that proteins can be radiolabelled by application of the agent to the nerve trunk or axonal processes, without involvement of protein synthesis, it should therefore be possible by this method to study the retrograde axoual transport of putative trophic factors which may be introduced to the axon by uptake at nerve terminals; this is in contrast to previous methods of measuring retrograde axonal transport, all of which have, of necessity, concentrated on cell body-derived proteins [2, 8]. However, a peculiarity of the early work by Fink and Gainer on rat sciatic nerve using the [3H]NSP label [6] was that only a wave of slowly transported proteins (3-6 mm/day) could be adequately distinguished moving in the retrograde direction. The same authors have recently shown that the major slowly transported protein in that wave is serum albumin [9]. We have used a similar technique and demonstrated waves of retrogradely * Current address: Department of Anatomy and Neurobiology. Washington University School of Medicine. St. Louis. MO 63110. U.S.A. 0304-3940/83/0000-0000/5
03.00 .~ 1983 Elsevier Scientific Publishers Ireland Ltd.
204
transported |JH]NSP-labelled protein travelling at two distinct faster rates in rat sciatic nerve [I 1]. Here we report on further attempts to characterize these waves. Female Wistar albino rats were anaesthetized with pentobarbitone (50 mg/kg i.p.)
in paraffin film and placed around approximately 5 mm of the nerve trunk and the wound closed. In some experiments |3HINSP was replaced by |t'51]N-succinimidyl 3-(4-hydroxy 5-iodophenyl)propionate (Bolton and Hunter reagent, 2000 Ci/mmol; Amersham). With both reagents the solvent in which it was supplied was blown off under a gentle stream of nitrogen; the saline was added before all the solvent had evaporated. In preliminary studies involving attempts to improve the efficiency of labelling mobile proteins with [3H]NSP, we applied up to ! pM unlabelled NSP (prepared according to the general method of Anderson et al. lID to the nerve 30 rain prior to application of [JH]NSP. in a number of experiments, the sciatic nerve was ligated 20 mm proximal to the cuff with a pair of silk thread ligatures tied 5 mm apart. Various times later the animals were killed by decapitation, the nerves removed and cut into 2 mm pieces. For analysis of the general distribution of radiolabelled proteins in the nerve, the pieces were soaked in 10070 trichloroacetic acid (TCA) overnight at 6°C, washed once more with TCA and dissolved in Protosol (New England Nuclear) for liquid scintillation counting (L$C). For analysis of the composition of the radiolabelled material the nerve pieces were homogenized in 60/d 2% (w/v) sodium dodecyl sulphate ($DS), 10070 (v/v) glycerol in water. The homogenatcs were then boiled for 3 min, 2-mercaptoethanol added to a final conccntration of 5010 (v/v), and the samples centrifuged at 13,000 g for 4 min. The supernatants wcrc applied to an 8-19e/o polyacrylamide vertical slab gel with a 507~ stacking gel, and electrophoresed for 16 h at 5 mA per gel, with bromophenol bV.ue as marker dye. Gels were fixed in 10o/0 (w/v) TCA, 3070 (w/v) sulphosalicylic a~id and stained with Coomassie Brillant Blue and, where [~H]NSP was used, cut into ! m m pieces which were dissolved in 0.5 ml 5007o (w/v) hydrogexi peroxide for LSC. Where the t-"~l-labelled reagent was used, gels were dried and pk:ced in contact with pre-flashed Fuji RX X-ray film for up to 6 days at - 7 0 ° C . Analysis of the distribution of the [3H]NSP-labelled proteins at various times after labelling produced typical waves of radioactivity within the nerves. A representative profile of radioactivity 3 h after labelling is shown in Fig. I A. In all 4 of the 3 h profiles studied there was a fast wave of protein moving at a mean rate of 7.4 mm/h, with a more clearly defined wave travelling at 4 mm/h. In order to determine if the waves of radioactivity remained distinct within the nerve during transport away from the cuff zone, we analyzed the distribution of radioactivity at later time points. At 4 h after radiolabelling only one wave could be distinguished clearly within each individual nerve (Fig. IB), with rather more variability in the position of the wavefront, which had reached a mean distance of
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Fig. I. The distribution of radiolabclled, TCA-precipitable protein in representative sciatic nerves proximal to a cuff containing 100/~Ci [JHINSP applied A, J h; B. 4 It; C. 8 h; D, 12 h, and E. 18 h previously.
20 mm. At 8 h. however, only one nerve (Fig. IC) out of 4 examined showed any evidence of increased radioactivity in the region of 32 mm from the cuff where a 4 mm/h transported wave would be expected. Twelve hour~ after radiolabelling0 all 4 nerves examined showed a further wave of radioactive proteins with a front at 16-20 mm from the cuff, i.e. with an apparent rate of 1.5 mm/h (Fig. ID). Again, however, at a later time point (viz. 18 h) only one out of 4 nerves showed the wave continuing to be transported as a separate entity (Fig. IE). When all the profiles from any one time point were averaged the resulting composite gave indistinct waves due to the variations in wave position. Furthermore, in individual profiles, the waves became very diffuse with increasing distance from the cuff zone. We considered that the lack of definition of the waves may have been due to their being masked by a high level of diffusion of unbound [3HINSP from the cuff and subsequent incorporation into proteins. We used three separate methods in an attempt to reduce this possible diffusion. (a) By application of ,nlabelled NSP to the cuff zone 30 min prior to application of the [3N]NSP. The aim of this was to occupy all the NSP-binding sites, transported proteins being replaced at the cuff area by retrograde transport before the radiolabel was applied. (b) By application of I% Triton X-100 to the cuff zone for I min prior to application of [~HINSP. The aim here was to improve permeability of the nerve, particularly perineurial barriers, to the radiolabel.
206
(c) By application of 4% bovine serum aJbumin to the nerve over a distance of 20 mm from the cuff zone at the time of application of [3HINSP. The aim was to "mop up" excess diffusing radiolabei. None of these methods led to sharper prof'des. In fact, in case (b) above the waves even less distinct, In a further series o f experiments, 60 mm lengths of sciatic nerve trunk were removed from the rat, manually desheathed and incubated in a two-compartment chamber, as described by McLean et al. IlO]. Five mm of the nerve trunk, either at the proximal or distal end, were exposed to 50/LCi [JHINSP and the nerves incubated for a further 3.5 h to allow anterograde or retrograde axonal transport respectively of the [~HlNSP-radiolahelled proteins to take place. Waves of radiolabelled protein were subsequently detected within the nerves (results not shown), but there was a marked variability in the positions of the waves and there was no distinction between the distance moved by the radiolabelled proteins in the anterograde and retrograde directions. Addition of I0-4 M vinblastine sulphate to the compartment containing the unlabelled portion of the nerve had no significant effect on the position or size of the waves. Two major cri. eria for identification of axonal transport, viz. the relatively higher rate of anteroggade transport compared with retrograde [3J, and the sensitivity to vinblastine [4], were therefore not fulfilled in these in vitro experiments. It is known that each wave of radiolabelled proteins undergoing anterogradc axonal transport after administration of radioactive aminoacid precursor to nerve cell bodies represents separate groups of proteins [12]. link and Gainer [6] identified both anterograde and retrograde slowly transported proteins by SDS gel electrophoresis of the waves of [~H]NSP-labelled material in the rat sciatic nerve. They were unable, however, to obtain suffici~ :ly high radiolabelling of fast transported proteins, in either direction, for accurate analysis after electrophoretic separation. Wc therefore collected the proteins which were apparently being transported at the 4 mm/h rate and at the 7.4 mm/h rate by tying ligatures on the nerve 20 mm proximal to the cuff. at appropriate times after application of [~H]NSP. The accumulated proteit~s were subjected to SDS-polyacrylamide gel electrophofesis and the r~'sults are shown in Fig. 2. Also shown are the electrophoretic profile of radiolabelled proteins at the cuff zone, and the profile of proteins radiolabelled with [~H]NSP in a homogenate of rat sciatic nerve, in all cases the most heavily labelled bands were of proteins with approximate molecular weight 68,000 and 19,000 daltons. Radiolabell,:d proteins at the cuff site (Fig. 2A) also included a less prominent 3 ! ,000 dalton protein which was absent in the profile of proteins collected from the 4 mm/h wave (Fig. 2B) but was just detectable in the 7.4 m m / h wave (Fig. 2(;). The only other differences between the proteins in the waves and the proteins under the cuff was a tendency for the ratio of 19,000 to 68,000 to be higher in the 7.4 mm/h proteins. The apparent split in th,: 68,000 peak seen in Fig. 2B and less markedly in 2C was also seen on some gels of proteins under the cuff. Radiolabell-
207
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Fig. 2. Electrophoretic analysis of radiolabelled proteins in rat sciatic nerve afler application of a cuff containing 100 ~Ci I~HJNSP. Proteins were separated on a di.~ontinuous 8-19% polyacrylamide-SDS gel. stained with Coomassi¢ Brilli-mt Blue, and the gels then sliced into I mm pieces, solubili/ed, and radioactivity determined by LSC. Estimated molecular weishts are shown in kilodaltons. Three diasrams ,,how proteins in a 2 mm piece from one representative nerve, taken front under the cuff (A) distal to a double ligature placed 20 mm proximal to the cuff at time points ~ as to accumulate those proteins moving at 4 mm/h (B) and tho~ proteins moving at 7.4 m m / h (C). I) shov,'s the radiolabelling pattern ~ f a homogenate of 2 mm sciatic nerve exposed to ~/4('i IJHJNSP and E is a densitontetrk" scan o f (,~oma~sie Brilliant Bluc stained proleins from 2 mm of unlabelled sciatic nerve (arbitrary units).
ing with 13HINSPof a homogenate of rat sciatic nerve (Fig. 2D), on the other hand, produced a labelling pattern which was much closer to thal of a densitometric scan of a Coomassie blue-stained gel (Fig. 2E), in which the most prominent peak is at 31,000, and a number of other proteins are clearly visible in addition to the 68,000 and 19,000 bands. We therefore confirm link and Gainer's observations [6] thai [JH]NSP radiolabels in vivo only a small number of selected proteins which can be detected in slices of polyacrylamide gels, with a protein of 68,000 molecular weigh[ predominating. One explanation for these findings was that the method of measurement of [3H]NSP-radiolabelled proteins in the gels was not sufficiently sensitive to detect other proteins which may have accumulated from the 4 mm/h or 7.4 mm/h waves. We therefore applied the IZSl-labelled analogue of NSP, viz. Bolton and Hunter reagent, to the nerve so that the more sensitive technique of autoradiography of the
20S
Fig. 3. Autoradiograph o f t2:lAabeBed proteins. 100 itC ~, I'~llBolton and Hunter reagent was applied in a cuff to the sciatic nerve. After 4 h the nerve was sectiotted into 2 m m pieces which were homogenized and dectrophoresecl on an
8- t9¢~ gradiempolyaa34mide-SDSgel (orion at top}. The gelsweredried, and exposed to LKB Ultrafilm for 16 days at - ?O°C. The segtion showlt is a 2 m m nerve piece tO m m proximal to the cuff, with the m a j o r labelled bands o f molecular weight 68,000 and 19,000 daltons.
gels could be employed (Fig. 3). Again two bands were predominantly labelled, the 19,000 protein and especially the 68,000 molecular weight protein. Very little '..abel could be visualized, even at the cuff zone, apart from these two bands, suggesting that our original findings with [~H]NSP were not as a consequence of insensitivity of technique, but due to a selectivity of binding of the labe; to these proteins. These two bands predominated even in those parts of the nerve containing the 4 mm/h attd 7.4 mm/h waves (Fig. 3) and even beyond these points (not shown). There was no evidence at any point in the nerve for any selective increase in individual proteins despite very intense radiolabelling of the 68,000 band. The wave-like appearance of the profiles of radiolabelled proteins travelling in a retrograde direction at various time points differed from the exponential distribution of radiolabeUing that would be expected if we were merely measuring diffusion of [~H]NSP away from the site of application. However, we are forced to conclude that, in our hands, there was no evidence to confirm the waves as representing the retrograde axonal transport of identifiable proteins. The tendency of [~H]NSP to label selectively only a small number of prominent proteins makes it useful, as has been demonstrated, for the measurement of the slow retrograde axonal transport of serum albumin [9]. However, this tendency limits its usefulness for the detection of retrogradely transported proteins in general. We are grateful to Ann McKay for technical assistance. The work was supported by a University of Liverpool research grant and postgraduate studentship to M.J.L.
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