The metabolism of nerve terminal glycoproteins in the rat brain

Brain Research, 130 (1977) 109-120

109

© Elsevier/North-Holland Biomedical Press

T H E METABOLISM OF NERVE T E R M I N A L G L Y C O P R O T E I N S IN T H E RAT BRAIN

O. K. LANGLEY and P. KENNEDY MRC Research Group in Applied Neurobiology, Institute of Neurology, London WC1 (Great Britain)

(Accepted November 3rd, 1976)

SUMMARY The fate of [3H]fucose in subcellular fractions has been followed for a period of three weeks after intraventricular injection in the rat brain. Isolated synaptosomal membranes were examined by gel electrophoresis. Seven fucosyl glycoproteins with molecular weights ranging from 123,000 to 31,000 were detected by scintillation counting of transversely sliced gels and their specific activities determined. The halflives of 6 of the membrane glycoproteins fell within the range 11-22 days. The smallest glycoprotein showed anomalous behaviour; its radioactivity did not fall significantly over the period of study. The results are discussed in relation to current concepts of fast axonal transport.

INTRODUCTION Two of the more intriguing aspects of the neurone concern the functional significance of proteins and glycoproteins located on the surface membrane of nerve terminals and the mechanisms whereby such molecules, that are synthesised close to the cell nucleus, are transported to such sites. Such glycoproteins on terminal membranes have been thought to determine the specificity of cellular interactions in the brain 2, a recognition function which has also been proposed for other tissues 17. Brain glycoproteins may also function both in learning processes 7 and in the transfer o f information from tissue sites outside the brain 4. While those glycoproteins which contain sialic acid confer a negative surface charge on membranes and may play a role in maintaining cell separation 20, neutral carbohydrate-containing macromolecules have less clearly defined functions 19. The chemical characterization o f individual glycoproteins that constitute nerve ending membranes remains an essential forerunner to understanding their function. A second problem resulting from the unusual morphology of the neurone,

110 namely axonal transport, has aroused considerable interest over the years. Recently the parameters of fast transport of cell components to the periphery have been defined both in the mammalian peripheral nervous system 2s and in the ciliary ganglion of the chicken 12. Data are presented here that relate to both aspects of normal neuronal behaviour. The fate of radioactively labelled fucose (a precursor of glycoproteins 3) has been followed after intraventricular injection in the rat. Previous investigations 32,a3 on the accumulation of radioactive fucose in the synaptosomal fraction of mouse brain demonstrated that incorporation does not occur directly at the nerve terminal, but that protein-bound fucose is transported to this site from the nerve cell body. In the present work individual labelled glycoproteins, subsequently transported and found in the membrane fractions of nerve terminals, have been studied by gel electrophoresis and their half-lives estimated. METHODS

Intraventricular injections L-[3H]fucose (Radiochemical Centre, Amersham) in 0 . 9 ~ saline (0.08 ml; 70 #Ci) was injected into the left lateral ventricles of male Wistar rats (250-300 g) under ether anaesthesia through a parietal burr hole.

Isolation of subcellular and sub-synaptosomal fraetions Synaptosomal membranes were isolated by the method of Cotman and Matthews 1°. Briefly, animals were killed by decapitation and individual entire forebrains were excised at intervals of 1 11-21 days after injection and homogenized in unbuffered 0.32 M sucrose (10 vol.) with three strokes of a Teflon/glass homogeniser (speed of rotation 1000 rev./min; clearance 0.25 mm). Two or more rats were used for each time interval. Neither brains nor subfractions were pooled at any stage in the isolation procedure. After removal of the nuclear pellet at low speed (1000 × g; 10 min) the crude mitochondrial pellet was isolated (12,000 × g; 20 min), resuspended in 0.32 M sucrose and centrifuged on a discontinuous gradient with 7.5 and 13 ~ Ficoll in 0.32 M sucrose (100,000 × g; 30 min). After centrifugation the uppermost interfacial layer (largely myelin) was discarded and the pellet was retained as the whole brain mitochondrial fraction. The interfacial layer above the 13 ~ Ficoll solution (synaptosomes) was aspirated, washed once in fresh sucrose and sedimented (100,000 × g; 30 min). A portion of this resuspended in 0.32 M sucrose was subsequently retained for assay, while the remainder was subjected to osmotic shock (at 4 °C for 90 min) by dilution (10-fold) with 6 m M Tris • HC1, pH 8.1. After this treatment particulate matter was pelleted (100,000 × g; 10 rain) and the supernatant (synaptosome 'soluble' fraction) kept. The pellet, after gentle resuspension in 0.32 M sucrose, was further fractionated (100,000 × g; 60 min) on a discontinuous gradient consisting of layers of 25 ~ and 37 ~ (w/w) sucrose to produce two membranous fractions (I and II) and an intrasynaptosomal mitochondrial pellet (lII). The microsomal fraction was obtained by centrifugation (100,000 × g; 90 min) of the postmitochondrial supernatant derived from the brain homogenate. The postmicrosomal supernatant was aspirated and retained as the 'brain soluble fraction'.

111

Analytical procedures Protein was assayed by the method of Lowry et al. 9'3 using bovine serum albumen as standard. Tritium was counted on washed TCA precipitates (final concentration of TCA, 10% w/v) after extraction of lipids with a sequence of ethanol, acetone and ether. Dried precipitates were dissolved in 1 ml Soluene-350 (Packard) and Permablend III (10 ml; Packard) was used as scintillant. A Packard Tri-carb liquid scintillation spectrometer 3320 was used for counting. Certain TCA soluble fractions were counted when testing for free fucose using a Triton X-100-toluene scintillant. Results for subcellular fractions and synaptosomal membranes were expressed as relative specific activity after dividing their specific activity (counts/min/mg protein) by that of the homogenate.

Gel electrophoresis Subcellular and sub-synaptosomal fractions were prepared for electrophoresis after protein precipitation (10% w/v TCA) and lipid extraction (ethanol, acetone, ether) by dissolution in Na2CO3 (0.05 M). Sodium dodecyl sulphate (SDS; 8 mg/mg protein) and 2-mercaptoethanol were subsequently added and the solutions heated at 90 °C for 2 min. The resulting clear solutions were dialysed against upper gel buffer containing 0.1% SDS and 0.05 % dithiothreitol and 2 M urea, and aliquots were then electrophoresed on 11.1% acrylamide gels (pH 9.5) using the discontinuous buffer system of Neville ~7, bromophenol blue being used as the tracking dye. After electrophoresis the gels were stained with Coomassie brilliant blue (0.035 % in 7 % acetic acid - 4 % methanol) for 4 h and destained by diffusion in large volumes of 7 % acetic acid containing 5 % methanol. Protein profiles of gels were then obtained by scanning at 550 nm using a Schoefel spectrodensitometer SD 3000. Scintillation counting was performed as for washed TCA percipitates on 1 mm transverse gel slices. The specific activities of the proteins contained within slices were calculated from the scintillation counts and the protein content calculated for each slice. For this, first the protein content of each slice, expressed as a percentage of the total protein present on the gel, was determined from the integration trace of densitometric scan of the Coomassie Blue stained gel. The total protein per gel was then calculated by dividing the radioactivity (counts/min) summed over the whole gel by the specific activity of the membrane fraction under investigation. Thus the actual protein content (in/~g) of each slice could be calculated and the specific activity of proteins contained within slices determined.

Electron microscopy The synaptosomaI fractions (prior to osmotic shock) and the three subsynaptosomal fractions were monitored by electron microscopy. Fractions were fixed in 5 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and pelleted by high speed centrifugation. They were postosmicated in 1 ~ osmium tetroxide in cacodylate buffer before dehydration in ascending alcohols and embedded in Epon. Thin sections were viewed in a Philips 301 electron microscope.

112 RESULTS

Isotope injection The efficacy of the injection technique in terms of isotope distribution was tested using a saline solution of Evans Blue. On removal of the forebrain 40 min after injection, Evans Blue was seen to be evenly distributed throughout the ventricular system. It had gained access to the basal subarachnoid cisterns through the fourth ventricular foramina. No trauma or distortion of the brain resulting from the injection could be discerned. A strict comparison of Evans Blue and fucose distributions may, however, not be valid due to differences in the size and cellular uptake of these molecules.

Purity of subcellular fractions The purity of the nerve ending preparation and membrane subfraction derived from it was monitored by quantitative electron microscopic techniques. Frequency distributions of various subcellular organelles were obtained by counting with the aid of a grid superimposed on electron micrographs of synaptosomal fractions not exposed to osmotic shock. Such fractions were estimated to consist of greater than 65 ~ synaptosomes which was within the range determined by the original authors 10. Contaminating elements included free mitochondria, myelin and membrane bounded vesicles. Synaptosomes and their contents were in general well preserved and occasionally the synaptic apparatus with postsynaptic membrane still attached was observed. The subsynaptosomal fractions I and II were made up largely of empty membrane systems of a size similar to intact synaptosomes, though smaller membraneous profiles were also present. Myelin was observed as a significant contaminant of fraction I, as also noted by the originators of the isolation scheme, though fraction II contained much smaller amounts of it. Fraction III consisted mainly of mitochondria, most of which appeared damaged or distorted. High speed centrifugation (100,000 × g; 30 min) of the 'soluble' fraction of osmotically disrupted synaptosomes resulted in the production of a pellet which in the electron microscope was seen to contain, in addition to large aggregates of dense fibrillary material, considerable numbers of membrane-bound vesicles ranging in size from about 30 to 70 nm in diameter. They were similar in size to the synaptic vesicles seen in intact nerve ending preparations and are presumed to be identical. Approximately 30 ~ of the total synaptosomal protein was released by hypoosmotic treatment. Fraction I constituted about 18 ~ and fraction II about 30 ~ of synaptosomal protein. Cotman's isolation procedure was modified here to achieve both a cleaner separation of subfractions and higher yield. Fraction II in the present scheme corresponds to combined fractions 2-4 of the original method, representing synaptosomal plasma membranes (SPM). The greater yield resulting from the change is obtained at the expense of a slight increase in mitochondrial contamination. A further modification reducing the concentration of the lower sucrose layer in the final centrifugation enabled the intrasynaptosomal mitochondrial fraction (III) to compact more satis-

113 factorily than in the original method. Thus aspirating the lower interfacial layer was easier.

Accumulation in subcellular fractions The specific activities of the various fractions relative to the brain homogenate (Fig. 1) illustrate that the microsomal fraction incorporates bound fucose more rapidly than any other fraction studied. The subsequent fall in this parameter reflects the passage of newly synthesised labelled glycoprotein to sites on subcellular organelles in the brain which are not sedimented in the microsomal pellet. The points in Fig. 1 represent means of two or more individual brains and the variations from the means were below 15 ~. While the relative specific activities of the fractions remain fairly constant after the initial fall, the actual specific activity of the whole homogenate drops by approximately 10 ~ between 2 and 21 days. A pertinent feature of these data is the low activity of 'soluble' fractions relative to those containing membranes. This is of particular interest in the case of nerve terminals where the relative activity of the 'soluble' fraction, isolated by osmotic shock and containing synaptic vesicles, never attains more than 37 ~ of the activity of the intact synaptosomes from which it is derived. Conversely, the membranes derived from synaptosomes (fraction II) are relatively more enriched with tritiated fucose. Gel electrophoresis The electrophoretic profiles of all fractions examined by SDS-acrylamide gel electrophoresis were complex; over 20 protein bands could be resolved and many were complimentary to both the whole brain soluble fraction, microsomal fraction and the synaptosomal plasma membrane fraction II (see Fig. 2). The latter two fractions showed only quantitative differences in the 50-60,000 MW range and

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Fig. 1. Specific activities of subcellular fractions after intraventricular [3H]fucose injection relative to the brain homogenate. Fractions were isolated as described in the text. ©, microsomal; *, synaptosomal fraction II; A , intact synaptosomes; 0 , brain soluble fraction; + , synaptosomal soluble fraction.

114

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Fig. 2. SDS-acrylamide gels stained with Coomassie blue. Fractions were isolated and prepared for electrophoresis as described in the text. Migration of proteins is downwards, a: whole brain soluble. b : microsomal, c: whole brain mitochondria, d: synaptosomal fraction I. e: synaptosomal fraction II. f: synaptosomal fraction III. m i n o r differences in p o l y p e p t i d e s o f M W lower t h a n 25,000. C o n s i d e r a b l e differences were f o u n d between fractions I a n d II. F r a c t i o n I c o n t a i n e d three intensely stained b a n d s o f low m o l e c u l a r weight t h a t were missing (or b a r e l y visible) in gels o f f r a c t i o n II. These were the m o s t p r o m i n e n t b a n d s in gels p r e p a r e d f r o m isolated myelin samples a n d p r o b a b l y reflect the extent o f myelin c o n t a m i n a t i o n in f r a c t i o n I. By contrast, f r a c t i o n I I c o n t a i n e d a d d i t i o n a l b a n d s o f m o l e c u l a r weight 25-30,000.

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Fig. 3. Spectrodensitometric scan (550 nm) of Coomassie blue stained gel of synaptic plasma membranes (fraction II) corresponding to gel e of Fig. 2. Direction of electrophoresis is to the right. Arrows indicate locations of fucosylglycoproteins on the gels determined by scintillation counting after transverse slicing (see text).

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Fig. 4. Specific activities of individual glycoproteins of synaptosomal plasma membranes (fraction II) after intraventricular [SH]fucose injection. Each graph refers to a single glycoprotein band located on electropherograms of synaptosomal subfraction II at different time intervals after fucose injection. a: M W 123,000. b: M W 85,000. c: M W 69,000. d: M W 56,000. e: M W 48,000. f: M W 36,500, g: M W 31,000.

Quantitative differences were also apparent in the 55-60,000 MW range. Fraction III was also distinctly different from fraction II but closely resembled whole brain mitochondria. The technique of transverse slicing of gels and subsequent scintillation counting afforded a means of location of fucosyl glycoproteins on gels which proved far more sensitive than PAS-staining methods considered highly specific for carbohydrates 31. Nevertheless, the resolution is related to the width of the slices (1 mm) and it was thus not always possible to resolve individual glycoproteins in regions of high molecular weight. Separate investigations in this laboratory have established the presence of at least 8 glycoproteins containing fucose in the 'soluble' fraction of whole brain with molecular weights ranging from more than 100,000 to 25,500. In the current studies 7

116 fucosylglycoproteins were distinguished with approximate apparent molecular weights of 123,000, 85,000, 69,000, 56,000, 48,000, 36,500 and 31,000. A typical scan of synaptosomal membrane proteins is illustrated in Fig. 3 in which the locations of the fucosyl glycoproteins are indicated.

Activity of individual glycoproteins The specific activities of 4 synaptosomal glycoproteins (in counts/min/#g protein) (Fig. 4) increased to a maximum by one day and thereafter declined at different rates. The patterns of activity for such glycoproteins of the same apparent molecular weight isolated from the microsomal fraction were quite different where maximal activity was realized within 3 h. The specific activities of two further glycoproteins, which reached a peak at two days, reflected a slower incorporation in the microsomal fraction. The pattern of accumulation of label in the smallest glycoprotein (Fig. 4g) was distinctly different, increasing during the first 4 days and subsequently declining very slowly. Half-lives calculated from the slopes of the lines produced by plotting the data on a log scale were 16, 13, 22, 22, 18, 11 days for glycoproteins a-f. DISCUSSION Synaptosomes from whole brain homogenates represent the purest neuronal preparations available and thus have been the subject of intensive scrutiny over the last few years a0. However, recent criticismsg, 22 of the purity of such material raise doubts as to the validity of experiments involving isolated nerve terminals. Contamination of nerve endings with glial membrane fragments has represented a major problem. It is known that some glial cell membranes sediment with the crude mitochondrial fraction (from which terminals are obtained) and have an isopycnic banding density similar to both that of synaptic plasma membranes and intact synaptosomes on sucrose gradients 8. Such contamination is considerably reduced in a method involving the isolation of synaptosomes on Ficoll-sucrose gradients 10. More recent preparative methodslS, 25 differ essentially from the method adopted here only in the number of washes given to the crude mitochondrial pellet (a procedure which lowers the level of microsomal contamination) prior to isolation of synaptosomes on Ficollsucrose gradients. The marked difference observed in the initial rates of fucose incorporation of the synaptosomal and microsomal fractions suggests that contamination with microsomal elements is not a significant feature of the nerve ending fraction (and membranes derived from it) as isolated here, and moreover justifies the use of a method providing higher yields, albeit at the cost of some loss in purity. While newer methods may provide synaptosomal membrane preparations with very low levels of both mitochondrial and microsomal contamination, the yields are less than onequarter of those obtained with the method adopted here, a factor which limits their application. The technique of gel electrophoresis enables interesting comparisons to be made of the constituents of the various subcellular fractions. The qualitative similarity of

117 the soluble, microsomal and SPM fractions agrees with published data 18, though the considerable quantitative differences found between the two former fractions contrast with earlier evidence 18. The close parallelism in the molecular weights of the 7 glycoproteins found in these three fractions is interesting and adds support to the belief that the glycoproteins present in brain soluble fraction originate in membranes from which they may be lost during homogenisation 14. The molecular weights of 4 of the SPM glycoproteins determined in this study (123,000, 69,000, 36,500, 31,000) correspond with those found by PAS staining of gels of highly purified terminal membranes 26. The additional bands found in the present study may represent fucose containing glycoproteins with lower carbohydrate content which are therefore less readily visualised with the relatively insensitive staining technique. In a consideration of the likely mechanism by which labelled fucose accumulates in nerve terminal membrane constituents, the possibility that free fucose is directly incorporated at these neuronal extremities should not be overlooked. Terminal glycoprotein synthesis has been reported 15 and the intraneuronal mitochondrion appears to be responsible for such synthesis 6. While earlier data appear to support terminal incorporation of certain carbohydrate¢ (e.g. glucosamine) more recent evidence confirms that for the neurone fucose is almost wholly incorporated at the level of the neuronal soma 11. Moreover the activity of fraction IlI containing mitochondria is never as high as that of intact synaptosomes, demonstrating that intraneuronal terminal mitochondria do not contribute to glycoplotein synthesis. The rapid incorporation rate noted for the microsomal fraction, relative to that of intact synaptosomes and their subfractions, most probably reflects the terminal addition of fucose to newly synthesised glycoproteins in the saccules of the Golgi apparatus 5 which sediment with the microsomal pellet. It is of interest to note that the specific activity of the SPM fraction never exceeds that of the microsomes. Brain microsomes are derived from both neuronal and non-neuronal cells and it is likely from these data that the neuronal microsomal elements have a lower specific activity. The contrast between the specific activity data for individual glycoproteins of the microsomal and SPM fractions is best interpreted in terms of the transport of a small proportion of glycoproteins from the total brain microsomal pool along axons to nerve terminals, the delay of about a day before maximal activity is reached in SPM glycoproteins representing the transport time. While it is not possible to calculate the rate of transport because of the wide variation in axonal length in the rat brain, it is likely that such glycoproteins undergo 'fast transport' as reported by other workers 3. The lower incorporation rates for SPM glycoproteins of MW 48,000 and 36,500 reflect similar anomalous behaviour in those microsomal constituents. The slower incorporation of fucose in the smallest glycoprotein (Fig. 4 g) and its slow decline relative to the others is intriguing and does not so easily fit into the concept of axonal transport. It is possible that the accumulated label in this glycoprotein represents re-utilization of fucose arising from the breakdown of other synaptosomal glycoproteins. Further information on the nature of the transport process may be derived from an examination of the activity levels of the 'soluble' fraction of terminals relative to intact synaptosomes. The low level of labelling of this fraction is an indication of

118 its non-involvement relative to membrane fractions in the transport process. Electron microscopy demonstrated the presence in this fraction of synaptic vesicles in addition to large dense aggregates, presumably of protein. Cell fractionation experiments with the chicken ciliary ganglion also indicate that the involvement of synaptic vesicles with newly synthesised glycoproteins is less than that of terminal membranes 11, while high resolution autoradiography suggests the reserve. The inherent problems of resolution encountered with autoradiographic techniques cast some doubt on such evidence. The data presented here demonstrate clearly that the bulk of the newly synthesised glycoproteins are transported bound to particulate matter which is isolated along with the membrane of osmotically disrupted nerve terminals. While it is not possible to ascertain the vehicle of transport from the present data, it has recently been suggested that the smooth endoplasmic reticulum may provide a means of rapidly conveying components along axons 13. Smooth endoplasmic reticulum ofsynaptosomes would be present in the SPM preparations and thus the data is in agreement with such views of the mechanism of fast axoplasmic transport. Half-lives of individual glycoproteins have not previously been calculated for mammalian nerve terminals though several reports provide data for groups of proteins and glycoproteins in brain subcellular fractions. In a variety of non-mammalian species the half-lives of [3H]fucosylglycoproteins have been found to range between 3 and 38 days3,16,24. In the rat using [14C]glucosamine as precursor, a half-life of only 0.4 days was foundlL The fucosyl glycoproteins examined here clearly behave quite differently and have half-lives more similar to those found for protein conveyed by slow transport 29. The possibility of re-utilization of fucose suggested by the unusual characteristics of the smallest glycoprotein studied (Fig. 4g) cast some doubt on the accuracy of estimates of half-lives. It is likely that the values given are over-estimates. While the specific activities of individual synaptosomal membrane glycoproteins drop considerably over the three-week period of investigation, that of the whole brain is reduced by only about 10% between 2 and 21 days following injections of fucose, suggesting that though glycoproteins may be lost from nerve ending membranes by a variety of mechanisms, the labelled fucose is re-utilized elsewhere following glycoprotein breakdown. As yet little is known of the nature of terminal membrane glycoproteins beyond their apparent molecular weights. Comparing both the half-lives of the glycoproteins and the times taken after intraventricular injection of precursor for individual glycoproteins to attain maximal activity in terminal membranes, gives an indication of whether the glycoproteins turn over as part of a unit or not. The differences found in these parameters suggest that apart from glycoproteins of MW 69,000 and 56,000 (Fig. 4c and d) they are independent of each other. Investigations of the chemical structure of such glycoproteins have in the past been hampered by difficulties experienced in their isolation and purificationL Current work, using techniques developed in isolating soluble brain glycoproteins21, is directed towards the separation of individual glycoproteins from synaptosomal membranes so that their chemical nature may be defined.

119 ACKNOWLEDGEMENTS We gratefully a c k n o w l e d g e t h e helpful advice and e n c o u r a g e m e n t o f P r o f esso r J. B. C a v a n a g h , the guidance o f Dr. D. N. L a n d o n with the electron m i c r o s c o p y an d the expert technical assistance o f Mrs. J. R. C u n n i n g h a m and Mr. P. M. Chandler. The w o r k was s u p p o r t e d in p a r t by grants f r o m the Multiple Sclerosis Society o f G r e a t Britain and f r o m the Brain R e s e a r c h Trust.

REFERENCES 1 Barondes, S. H., Further studies of the transport of protein to nerve endings, J. Neurochem., 15 (1968) 343-350. 2 Barondes, S. H., Brain glycomacromolecules and interneuronal recognition. In F. O. Schmidtt (Ed.), The Neurosciences: 2nd Study Program, The Rockefeller Univ. Press, New York, 1970, pp. 747-760. 3 Bennett, G., Di Giamberardino, L., Koenig, H. L. and Droz, B., Axonal migration of protein and glycoprotein to nerve endings. II. Radioautographic analysis of the renewal of glycoproteins in nerve endings of chicken ciliary ganglion after intracerebral injection of [aH]fucose and [aH]glucosamine, Brain Research, 60 (1973) 129-146. 4 Bogoch, S., Brain glycoprotein 10B: further evidence of the 'sign-post' role of brain glycoproteins in cell recognition, its change in brain tumor and the presence of a 'distance factor', Advanc. exp. Med. Biol., 32 (1972) 39-52. 5 Bosmann, H. B., Hagopian, A. and Eylar, E. H., Cellular membranes: The biosynthesis of glycoprotein and glycolipid in Hela cell membranes, Arch. Biochim., 130 (1969) 573-583. 6 Bosmann, H. B. and Hemsworth, B. A., lntraneural mitochondria: incorporation of amino acids and monosaccharides into macromolecules by isolated synaptosomes and synaptosomal mitochondria, J. biol. Chem., 245 (1970) 363-371. 7 Brunngraber, E. G., Biochemistry, function and neuropathology of the glycoproteins in brain tissue, Advanc. exp. Meal. Biol., 32 (1972) 109-133. 8 Cotman, C., Herschman, H. and Taylor, D., Subcellular fractionation of cultured glial cells, J. NeurobioL, 2 (1971) 169-180. 9 Cotman, C., Mahler, H. R. and Anderson, N. G., Isolation of a membrane fraction enriched in nerve-end membranes from rat brain by zonal centrifugation, Biochim. Biophys. Acta (Amst.), 163 (1968) 272-275. 10 Cotman, C. W. and Matthews, D. A., Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization, Biochim. Biophys. Acta (Amst.), 249 (1971) 380-394. 11 Droz, B., Renewal of synaptic proteins, Brahl Research, 62 (1973) 383 394. 12 Droz, B., Koenig, H. L. and Di Giamberardino, L., Axonal migration of protein and glycoprotein to nerve endings. I. Radioautographic analysis of the renewal of protein in nerve endings of chicken ciliary ganglion after intracerebral injection of [3H]lysine, Brain Research, 60 (1973) 93-127. 13 Droz, B., Rambourg, A. and Koenig, H. L., The smooth endoplasmic reticulum: structure and role in the renewal of axonal membrane and synaptic vesicles by fast axonal transport, Brain Research, 93 (1975) 1-13. 14 Dutton, G.R. and Barondes, S.H., Glycoprotein metabolism in developing mouse brain, J. Neurochem., 17 (1970) 913 920. 15 Festoff, B. W., Appel, S. H. and Day, E. D., In vitro incorporation of 14C-glucosamine into membranes of rat brain synaptosomes, Fed. Proc., 28 (1969) 734. 16 Forman, D. S., Grafstein, B. and McEwen, B. S., Rapid axonal transport of [aH]fucosyl glycoproteins in the goldfish optic system, Brain Research, 48 (1972) 307-342. 17 Gesner, B. M. and Ginsburg, V., Effect of glycosidases on the fate of transfused lymphocytes, Proc. nat. Acad. Sci. (Wash.), 52 (1964) 750-755. 18 Gurd, J. W., Jones, L. R., Mahler, H. R. and Moore, W. J., Isolation and partial characterization of rat brain synaptic plasma membranes, J. Neurochem., 22 (1974) 281-290.

120 19 Hughes, R . C . , Glycoproteins as components of cellular membranes. Progr. Biophys. molec. BioL, 26 (1973) 189-268. 20 Langley, O. K., Sialic acid and membrane contact relationships in peripheral nerve, Exp. Cell Res,, 68 (1971) 97-105. 21 Langley, O. K., In preparation. 22 Leviton, 1. B., Mushynski, W. E. and Ramirez, G., Highly purified synaptosomal membranes from rat brain, J. biol. Chem., 247 (1972) 5376-5381. 23 Lowry, O, H., Rosebrough, N. J., Farr, A. C. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-272. 24 Marko, P. and Cu6nod, M., Contribution of the nerve cell body to renewal ofaxonal and synaptic glycoproteins in the pigeon visual system, Brain Research, 62 (1973) 419-423. 25 Morgan, I. G., Wolfe, L. S., Mandel, P. and Gombos, G., Isolation of plasma membranes from rat brain, Biochhn. Biophys. Acta (Amst.), 241 (1971) 737-751. 26 Morgan, I. G., Zanetta, J. P., Breckenridge, W. C., Vincendon, G., and Gombos, G. The chemical structures of synaptic membranes, Brain Research, 62 (1973) 405-411. 27 Neville, D. M., Molecular weight determination of proteindodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system, J. bioL Chem., 246 (1971) 6328-6334. 28 Ochs, S., The dependence of fast transport in mammalian nerve fibers on metabolism, Acta neuropath. (BerL.I, Suppl. V (1971) 86-96. 29 Von Hungen, K., Mahler, H. R. and Moore, W. J., Turnover of protein and ribonucleic acid in synaptic subcellular fractions from rat brain, J. biol. Chem., 243 (1968) 1415-1423. 30 Whittaker, V. P., The use of synaptosomes in the study of synaptic and neural membrane function. In G. D. Pappas and D. P. Purpura (Eds.), Structure and Flmction of Synapses, Raven Press, New York, 1972, pp. 87-100. 31 Zacharius, R. M., Zell, T. E., Morrison, J. H. and Woodlock, J. J., Glycoprotein staining following electrophoresis on acrylamide gels, Analyt. Biochem., 30 (1969) 148-152. 32 Zatz, M. and Barondes, S. H., Fucose incorporation into glycoproteins of mouse brain, J. Neurochem., 17 (1970) 157-163. 33 Zatz, M. and Barondes, S. H., Rapid transport of fucosyl glycoproteins to nerve endings in mouse brain, J. Neurochem., 18 (1971) 1125-1133.