ht. J. Devl. Neuroscience,

07365748/84$03.00+0.00 Pergamon Press Ltd. @ 1984ISDN

Vol. 2, No. 3, pp. 287-299,1984.

Printed in Great Britain

PROTEIN SYNTHESIS IN CELLS ISOLATED FROM THE DEVELOPING RAT CEREBELLUM N. J. PATEL,* MRC Developmental

J. CoHENt and R. BALAZSS

Neurobiology Unit, Institute of Neurology, London WClN 2NS, U.K. (Accepted 26 December

1983)

Abstract-The rate of protein synthesis was estimated in structurally preserved perikaryal preparations from &day-old rat cerebellum under conditions which overcome the problems of intracellular compartmentation. The rates were lower than the in viva estimates at comparable ages, but they were of similar magnitude, and very much higher than previous estimates on isolated cells. Protein synthesis rate depended on the cell type. When expressed per cell the rank order in the preparations enriched in the indicated classes of cells was: Purkinje cells > astrocytes > granule cells in the S, G2 and M phase of the cell cycle > granule cells in Gl and GO. However, after normalizing the results for size differences between cell types, by expressing the rates in terms of unit protein or as a percentage replacement of the protein bound amino acid, astrocytes and replicating granule cells displayed greater rates than the Purkinje cells. The resolution of labelled proteins using SDS-PAGE indicated marked differences in the rate of synthesis of particular proteins. The results were consistent with the view that certain polypeptides are uniquely expressed in particular cell classes. Key words: Separated cells, Protein synthesis.

Early studies on the incorporation of labelled amino acids into brain proteins indicated relatively high rates, which at that time were unexpected in view of the fact that most of the cells in the CNS are not renewed by replication. 2o Furthermore autoradiographic investigations have shown that the labelling of nerve cell bodies is very high in comparison to other structures and cell types in the neuropil.‘*‘4*” Factors other than the rate of protein synthesis, such as differences in the labelling of the precursor pools may, however, have contributed to these results. Attempts have been made to overcome these problems using biochemical techniques and preparations enriched in neuronal and glial perikarya, thus permitting a better control over the behaviour of precursor pools. 5,6,22,32,36 Simultaneously new approaches have been developed to deal with the problem of compartmentation of the amino acid poo1s,18*23.26*34 t h at appears to exist even in relatively homogeneous populations of cultured cells.24*37 The detailed studies of Dunlop, Lajtha and their co-workers (for review see ref. 17) have shown that valid estimates of the rate of protein synthesis can be obtained either in vivo or in brain slices by flooding the precursor pools with high concentrations of a single radioactive amino acid. This ensures that the specific activity of the amino acid is constant throughout the different compartments of the cell and is comparable to that found in the plasma or the incubation medium. Nevertheless, it has not yet been unambiguously resolved whether the differences observed by autoradiography in the protein labelling of neuronal and glial cells are a consequence of real differences in the actual rates of protein synthesis within these cell types. When this question was examined previously using preparations of isolated perikarya, some of the investigations indicated that the rate of incorporation of labelled amino acids into protein was much higher in neurons than in glia,5,6,22,35,36whilst in other studies the difference between the two cell types when derived from the brain of young animals, was equivocal, whereas in preparations from adult animals it was the glial fraction which showed higher labelling. 27 It would appear that the rates in these earlier studies were much lower than those obtained in vivo or in incubated slices of young brains (Table 3). These rates, however, could only be calculated from the specific radioactivity of the precursor which was added in tracer

* Present address: Department of Molecular Endocrinology, Middlesex Hospital Medical School, Mortimer Street, London WlP 7PN. t Present address:Department of Zoology, University College, Gower Street, London WClE 6BT. $ Author to whom correspondence should be addressed. 287

N. J. Pate1 et al.

288

amounts to the medium. In addition it is also possible that the preservation of the isolated cells was impaired.28,39 To resolve these questions we have now studied the rate of protein synthesis using preparations, developed in our laboratory, of perikarya from early postnatal cerebella. The structural integrity of the various classes of separated cells and their metabolic competence have already been documented.9~‘0~40 Furthermore the rate of protein synthesis was estimated using the technique of Dunlop et al. l5 to overcome the effects of the possible compartmentation of the amino acid pools. The polypeptides from the different cell type enriched preparations were also resolved using SDSpolyacrylamide gel electrophoresis (SDS-PAGE). As the Coomassie-blue stained profiles were apparently very similar it was also examined whether cell type dependent differences are detectable in the newly synthesized proteins.

EXPERIMENTAL

PROCEDURES

Preparations enriched in cell types

A suspension of the cerebellar cells, representing over 30% of the cells in the cerebellum, was obtained by dissociating the tissue from &day-old Porton rats by mild trypsinization. This suspension was then fractionated by sedimentation under gravity through a gradient of bovine serum albumin (BSA)9 to give two granule cell enriched fractions: ‘B’ containing differentiating granule neurons and external granule cells in the Gl phase of the cell cycle, and ‘C’ containing replicating granule neurons in the late S and G2 phase. Fractions enriched in astrocytes (‘D’) and Purkinje cells (‘E’) were prepared by the same technique from cerebella of rats that had previously been treated with hydroxyurea (2 g/kg body wt).10,40 The proportion of dominant cell types in the various fractions was as follows (for review see ref. 21): 7080% granule cells, identified by electron microscopy in ‘B’ and ‘C’, 30-40% [3H]thymidine labelled or mitotic external granule cells in ‘C’, about 50% glial fibrillary acidic protein (GFA) positive cells in ‘D’, although the proportion of astrocytes was higher, as indicated by previous biochemical studies” and by immunocytochemistry using anti-vimentin serum (generous gifts from Dr. D. Pauline of the Pasteur Institute or Prof. S. Fedoroff, Saskatoon), 80% Purkinje cells, assessed by immunofluorescence with the Purkinje cell antiserum, in ‘E’. Incorporation of radioactive leucine

The cell preparations were washed twice in bicarbonate buffered Krebs Ringer solution (KRB) to remove the BSA and then resuspended in the same solution. After determining the number of cells with a Coulter Counter (Coulter Electronics) an appropriate number of cells, usually 2 x 106, were pre-incubated in a volume of 0.5 ml under an atmosphere of 95% 02/5% CO2 in a shaking water bath at 37°C. After 30 min an equal volume of KRB containing labelled leucine was added. When inhibitors of protein synthesis were used these were also added at this stage. For the quantitative estimation of protein synthesis rates the precursor was L[1-‘4C]leucine (Radiochemical Centre, Amersham) at a final concentration of 1.0 mM and a specific radioactivity (SA) of 7.6 mCi/mol. The use of the 1-14C labelled compound ensured that leucine metabolism did not yield labelled intermediates and consequently, all the protein bound radioactivity was in leucine proper. In some experiments, indicated in Results, L[4,5-3H]leucine was also used. At the stated time points 100 ~1 samples were added to 5.0 ml of trichloroacetic acid together with 200 ~1 of 0.2% (w/v) BSA. After 60 min at 4°C the pellets obtained by centrifugation were washed twice with 5.0 ml of 5% trichloroacetic acid. At the first wash the suspension was heated to 90°C for 15 min before centrifugation. Finally the precipitate was dissolved in 0.3 ml of NCS solubilizer (Amersham, Searle), neutralized with 0.2 ml of 1.OM acetic acid in toluene and the radioactivity content measured by liquid scintillation spectrometry. Determination of cell protein and protein bound leucine content

Cell preparations incubated as for the leucine incorporation, but without the radioactive leucine, were precipitated with 20% trichloroacetic acid. The pellet was washed twice with the same solution and then dissolved in 1.0 N NaOH. An aliquot was taken for determining the protein content3’

289

Protein synthesis in cells from developing rat cerebellum

and the rest of the sample was hydrolyzed in a sealed tube at 120°C with 6 N HCl. The acid was removed by rotary evaporation under vacuum and the residue was dissolved in lithium citrate buffer (pH 2.2). Norleucine solution was then added to the sample and the amino acid content was determined using the Rank Hilger Chromospec with a fluorimetric attachment and a programmed gradient elution system with lithium citrate buffers and the o-phtalaldehyde reaction.25 Amino acid concentrations were calculated from peak area estimations performed by the analogue-digital converter and printed out via the integral microcomputer (Digico Ltd). Absolute values were obtained by reference to quantitative chromatograms of mixtures of pure amino acids including norleucine. Sodium dodecyl sulphate polyacrylamide gel electrophoresis

(SDS-PAGE)

of proteins

Proteins of the different cell type enriched preparations were resolved using SDS-PAGE2 (continuous 7-20% gradient of polyacrylamide). Electrophoresis was carried out for 3.5 h at 4°C using a constant voltage of 350 V. The gel was calibrated for molecular weights using standards ranging from 14,000 to 94,000 (Pharmacia) which were run in one of the channels of the gel. The gels were fixed and stained in a 0.05% (w/v) solution of Coomassie-blue in a mixture of acetic acid, methanol and water (1: 5 : 5). The same solvent was also used for destaining the gel, and the relative mobilities of the molecular weight standards were calculated. Differences in newly synthesized proteins of the different cell types were examined after incubating two equal portions of each cell fraction in the presence of either [ l-14C]leucine (SA 59 Cilmol) or [4,5-3H]leucine (SA 234 Cilmol) as described above. In both cases the leucine concentration was adjusted to 42 p,M. After 2 h the labelling was terminated by the addition of 2.0 ml of 1.0 mM unlabelled leucine solution. The cells were then pelleted by centrifugation and redissolved in the gel sample buffer. Samples from a 3H labelled preparation of one cell type were then mixed with the i4C labelled preparation from another cell type and the converse mixture was also prepared routinely. In each case the ratio of the 3H:14C counts was app roximately 3. The samples were then analyzed by SDS-PAGE (see above). The destained gels were separated into the appropriate channels using a straight edge and scalpel. The strips were then cut into slices 1.0 mm thick using a Mickle gel slicer. Proteins from the gel slices were solubilized by incubating the slices in tightly capped scintillation vials with 700 pl of a 90% aqueous solution of NCS solubilizer for 16 h at 50°C. The vials were then allowed to cool and counted in a liquid scintillation spectrometer after the addition of 400 p.1of 1.0 M acetic acid in toluene and 10.0 ml of scintillator solution. The 3H or 14Ccontent of each slice was expressed as a percentage of the total radioactivity for the respective isotope recovered from the entire strip. RESULTS Incorporation

of labelled leucine in the total cell suspension

The incorporation of [3H]leucine into trichloroacetic acid precipitated material was linear for about 1 h and was inhibited by over 90% by 50 &ml of cycloheximide when the precursor was used in a relatively low concentration (Fig. 1). In contrast to the effect of this inhibitor of eukaryotic ribosomal protein synthesis, chloramphenicol, which blocks protein synthesis in mitochondria and bacteria,7 had relatively little influence on the incorporation of f3H]leucine. The rate of incorporation was also linear when leucine concentration was increased to 1 .O mM (Fig. 2). At this concentration, cycloheximide inhibited only 60% of the incorporation of [3H]leucine into trichloroacetic acid-precipitable material. When [1-“Clleucine was used as the precursor, however, the inhibition was as complete at 1.0 mM as it was at 16 p,M concentration of the amino acid. On the basis of these findings, which are in keeping with the observations of Dunlop et al. l5 on brain slices, the [l-14C] labelled precursor was used to measure the rate of protein synthesis at a high precursor concentration, so that the SA of the amino acid precursor in the tissue could be considered as being similar to that found in the medium. The rate of protein synthesis calculated on the basis of the SA of the amino acid in the medium, at a leucine concentration of 16 PM, was about half the estimate obtained at 1 mM. This indicated that, unless the SA of the intracellular leucine is continuously monitored when the precursor is used in tracer amounts, it is preferable to estimate the rate under ‘flooding’ conditions.

290

N. J. Pate1 et al.

0

30 Time (mln)

60

Fig. 1, Incorporation of radioactivity into the proteins of a total cell suspension incubated with 16 JLM [“Hlleucine (312 Ci/mol) in the presence of 250 &ml chloramphenicolO,50 &ml cycloheximide 0, and in the absence of any inhibitor A.

Time

(mln)

Fig. 2. The incorporation of radioactivity into the proteins of a total cerebellar cell suspension incubated in the presence of 1.0 mM [3H]leucine 0, 1.0 mM [t4C]leucine cl, 1.0 mM [3H]leucine+50 t@nl cycloheximide 0, 1 .O mM [t4C]leucine + 50 &ml cycloheximide n

Rates of protein synthesis

The results in Table 1 show that the different cell types have remarkably different rates of protein synthesis. When compared to the total cell suspension the rate per lo6 cells was about 4 times higher in the fraction containing the Purkinje cells (‘E’); about 2.4 times higher in the fraction containing the astrocytes (‘D’); and about 1.4 times higher in the fraction enriched in the external granule neurons in the S phase (‘C’); whereas fraction ‘B’ containing granule cells in Gl and GO had a relatively low rate. Table 2 gives the protein, and the protein bound leucine contents of the preparations. A comparison of these results with those in Table 1 showed that the rank order of these preparations, in terms of protein content and protein synthesis rates per cell, is similar, whilst the proportion of leucine within proteins of the different cell types is relatively constant. Protein synthesis rates were also expressed in Table 1 per unit protein and as a percentage replacement value of the protein bound leucine. These permit the comparison of the rates of the different cell types independently of their size or of the precursor used. It should be noted that on the

Protein synthesis in cells from developing rat cerebellum

160 K

39 K

21.5 K

Fig. 3. Electrophoretic patterns of the polypeptides from different cell types after separation by SDSPAGE. Proteins were extracted from each of the cell fractions and separated as described in Experimental Procedures. The samples run on the gel were: molecular weight markers (a), polypeptides from fractions E (b), D (c), and C (d) as well as polypeptides from the unseparated total cell suspension (e). The gel was stained with Coomassie-blue.

291

293

Protein synthesis in cells from developing rat cerebellum Table

1. Rates of protein

synthesis

of fractions

nmol Leu/lO’cells Total cell suspension (TCS) Purkinje cells (‘E’) Astrocytes (‘D’) Replicating granule cells (‘C’) Granule cells (‘B’)

enriched

per h*

in cerebellar

nmol Leu/mg protein

0.30 * 0.054 (7) 1.10?0.270(3) 0.70? 0.062 (3) 0.49 %0.091(5) 0.20 2 0.073 (4)

cell types per ht

5.773 6.490 8.208 8.140 4.762

%/n

t

0.701 0.747 0.896 0.884 0.531

The cell preparations here and in Tables 2 and 3 and Figs 14 refer to fractions enriched in the indicated class of cells as described in Experimental Procedures. * The values represent the mean ? S.E.M. of the number of separate determinations indicated in parentheses. t These values were calculated using the estimates here and in Table 2.

Table 2. Protein

content

of cell fractions and leucine content hydrolysates ug protein/lo6

cells

of protein

nmol Leu/mg protein

Total cell suspension (TCS) Purkinje cells (‘E’) Astrocytes (‘D’) Replicating granule cells (‘C’) Granule cells (‘B’)

51?3.8(4) 183 + 8.1 (4) 85 + 8.1(5) 60 f 4.8 (4) 42%4.0(3)

823 2 20.1(21) 869a 117.7 (8) 916t 16.7 (4) 921+ 22.5 (4) 897? 114.2 (6)

Each value represents the mean? nations indicated in parentheses.

S.E.M.

of separate

of the number

determi-

basis of these estimates the fractions enriched in astrocytes (‘D’) or in external granule neurons in S and G2 phase (‘C’) are more active than fraction ‘E’, which is enriched in the Purkinje cells that are by far the largest cells in the cerebellum. Differences

in the newly synthesized proteins of the different cell types

Proteins of the different cell type enriched preparations were resolved using SDS-PAGE. The Coomassie-blue stained polypeptide profiles of the different classes of cells were remarkably similar (Fig. 3). To examine whether, in spite of these similarities, unique features in the newly synthesized proteins are detectable we examined the labelled polypeptide profiles after incubating the various types of cells in the presence of radioactive leucine. Significant labelling of proteins over a wide range of molecular weights was detected in case of each preparation. For the comparison of the different classes of cells, a dual labelling technique appeared to offer definite advantages in reducing the experimental error. As described in Experimental Procedures aliquots of each cell type enriched preparation were incubated in the presence of [3H]leucine or [‘4C]leucine and the 3H labelled proteins of one cell type were mixed with the 14Clabelled proteins from another cell type (and vice versa) before separating the proteins by SDS-PAGE. The gel was sliced and the 3H and r4C contents in each slice were expressed as a percentage of the total “H or 14Ccontent in the strip. Proteins at a specific position on the gel, if synthesized at different rates by the two cell types, would thus be revealed by an isotope ratio which deviates from 1. To safeguard against the appearance of spurious differences, the mixed proteins of two cell types labelled by reversing the radioactive precursors were also analyzed. If the differences in the synthesis rate of polypeptides at a given molecular weight range were genuine the 3H: 14C ratio should be reversed when the isotopes were reversed. The results in Fig. 4a-c showed evident differences in the labelling of certain polypeptides in the various cell type enriched preparations compared. In the case of the fractions containing Purkinje cells (‘E’) and astrocytes (‘D’) particularly great differences emerged in the ratio of polypeptides labelled by 14C and [3H]leucine in the molecular weight range of 17,000-18,500 and 25,000-27,000 (Fig. 4a). The comparison of the fractions enriched in replicating granule cells either in the S and G2 phase of the cell cycle (‘C’) with the astrocyte and Purkinje cell fractions (Fig. 3b and 3c, respectively) showed many marked differences over a wide range of molecular weights. In particular the rate of synthesis of polypeptides in the relatively low molecular weight range was

N. J.Pate1 et al.

294

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Fig. 4. Differential protein synthesis by preparations enriched in various cell types. Polypeptides were resolved using SDS-PAGE (7-20% gradient gels) after incubating for 2 h the indicated cell fractions at 37°C in the presence of 42 pM leucine, either [1-t4C]leucine (SA 59 Cilmol) or [4,5-3H]leucine (SA 234 Ci mol) (and vice versa), and mixing the 3H labelled cells dissolved in the gel sample buffer from one preparation with the t4C-labelled cells from another fraction (see text). The gels were cut into 1 mm thick slices, the radioactivity content of which was then determined (the estimates were at least 50 times the background). The 3H or 14Ccontent of each slice was expressed as a percentage of the total radioactivity for the respective isotope recovered from the entire gel strip and the ratio of these values for the two isotopes in each slice is plotted against slice number and molecular weight, which was determined using standards run in one of the channels of the gel. The comparison of the protein labelling of fraction D and E, C and D and C and E is given in (a), (b) and (c) respectively.

higher in fraction ‘C’ than in the other preparations. It is likely that these proteins would include histones. In addition evident differences were observed between fraction ‘C’ and either of the other cell types in the molecular weight range of 18,500-23,000 and 26,500-31,000. Furthermore the labelling of relatively high molecular weight polypeptides (>80,000) was more marked in the astrocyte (‘D’) and Purkinje cell (‘E’) fractions than in the preparations enriched in replicating granule cells (‘C’).

much

DISCUSSION The present study showed that the cells isolated from 8-day-old rat cerebellum retain a high capacity for protein synthesis even after the relatively long separation procedure. The incorporation of radioactive leucine, which proceeded linearly for an hour reflected mainly ribosomal protein synthesis in the cells since it was inhibited by over 90% by cycloheximide, and hardly at all by chloramphenicol which is known to block mitochondrial and bacterial protein synthesis.’ The rates were estimated in the presence of 1.O mM labelled leucine which permitted the calculations of the incorporation rate from the specific radioactivity of the added precursor.” Table 3 shows that the observed rates were much higher than previously published estimates for isolated cells. For example, the estimates (calculated by us from published data) for the neuronal

296

N. J. Pate1 et al.

fraction from lO-day-old rat brain,35 or from adult rabbit brain12 are only about 0.1 or 1% of the values in the present work for the total cerebellar cell suspension. There are several factors which may have contributed to these differences. It has been found that in the lo-day-old rat the rate of protein synthesis in the cerebellum is about 60% higher than in the forebrain (Table 3). Furthermore, in earlier studies the precursors were used in trace amounts and since the SA of intracellular amino acids was not determined, our calculations, based on the SA in the medium, probably underestimated the rates. The discrepancy between our results and previous observations is, however, so large that it cannot be fully accounted for by these factors. For instance, in our experiments where labelled leucine was used in either a high or low concentration (Fig. 1 and Table 1) and the rates calculated from the SA of the leucine in the medium, the values differed only by a factor of two. The most likely explanation for the discrepancy must therefore be that the impaired structural preservation of the earlier preparations, especially their lack of intact plasma membrane, was detrimental to the protein synthesizing machinery of the cells. Table 3. Comparison

of estimates

of rates of brain protein synthesis preparations

obtained

in viva and in virro including

cell type enriched

Kate (A) Estimates obtained for rat brain either in viva or in vitro under conditions concentration of the labelled precursors Braincortex (PlO) Braincortex (adult) Cerebral hemispheres (PlO) Cerebral hemispheres (adult) Cerebellar slices (PI()) Cerebellum (adult) Cerebellar slices (adult) Brain cortex slices (P7) Brain cortex slices (P14) Brain cortex slices (P2 1) Isolated neurons (PI 1) C‘erebellar cell suspension (P8) Separatedcerebellar cell types (P8)

is maintained

at high (flooding)

The estimates days

rat) (10 I*.M[;‘H]lyyine; 137 Ci/mol) rat) (10 FM[ Hllysme; 137 Ci/mol) rabbit) (100 p,M[3H]leucine; 100 Ci/mol) (4 pM[‘4C]leucine; 2.51 Ci/mol)

were obtained

from the references

as indicated.

Reference

I.46 0.65 2.1 II.62 2.34 0.70 11.04 0.x2* II. 1x* 0.06* 0.01_%0.3h* 0.70 O.S-o.9

33 33 15 IS I6 I6 16 31 31 31 32 Present study Present study

when the levels

(B) Estimates obtained using tracer amounts of precursort Neuronal fraction (adult Neuropil fraction (adult Neuronal fraction (adult Synaptosomes (rat P18)

(“h/h)

The age is given in brackets

1.3x10 3 O.6x 10 i 10.4 x 10 -j I .s x 10~ 1

3s 35 22 3

where P stands for postnatal

’ These values were calculated using our average values for leucine and lysine content of protein. i.e. 898 and 759 nmoli mg protein. respectively. t These values were calculated from the published data by assuming that the specific activity of the precursor at the site of synthesis was the same as that in the medium. Protein synthesis rates in the present preparations were of the same order of magnitude as observed in vivo (Table 3). The estimate for the total cell suspension (0.7% h-‘) was, nevertheless, still lower than the values obtained for the cerebellum in vivo or in slices at a comparable age, in spite of the fact that our preparations contain perikarya where most of the protein synthesis is thought to occur. It is possible that the amputation of the processes from the perikarya, which inevitably occurs during tissue dissociation, may have lead to a loss of some cell constituents essential for maximal protein synthesis. Poduslo & McKhann3* have observed a marked increase in the low rate of protein synthesis when their preparations of neural perikarya were incubated in complete tissue culture medium. It is also possible that protein turnover in vivo is stimulated by neuronal activity, the absence of which, during the incubation of the perikarya, may have contributed to the relatively low rate observed. Further experimental work is, therefore, still needed to establish whether conditions can be found that can increase the rate of protein synthesis in preparations of perikarya to the levels observed in the organized tissue. The structural preservation of the perikarya in the preparations enriched in different cell types. was satisfactory.’ This permitted the meaningful comparison of the rates of protein synthesis be-

Protein synthesis in cells from developing rat cerebellum

297

tween neurons and glia. It is generally believed that the rate is much higher in nerve cells than in glia, but much of the supporting evidence for this has come from autoradiographic studies which, because of the problems of precursor compartmentation, are subject to severe reservations. Previous studies with isolated cells have indicated, with one exception,27 that the rate of protein synthesis in neurons is about double that in the glia, but, as indicated above, the reported values were very low. We have attempted in this study to overcome the problems both of inter-cellular and intracellular compartmentation, by using fractions enriched in particular cell types and a high concentration of labelled precursor. We have satisfied ourselves by autoradiographic studies that the incorporation of the label is not confined to a small sub-population within any of the fractions but is representative of the whole preparation (unpublished results). Under these experimental conditions, the fraction enriched in Purkinje cells, which in autoradiographic studies had shown outstandingly high labelling,’ did indeed display higher synthesis rates (per cell) than any of the other preparations (Table 1). However, when the results were normalized to take into account the fact that the Purkinje cells are much larger than any of the other perikarya studied, the results, in terms of either unit protein or percentage replacement of the protein bound leucine, showed that both the external granule cells in S phase (‘C’) and the astrocytes (‘D’) had higher rates of protein synthesis than the Purkinje cells. The latter finding was particularly surprising in view of the very low labelling of the neuropil observed in autoradiographic studies. It cannot be excluded, however, that the rate in the astrocyte preparation was unusually high as a result of some hypertrophy following hydroxyurea treatment. However, it is worth mentioning that the rate of protein synthesis reported by White & Hertz38 for cultured astrocytes from mouse cerebral hemispheres is also relatively high and of the same magnitude as observed in the present work. The granule cells of fraction B displayed a rate of incorporation of labelled leucine per lo6 cells which was only 18% of that for the Purkinje cells. This is in keeping with the findings of Altman,’ that the grain density in the internal granule layer is only about 13% of that found in the Purkinje cell layer. Although the difference was much reduced when the results were expressed in terms of percentage replacement values, it is evident that the rates in the various classes of neurons are not the same. In this context it should be mentioned that the preparation enriched in external granule cells in S and G2 phase (‘C’), exhibited a far higher (70%) rate of protein synthesis than the external granule cells in Gl phase and differentiating granule cells (‘B’) irrespective of the way the results were expressed (Table 1). Although the comparison of the different cell type enriched preparations revealed significant differences in the rate of protein synthesis, the polypeptide profiles obtained after SDS-PAGE and Coomassie-blue staining were apparently similar. Furthermore it was difficult to assess from the profiles of polypeptides labelled after incubating the different preparations with radioactive amino acids the differences in the synthesis rate of certain proteins. The double isotope labelling technique combined with the resolution of the polypeptides by SDS-PAGE provided a means to detect convincingly such differences (Fig. 4). The design of the experiments safeguarded against certain artefacts. Thus to ensure that the radioactivity incorporated into the proteins was primarily in the same amino acid, [l-14C] and [3,4-3H]leucine were used as precursors. Leucine metabolism by pathways other than incorporation into proteins would result in the loss of radioactivity predominantly either as 14C02 or as 3H20, while labelled leucine metabolites would be greatly diluted by the endogenous amino acid pools. The authenticity of the differences was also confirmed by reversing the radioactive precursor used (Fig. 4). Nevertheless the evident cell type dependent differences observed in the labelling of certain polypeptides do not necessarily indicate that unique proteins have been revealed, as they may relate to differences in the rate of labelling of the same proteins. Further limitations of the technique include the possibility that differences in intracellular compartmentation between cell types may have contributed to the observed differences in the labelling of the polypeptides, as the precursor concentration could not be maintained at a high ‘flooding’ concentration, if sufficient radioactivity were to be incorporated into the gel slices. In addition, only proteins with a relatively rapid turnover would be sufficiently labelled during the 2 h experimental period and this limits the protein species which can be compared. In one studyz9 it has been calculated that only about 10% of the brain proteins have a half life of

Protein synthesis in cells isolated from the developing rat cerebellum.

The rate of protein synthesis was estimated in structurally preserved perikaryal preparations from 8-day-old rat cerebellum under conditions which ove...
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