Brain Research, 150 (1978) 117-133 © Elsevier/North-Holland Biomedical Press

117

N E U R O N A L , N O N - N E U R O N A L A N D H Y B R I D F O R M S OF ENOLASE IN BRAIN: S T R U C T U R A L , I M M U N O L O G I C A L A N D F U N C T I O N A L COMPARISONS

PAUL J. MARANGOS*, ATHANASIOS P. ZIS**, REENA L. CLARK and FREDERICK K. GOODWIN Clinical Psychobiology Branch, National Institute of Mental Health, Bethesda, Md. (U.S.A.)

(Accepted November 3rd, 1977)

SUMMARY Three forms of the glycolytic enzyme, enolase [2-phospho-D-glycerate hydrolase (E.C. No. 4.2.1.11)] have been prepared from rat whole brain extract. The most acidic enolase form is neuron specific enolase (NSE) which had previously been designated neuron specific protein (NSP). The least acidic form designated non-neuronal enolase (NNE) has been purified and compared structurally, immunologically and functionally to NSE. N N E is a dimer of 86,500 M.W. consisting of two very similar subunits. The data establish that N N E is larger than NSE which has been shown to be composed of two apparently identical 39,000 molecular weight subunits (78,000). N N E is less acidic than NSE having a pI of 5.9 compared to the value of 4.7 for NSE. Structural and immunological analysis establishes that the N N E subunit is distinct from the NSE subunit, and are therefore products of two separate genes. The structural designation of NSE is (Y7) and that o f N N E (a'a'). NSE is strictly localized in neurons indicating that the gene coding for the 7 subunit is only expressed in neuronal cells. The intermediate brain enolase form has been partially purified; structural and immunological evidence indicate that it is a hybrid molecule consisting of one N N E subunit and one NSE subunit (a'y).

INTRODUCTION A neuron specific protein (NSP) has been isolated and characterized from several species 9-1a. The neuronal localization of this protein has been established by both * Reprint requests to Paul J. Marangos, Clinical Psychobiology Branch, N1MH, 9000 Rockville Pike, Building 10, Room 4S239, Bethesda, Maryland 20014. ** A. P. Zis is an MRC Fellow (Canada).

118 radioimmunoassay TM and immunocytochemistry ts. NSP has been shown to be homulogous to the bovine 14-3-2 protein~2,1:~ originally described in the pioneering work of Moore concerning the isolation of brain specific proteins t l.t:, Recently, it has been determined that NSP (14-3-2) represents a neuronal form of the glycolytic enzyme enolase [2-phospho-o-glycerate hydrolase (E.C. No. 4.2.1. I 1)] J,tz. Due to the recent realization of a functional role for this protein,a more appropriate designation would be neuron specific enolase or NSE. Three forms ofenolase activity ca n be resolved upon fractionation of a rat brain extract on DEA E cellulose 5,r'. The most acidic isoenzyme (eluting at 0.3 M NaCI) has been demonstrated to be NSE. The least acidic form elutes before the addition of NaCI and has been designated non-neuronal enotase (NNE) since immunohistochemical techniques have shown it to be absent in neurons 22. NSE appears at a point coincident with neuronal functional maturation a,1 indicating that the neuronal enolase function is a highly differentiated one and suggesting that NSE might have different functional properties than non-neuronal enolases. An interesting question that arises is whether the neuronal forms of enolase have unique functional properties that suit them to their neuronal localization. This report describes the first isolation of rat brain NN E. The properties of this protein are described and compared to those of NSE. The partial purification and characterization of the intermediate enolase form is also described with the evidence presented indicating that it is a hybrid of NSE and NN E. Analysis of the data establishes that NSE and NNE are distinct gene products. MATERIALS AND METHODS

Tissue preparation Male Sprague-Dawley rats (120-160 g) were sacrificed by decapitation and the whole brain removed. The tissue was minced in 3 vols. of 10 m M Tris phosphate buffer pH 7.4 containing l m M MgSO4 (buffer A) at 0 °C followed by homogenization in a teflon-glass apparatus. The homogenate was centrifuged at 15,000 × g for 20 rain. The resulting supernatant was centrifuged at 100,000 × g for 1 h. This supernatant is referred to as the crude soluble fraction. All protein determinations were performed according to the method of Lowry 7.

Column chromatography DEAE cellulose (Whatman preswollen 52) was used exclusively for ion exchange chromatography. The cellulose was cycled through 0.5 M HCI and 0.5 M NaOH followed by equilibration in Buffer A prior to use. A zero to 0.5 M NaCI gradient was used to elute the columns with the total gradient volume being at least 10 x the column void volume. Gel filtration was performed using sephadex G-150 superfine (Pharmacia) equilibrated in buffer A.

Isoelectric focusing Preparative isoelectric focusing was performed in a LKB 440 ml column using

119 LKB ampholine at a final concentration of 2 ~. All columns contained 0.025 ~ flmercaptoethanol. Focusing time varied from 42-48 h with final voltages of 900-1000 V. The focusing temperature was 4 °C.

Gel electrophoresis Analysis of the undissociated enolase forms was done using the Tris-glycine gel system16. The upper and lower gel pHs were 6.7 and 8.9 respectively. The lower gel concentration was 7 ~. These gels were stained with 0.5 ~ amido black. Gels were run at 3 mA/tube at 12 °C for 2-3 h. The SDS gel electrophoresis system utilized was the Tris-glycine system6,8 using a stacking gel. Samples were dissociated in Buffer A supplemented with 1 ~ SDS and 1 ~ fl-mercaptoethanol. The samples were incubated at 70 °C for 20 min prior to electrophoresis. The lower gel concentration was 10 ~ acrylamide. Run time was 3 h and the gels were stained in a 0.25 ~ coomassie brilliant blue R 250 solution (methanol: acetic acid: water, 2.5:7.5:90). Eight molar urea gel electrophoresis was performed at pH 8.9 (lower gel). The urea gels were identical to the Tris-glycine gels with the exception that both upper and lower gels were adjusted to 8 M urea (Schwarz-Mann ultrapure). Samples were incubated in 8 M urea, 1 ~ fl-mercaptoethanol at 50 °C for 30 min prior to electrophoresis. Run time was routinely 4-4.5 hours at 3 mA/tube and 12 °C. The gels were stained with 0.5 ~ amido black.

Immunologicalprocedures Anti-NSE (rat) was raised in New Zealand white rabbits as previously described 9, as was the anti-serum to NNE. Visible precipitin lines were observed on ouchterlony plates (Cordis) 5 weeks after the intradermal injection of 500 #g of NNE. The NNE solution (Buffer A) was thoroughly mixed with 1 vol. of Freunds complete adjuvant prior to injection. The radioimmunoassay of NSP-R was performed as previously :lescribed 10. The assay range was 5 ng to 200 ng. Immunoprecipitation experiments were done using both anti-NNE and antiNSP serum. The enolase forms used in these experiments (approximately 25 units of activity/ml) were the respective DEAE cellulose column fractions. The enzyme fractions were incubated for 2 h at 22 °C with the indicated sera, after which time they were centrifuged at 25,000 × g for 30 min. The resulting supernatant fraction was assayed for enolase activity.

A.A. analysis Performic acid oxidation of each form was done prior to hydrolysis in order to obtain values for cysteine. Hydrolysis was performed in 6 N HCI at 110 °C for 24, 48 and 72 h. The values for serine and threonine were obtained by extrapolation and for the remaining residues by averaging each of the three time points. Amino acid analysis was performed on a Beckman amino acid analyzer.

Sedimentation equilibrium High speed equilibrium centrifugation was performed according to the procedure

120 o f Yphantise~L The high speed e q u i l i b r i u m - r u n s were allowed to come to equilibrium a n d p h o t o g r a p h s were t a k e n over a p e r i o d o f 20-55 h with s t a n d a r d K o d a k metallog r a p h i c plates. All plates were measured with a N i k o n M o d e l C c o m p a r a t o r .

Enzyme assay and kinetic analysis Enolase activity was assayed using the direct s p e c t r o p h o t o m e t r y assay as previously described 12. A n enzyme unit is defined as t h a t a m o u n t o f enzyme yielding I /~mole o f p r o d u c t per m i n u t e at 25 °C. A s s a y s were linear for at least 2 rain. Kinetic p a r a m e t e r s were d e t e r m i n e d by reciprocal plots o f velocity versus substrate c o n c e n t r a tion at 30 °C. In all cases at least 4 substrate c o n c e n t r a t i o n s (done in triplicate) and 5 M g z~ c o n c e n t r a t i o n s were used to generate each L i n e w e a v e r - B u r k e plot. Plots were d r a w n using the m e t h o d o f least square. RESULTS

Brain enolase form preparation N N E was isolated using a p r o c e d u r e similar to that previously described for the

50 MUSCLE 2c

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54

ELUTION VOLUME (WASH)

162

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216

245

E L U T I O N VOLUME (GRADIENT}

Fig. I. DEAE cellulose chromatography of brain, liver and muscle enolase. In each case 2-5 ml of the respective crude soluble fraction was chromatographed on a 1.5 × 50 cm column. Enolase assays were done at 22 °C. Each column was washed with 2 column volumes of buffer A after sample addition and then eluted with a 0--0.5 M NaCl_gradient.

121 preparation of NSE 9. This protocol involves obtaining the crude soluble fraction of brain tissue, ammonium sulfate fractionation, DEAE cellulose ion exchange chromatography, septtadex G-150 gel filtration and preparative isoelectric focusing. The ammonium sulfate fraction used is the material precipitated between 40 and 60 ~ salt saturation and is referred to as the P60 fraction. All preparations were performed in Buffer A. Brain tissue (2-300 g) was routinely processed yielding milligram quantities of the protein. The DEAE cellulose elution pattern of rat brain extract is illustrated in Fig. 1. The three enolase activity peaks are labeled along with the percent of the total activity. A very similar pattern is obtained when the P60 fraction is chromatographed on this column. The elution profile clearly indicates that N N E is much less acidic than NSE since it is not retarded by the DEAE cellulose. The intermediate activity peak is designated as hybrid. Comparison of the brain enolase profile with that of muscle and liver reveals that N N E behaves in a similar manner to the muscle and liver enzymes under these conditions. Each of the 3 DEAE peaks from brain were further processed in an effort to obtain the respective enolase species in a pure state. Since NSP has been previously purified only the results pertaining to NNE and the hybrid will be discussed. Fig. 2 illustrates the profiles obtained when the sephadex G-150 fraction of NNE

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Fig. 2. Isoelectric focusing of NNE and hybrid enolase. The G-150 fraction of NNE was focused in a pH 5-7 (440 ml) gradient and the hybrid G-150 enolase fraction in a pH 3-10 gradient (110 ml). In each profile x-x-x represents the enolase activity profile and o--o--othe pH gradient.

122 TABLE 1 Preparation o]'rat brain enolase enz.vme /brms

The data presented is representative of a typical preparation where 350 g of rat brain tissue is processed. Enzyme activity was measured at 25 °C. Fraction

Soluble P6o DEAE cellulose NNE Hybrid NSE G-150 NNE Hybrid NSE lsoelectric focusing NNE NSE

Specific activity (Enzyme units~ragprotein)

1.06 2.13

Total units

4725 2940

20.0 21.5 10.0

650 710 640

33.5 31.0 18.0

540 600 550

61.5 65.0

255 305

and the hybrid form are electrophoresed. A pH 5-7 gradient was utilized for NNE and a 3-10 gradient for the hybrid. NNE focuses as a single symmetrical peak and has an isoelectric point of 5.9. Focusing of the hybrid species yielded two major activity peaks with isoelectric points of 4.7 and 5.6 respectively. A minor species with a pI of 5.1 was also observed. As will be detailed below, it has been determined immunologically that the 4.7 peak is NSE, the 5.6 peak N N E and the 5.1 peak the hybrid. Since the hybrid form is apparently labile during isoelectric focusing the purification of this enzyme form has not been accomplished. Therefore, further characterization of the hybrid has been performed with either the respective DEAE or G-150 fraction. Table I summarizes the purification procedures used in the preparation of the rat brain enolases. High specific activities are obtained for each enzyme form indicating that each preparation is relatively free of contaminating proteins. The partial purification of the hybrid form is also summarized. Structural properties o f N N E

The electrophoretic behavior of native N N E is compared to that of NSE in Fig. 3. N N E behaves as predicted by the DEAE and isoelectric focusing experiments in that it has a much lower mobility indicating that it is less acidic than NSE. Examination of the N N E profile reveals a diffusely stained region immediately below the major band; this probably represents poor stacking since homogeneous profiles were observed for N N E on the subunit gels and during sedimentation equilibrium. Subunit analysis was performed electrophoretically comparing N N E to NSE, which has previously been characterized as a dimer (m.w. 78,000) consisting of two apparently identical subunits of m.w. 39,0009. Fig. 4 illustrates the profiles obtained when N N E and NSE are dissociated and electrophoresed in the presence of 8 M urea.

123

Fig. 3. Gel electrophoresis of native NNE and NSE. The gels from left to right contain 10/~g NSE, 5 #g NNE, 5 #g NSE + 5/~g NNE. All samples were prepared in the Tris-glycine running buffer supplemented with 1 ~ fl-mercaptoethanol. This gel system resolves protomer units on the basis of size and net charge since it does not alter the charge identity of proteins. As previously demonstrated the NSE subunit runs as a single band in this system 9. The N N E subunit is very different from that of NSE as evidenced by its marked mobility difference, indicating that it is less acidic than the neuronal enolase subunits; this finding is consistent with the properties of the native dimers. Examination of the profiles clearly shows that NSE does not share a c o m m o n subunit with NNE. Close examination of the N N E profile reveals that a very close doublet band is present suggesting that N N E consists of two structurally distinct subunits. For the sake of clarity in this report, the doublet will be considered as one subunit type. If the N N E subunit is different compared to liver enolase then a fourth genetic locus would exist for enolase in rat. The question of whether N N E is distinct from liver

124

Fig. 4. 8 M urea gel electrophoresis of rat brain enolase forms, Each enzyme form was dissociated and electrophoresed as described in methods. The gels from left to right contain 10 pg NNE, 5/~g NSE, 10/~g NNE t- 5 l,g NSE and 25/*g of the hybrid G-150 fraction. enolase 1 and is composed of two distinct subunits is presently under investigation. The hybrid G-150 fraction contains bands corresponding to both the NSE and N N E subunits suggesting that the hybrid enolase does contain a subunit from each form. The size of the N N E subunit was estimated by comparing its mobility on SDS gels to that of NSE (m.w. 39,000) (Fig. 5). It can be seen that the N N E subunit is larger than the NSE subunit; the difference in mobility observed corresponds to a difference in subunit molecular weight of about 5,000 daltons, indicating that the N N E subunit has a m.w. of approximately 44,000. The N N E subunit is therefore distinct from the neuronal enolase subunit in both size and charge. The electrophoretic profile of the partially purified hybrid enolase form (G-150 fraction) contains protein bands corresponding to both the NSE and N N E subunit, again indicating its hybrid nature.

125

Fig. 5. SDS gel electrophoresis of rat brain enolases. The gels from left to right contain 5/~gNNE, 5 pg NSE, 2/~g NNE + 2/~g NSE and 20/~g of the hybrid G-150 fraction. Sedimentation equilibrium analysis was performed on the purified NNE preparation with the results shown in Fig. 6. A linear plot was obtained indicating that the preparation is homogeneous. The calculated molecular weight is 86,500; this, along with the SDS gel results, indicates that N N E is a dimer consisting of two very similar, if not identical subunits. The amino acid composition of the two enolase forms was determined with the results shown in Table II. Comparison of N N E to NSE reveals that significant differences exist in the amount of several amino acids (lysine, glutamic acid, leucine and isoleucine). The ratio of acidic to basic amino acids is slightly higher in NSE than in N N E (1.70 compared to 1.52). This finding is consistent with the chromatographic and electrophoretic behavior of the respective proteins.

Immunological results Antisera were raised against purified NSE and N N E and used to immunologically characterize each brain enolase form. Fig. 7 illustrates the patterns obtained when each enzyme form is titrated with anti-NSE and anti-NNE serum. N N E and NSE areimmunologically distinct since antisera to each preparation is totally unreactive with the other.

126

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I 50.6

I 50.8

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Fig. 6. Sedimentation equilibrium of NNE. Approximately 25 t~g of NNE was centrifuged at 26,000 rev./min in buffer A supplemented with 0.05 ~ fl-mercaptoethanol.

TABLE II Amino acid analysis o f N N E and N S E One milligram of N N E and NSE was oxidized with performic acid and then split into 3 equal aliquots which were acid hydrolyzed for 24, 48 and 72 h. The values for threonine and serine were obtained by extrapolation to zero. All other values were averages of the 3 time points. Amino acid Mole (%)

Lys. His. Arg. Cys. Asp. Meth. Thr. Ser. Glu. Pro. Gly. Ala. Val. lie. Leu. Tyr. Phe.

NNE

NSE

8.72 1.24 3.95 1.38 11.38 1.77 4.30 6.53 9.85 4.07 8.80 11.04 7.99 6.06 8.39 2.04 3.93

6.85 1.58 4.69 1.28 11.20 1.57 4.00 5.88 11.22 3.73 9.60 10.56 7.85 5.00 9.72 1.97 3.27

127 NNE I00 >..

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Neuronal, non-neuronal and hybrid forms of enolase in brain: structural, immunological and functional comparisons.

Brain Research, 150 (1978) 117-133 © Elsevier/North-Holland Biomedical Press 117 N E U R O N A L , N O N - N E U R O N A L A N D H Y B R I D F O R M...
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