Journol of NeuroihenuslrvLippincoti—Raven Publishers, Philadelphia

1996 International Society for Neurochemistry

Enolase Activity and Isoenzyme Distributionin Human Brain Regions and Tumors

Joan Joseph, *Féljx F. Cruz-Sánchez, and José Caneras

Unit of Biochemistry, Faculty of Medicine, University of Barcelona, and5Neurological Tissue Bank, Department qf

Medicine (Neurology), Hospital Clinic i Provincial, University of Barcelona, Barcelona, Spain

Abstract: The distribution of enolase (EC 4.2.1.11) activ-ity and isoenzymes in various regions of human brain atdifferent ages (from 23 weeks of gestation to 95 years)and in brain tumors has been determined. Total enolaseactivity increased in all regions with age. No significantdifferences were found in the relative proportions ofc~a-,cry-, and yy-enolase isoenzymes in the various brainregions, determined by agarose gel electrophoresis. Typecsa-enolase was the predominant isoenzyme, and cry-enolase represented a substantial proportion of the totalenolase activity. Astrocytomas, anaplastic astrocytomas,glioblastomas, and meningiomas possessed lower eno-lase activity than normal brain. Among astrocytic tumors,total enolase activity correlated with malignancy. Astrocy-tomas possessed the lowest and glioblastomas the high-est enolase activity. All tumors possessed a higher pro-portion of aa-enolase and a lower proportion of yy-eno-lase than the normal human brain. Among astrocytictumors, glioblastomas were the tumors with the highestproportion of aa-enolase and lowest proportion of yy-enolase. Key Words: Enolase activity—Enolase isoen-zymes—Human brain—Brain tumors— Development.J. Neurochem. 66, 2484—2490 (1996).

Enolase (2-phospho-D-glycerate hydro-lyase, EC4.2. 1. I I) is a glycolytic enzyme that catalyzes the in-terconversion of 2-phosphoglycerate and phosphoenol-pyruvate (for a review, see Wold, 1971). Enolasemol-ecules are dimers composed of three distinct subunitscoded by separate genes and designated a (liver), ~(muscle), and y (brain). The three homodimers aa,~8, and yy, and the two heterodimers a13 and ay,have been found in the extracts of mammalian tissues.The act isoenzyme, usually referred to as liver enolaseor nonneuronal enolase (NNE), is the most commonform. ~8- and c~8-enolaseare found predominantly inskeletal muscle and heart. ‘yy and ay enolases,fre-quently designated as neuron-specific enolases (NSE),are located mainly in nervous tissue and neuroendo-crine cells. In addition, they are present in plateletsand in some other tissues but at a lower level. The ~yhybrid has not been found, probably because the ~-

and y-enolase subunits are not expressed in the sametissues (for a review, see Taylor et al., 1983).

Mainly yy-, aa-, and ay-enolase isoenzymes havebeen detected in mammalian adult brain. The /3,8 andthe a/3 isoenzymes also havebeen found at much lowerlevels. y-Type enolase subunit has been found in neu-rons and a-type enolase subunit in glial cells. A fewneurons and glial cells seem to contain both a and ysubunits in adult mammalian brain. In the developingbrain, matrix cells possess NNE, and a switch-overfrom a- to y-enolase subunit expression occurs in neu-rons linked to their final functional differentiation (fora review, see Marangos and Schmechel, 1987).

Numerous studies of the immunocytochemical lo-calization of enolase subunits in human brain and inits tumors have been published, and NSE has beenused as a tumor marker (for reviews, see Royds etal., 1985; Gerbitz et al., 1986; Kaiser et al., 1989).However, the biochemical data on the distribution ofenolase activity and isoenzymes are incomplete andcontradictory with some results from immunohisto-chemistry. To our knowledge, only three studies(Odelstad et al., 1981; Chen and Omenn, 1984; Grosset al., 1990) have been published on enolase isoen-zymes in human brain during development, one ofwhich reports the distribution of enolase isoenzymesin various regions of the brain at early stages (up to3 months postnatal). No data exist for later ages. Fourstudies have been reported on the determination ofenolase isoenzymes in human brain tumors (Odelstadet al., 1981, 1982; Beemer et al., 1984; Van den Duelet al., 1986). The present work was designed to deter-mine the distribution of enolase activity and isoen-zymes in different regions of human brain at differentages up to senescence, as well as their changes insome brain tumors, as a first step to study the possible

Received November 21, 1995; revised manuscript received Janu-ary 30, 1996; accepted January 30, 1996.

Address correspondence and reprint requests to Dr. J. Carreras atUnitat de BioquImica, Facultat de Medicina, Universitat de Barce-lona, Av. Diagonal, 643, 08028 Barcelona, Spain.

Abbreviations used: NNE, nonneuronal enolase; NSE, neuron-specific enolase.

2484

ENOLASE ISOENZYMES IN HUMAN BRAIN 2485

alterations of the expression of enolase genes in neo-Plastic tissues.

MATERIALS AND METHODS

MaterialsEnzymes, substrates. cofactors, and biochemicals were

purchased from either Boehringer (Mannheini, Germany) orSigma (St. Louis, MO, U.S.A.). /3-Mercaptoethanol wasfrom Merck (Darmstadt, Germany) and bovine serum albu-ittin was from Calbiochem (La Jolla, CA, U.S.A.). Otherchemicals were reagent grade. Agar noble was obtained froml)ifco Laboratories (Detroit, MI, U.S.A.) and agarose gelswere from Ciba—Corning (Palo Alto, CA, U.S.A.).

Tissue samplesTissue samples were obtained from the Neurological Tis-

sue Bank, ‘Hospital ClinIc.” University of Barcelona. Sam-ples from cerebral cortex (superior frontal gyrus), nucleuseaudaius (anterior), cerebral white matter (centrum semi-ovalc), and cerebellar hemisphere were used as control. Theeases were aged 23 weeks of gestation, 2, 8, It, and 16months, and 23, 41, 48. 52, 65, 67, 74, 82, and 95 years.l,ipht were males and six females. The postmortem delaywas I —12 h. Tumor tissues were obtained from materialused (or biopsy during surgery. Tumors were supratentorialand (here were five meningiomas, five low-grade astrocyto-urns, six anaplastic astrocytomas, and seven glioblastomasaccording to the last WI-lO’s brain tumor classification(Kleihnes et al., 1993). The mean age of patients sufferingtoni tumors was 58.6 years.

‘I’issue extractionI’tssue extracts were prepared by homogenization in three

volunies (wt/vol) of cold Tris-HCI buffer (20 mM Tris, ImM EDTA, 1 mM /3-mercaptoethanol, pH 7.5) with a Poly-ton homogenizer (Luzern, Switzerland) (position 5, 20 s).

(‘ellular debris was removed by centrifugation (12,500 g,9) mm. 4°C),and the supernatants were used foi- the assayof enolase activity and isoenzymes.

Eiri.yme assaysEnolase activity was measured spectrophotometrically at

30°Caccording to Bergmeyerand GraM (1983). The assayis based on the formation of ATP linked to the disappearanceof NADI-I via pyruvate kinase and lactate dehydrogenase.Flie reaction mixture contained 50 mM Tris-HCI, 0.5 mMl1)TA, 10 mM MgCl2, 2.5 mM ADP, 0.25 mM NADH, ItiM glycerate 2-phosphate, lactate dehydrogenase (12.5 U/

iii!). pyrLivate kinase (0.8 U/nil), pH 7.4. Enzyme activitieswet-c expressed as units per gram of wet tissue and as units

icr milligram of protein (I unit = 1 Fmol of substrate con-verled per minute). Protein was determined by the method ofUradlord (1976), using bovine serum albumin as a standard.

lsoenzyme analysisEtiolase isoenzymes were separated by electrophoresis in

igarose gels (Corning, cat. no. 470104). Electrophoresiswas performed at 25°C in Tris-sodium barbital buffer, pH5.5 (Electra HR Buffer, eat. no. 5805), for 20 mm at 95 V.Intilase isoetizymes were stained using a mixture containing50 tiM Tris-HCI, 2 mMMgCI2, 20 mMKCI, 2 mM glucose,00 pM ADP, 20 mM AMP, 500 FM NADP, 1.2 mM glycer-ale 2-phosphate. pyruvate kinase (4 U/mI), hexokinase (7ti/mI). and glucose-6-phosphate dehydrogenase (3.5 U/

ml), pH 7.4. After electrophoresis was completed, the agar-ose gel plate was covered with 14 ml of staining mixture(freshly prepared) containing 1%- (wt/vol) agar gel, andincubated at 37°Cfor 20 mm, in the dark. The gels were airdried and scanned under ultraviolet light (265 nrn) using aShimadzu CS-9000 densitometer. Linearity in the quantita-tion of bands was determined by applying different aliquotscontaining increasing amounts of enolase. The method wasfound to be linear (correlation coefficient of 0.99) up to 0.5mU of enolase, with a lower detection limit of 0.08 mU ofenolase.

Statistical analysisA one-way analysis of variance with repeated measures

was employed for statistical evaluation and used to compareenolase activity and isoenzyme levels among different tumorgroups and control. When a significant F ratio was obtained(p < 0.05), the difference between means was located usingthe Tukey test. Values are reported as means ±SEM.

RESULTS AND DISCUSSION

Distribution of total enolase activity in humanbrain at different ages

Table 1 summarizes the levels of total enolase activ-ity in various regions of human brain at different ages.As shown, enolase activity increases with age. In allbrain regions investigated, the levels of enolase activityper gram of wet tissue rose progressively from 23weeks of gestation, reaching adult levels at 11—16months postnatally. When expressed as units of activ-ity per milligram of protein extracted, the enolase con-tent in all brain regions increased up to 40 years ofage. The distribution of total enolase activity in humanbrain has been found to be nonuniform. In most cases,cerebellum contained higher enolase levels than otherbrain regions, but a clear pattern of enolase distributioncannot he established.

In agreement with our results, only small differencesin total enolase activity were found among variousregions of the brain of a 21-week-old human fetus, anda developmental increase in the enolase activity of thevarious regions was observed from the 21St gestationalweek to the third month of postnatal life (Gross et al.,1990). The levels of total enolase activity found byus in human adult brain are similar to those reportedby others (Taylor et al., 1983; Pahlman et a!., 1984;Van den Dod et al., 1986; Batandier et al., 1987; Vial-lard et al., 1988).

Distribution of enolase isoenzymes in humanbrain at different ages

It has been shown by immunological techniques thata-type and y-type enolase subunits are present in adulthuman brain at similarconcentrations (Marangos et al.,1980; Kato et al., l983a; Tayloret al., 1983; Tsutsui etal., 1987). Type /3 subunit has been detected also, butat much lower levels (Kato et al., l983b; Taylor etal., 1983; Tsutsui ci al., 1987). By electrophoresis andby ion-exchange chromatography, the aa-, cry-, andyy-enolase isoenzymes have been found in extracts of

.1. Neuroc/u’nr, Vol. 66, No. 6, /996

2486 J. JOSEPH ET AL.

TABLE 1. Levels of total enolase activity in various regions of human brain at different age.s

Brain region Activity”

Age

23 w 2 iii 8 m II m 16 ni 23 y 41 y 48 y 52 y 65 y 67 y 74 y 82 y 95 y

Cortex U/gU/mg

2.80.4

(0.30.9

II0.7

IS0.7

201.0

130.6

232.6

211.3

222.1

161.4

132.7

191.2

271.1

141.1

Nucleus caudatus U/gU/mg

3.20.4

12.41.1

181.2

12.40.9

261.1

13.30.6

231.5

191.4

262.1

261.8

192.1

302.0

170.9

231.3

White matter U/gU/mg

2.40.4

12.20.8

70.9

14.30.9

160.9

10.6t).6

(81.3

201.7

141.5

IS1.4

192.1

161.3

140.8

141.2

Cerebellum (hemisphere) U/gU/mg

4.80.7

13.60.9

240.9

211.0

241.3

32.5

322.7

341.4

292.3

292.1

262.6

262.1

240.8

332.3

w, weeks of gestation; m, months (postnatal); y, years (postnatal).Levels are expressed as units of activity per gram of fresh tissue and as units per milligratii of extracted protein.

adult human brain, but the data were very incomplete(Chen and Giblett, 1976; Pearce et al., 1976; Beemeret al., 1984; Chen and Omenn, 1984; Van den Doel etal., 1986; Viallardetal., 1986; Batandieretal., 1987).

We have determined the distribution of enolase iso-enzymes in various regions of human brain at differentages by agarose gel electrophoresis. Figure 1 showssome of the electrophoretograms and Table 2 summa-rizes the results obtained. All specimens presentedthree electrophoretic bands corresponding to the yy-,ay-, and aa-enolase isoenzymes, and no significantdifferences were observed in the relative proportionsof the three isoenzymes. In all samples, aa-type eno-lase was the predominant isoenzyme, and cry-type eno-lase represented a substantial amount of the total eno-lase activity.

Previous studies (Parsons et al., 1981; Nishimura et

FIG. 1. Electrophoretograms of enolase isoenzymes in extractsof various regions of normal human brain at different ages: (A)23 weeks of gestation; (B) 11 months postnatal; (C) 23 years;(D) 52 years; (E) 74 years; and (F) 95 years. 1, cortex; 2, whitematter; 3, nucleus caudatus; 4, cerebellum (hemisphere).

al., 1985) have shown that in humans, as in the rat(for a review, see Marangos and Schmechel, 1987),during differentiation of neurons there is a transitionfrom NNE to NSE. The relative NSE activity in humanfetal brain has been shown to increase with develop-ment (Odeistad ci al., 1981; Muller ci al., 1985), andit has been concluded that the switch-over from a- toy-enolase subunits most probably takes place in thephylogenetically old regions of fetal brain before the21st gestational week, and in the phylogeneticallyyounger regions between the 21st and the 28th gesta-tional week. During this period the ratio between NNEand NSE increased continuously in the phylogeneti-cally old regions, whereas it showed a steep decreasein the phylogenetically young cortex regions (Grosset al., 1990). Our results show that the relative propor-tions of enolase isoenzymes remain essentially con-stant in the various regions of the brain during allpostnatal life, which indicates that the relative expres-sion of a- and y-enolase subunits does not changepostnatally.

The presence of ay-type enolase in human adultbrain reported here represents a discrepancy with thedata obtained from localization studies. In humans,using immunohistochemi stry, y-type enolase subunithas been found only in neurons. Type a subunit hasbeen detected in astrocytes, ependymal cells, capillaryendothelial cells, Schwann cells, and arachnoidal endo-thelial cells. Oligodendrocytes showed very weak astaining and neurons did not contain a-enolase (Roydset al., 1982; Van den Dod et al., 1986). These studiesimply that brain extracts would contain aa- and yy-enolase but not the hybrid ay isoenzyme, as no cellpopulation has been found with both a- and y-enolasesubunits. The same discrepancy between the cyto-chemical and the biochemical results was observed inthe rat (for a review, see Marangos and Schmechel,1987), where it was suggested that the ay-enolase wasan artefact of homogenization (Marangos andSchmechel, 1987). However, studies of the composi-

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ENOLASE JSOENZYMES IN HUMAN BRAIN 2487

TABLE 2. Distribution of enolase isoenzymes in various region.c ot human brain at di/jerent ages

Brain regionEnolase

isoenzyme”

Age

23 w 2 m 8 m ii m 16 m 23 y 41 y 48 y 52 y 65 y 67 y 74 y 82 y 95 y

(‘ottex an

ayyy

413425

382636

363034

442531

482824

462826

443521

403030

383131

502525

363331

412930

383230

383230

Nucleus caudatus anayyy

373528

393130

353035

432730

502624

462925

602515

433027

423127

432730

403426

413029

413227

423226

White matter anayyy

373627

392734

363034

432631

542521

472825

572617

422830

472924

572221

413128

482626

453025

522523

(‘crehellum (hemisphere) an 40 39 37 39 37 45 36 39 41 46 38 36 36 41nyyy

3327

2635

2934

2734

3330

2629

4024

3130

2930

2430

3527

3133

3331

3029

w, weeks of gestation; m, months (postnatal); y, years (postnatal).The results are expressed as percentage of the total enolase activity on electrophoresis.

lion of enolase isoenzymes in differentiating neuronalcell lines under various culture conditions supportedthe existence of the ay-enolase as such in the cells(Legault-Demare et al., 1981), and experiments withthe purified enzymes from mouse (Keller ci al., 1981),rat (Jørgensen and Centervall, 1982), and human(Shimizu et al., 1983) strongly suggested that the hy-brid cannot be artefactually produced during the ho-ulogenization procedures. In situ hybridization experi-inents demonstrated the presence of y-enolase mRNAin neurons but not in glial cells of human adult brain(Schmechel et al., 1987). However, recent in situ hy-hridization studies have shown that many rat brainneurons coexpress a- and y-enolase transcripts, givingthem the possibility to synthesize the ay hybrid (Ka-tagiri ci al., 1993; Watanabe et al., 1993; Keller etal., 1994). The discrepancy between the results frommRNA studies and studies of peptide localization inneurons could reflect the presence of untranslated a-enolase mRNA. It could also represent the rapid cellu-lar turnover of the peptide coded by the mRNA (Sil-verman, 1992). But it has been suggested that the mostlikely explanation is that, for some unknown reasons,antibodies directed against the a-enolase subunit dorecognize the aa dimer in nonneuronal cells, but notthe ay hybrid in neurons (Keller et al., 1994).Schmechel et al. (1987) suggested that could be due todifferences in the disposition of NSE after translation.Jørgensen andCentervall (1982) indicated that the im-munochemical difficulties with the demonstration ofct-type enolase subunits in neurons could be related todenaturation induced by the fixatives used.

Distribution of total enolase activity in braintumors

Table 3 summarizes the levels of total enolase activ-ity in astrocytomas, anaplastic astrocytomas, glioblas-tomas, and meningiomas. As shown, all groups of tu-

mors possess lower enolase activity than normal braintissue. Among the astrocytic tumors, the enolase activ-ity correlates with degreeof malignancy. Astrocytomasare the tumors with lowest enolase activity, anaplasticastrocytomas possess intermediate values, and glio-blastomas are the tumors with highest enolase activity.The total enolase activity of meningiomas is not sig-nificantly different from that of astrocytic tumors.

These results are in agreement with previous datashowing that the enolase activity in astrocytomas, oh-godendrogliomas, and ependymomas was lower thanin normal brain (Van den Dod et al., 1986). Thehigher enolase activity in tumors with highest malig-nancy could result from the increase in the levels ofaa-enolase, as discussed below.

Distribution of enolase isoenzymes in braintumors

The proportion of enolase isoenzymes in extractsof brain tumors has been determined by agarose gelelectrophoresis. Figure 2 shows the electrophoreto-grams of some tumors and Table 3 summarizes thedata obtained. As shown, all groups of tumors tend topossess a higher proportion of aa-enolase and a lowerproportion of yy-enolase than normal brain, althoughonly the differences between normal brain and glio-blastomas, and between normal brain and meningio-mas are statistically significant. Among the astrocytictumors, glioblastomas are the tumors with the highestproportion of aa-enolase and the lowest proportion ofyy-enolase. Meningiomas possess a higher proportionof aa-enolase and a lower proportion of yy-enolasethan glioblastomas.

These results are in agreement with previous studiesof the presence of enolase subunits and enolase isoen-zymes in brain tumors. Although an early immunohis-tochemical study detected a-enolase staining but noty-enolase staining in brain tumors (Royds ci al.,

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2488 J. JOSEPH ET AL.

TABLE 3. Total enolase activity and isoenzymes in human brain tumors

TumorCaseno.

Survivalperiod

(months)

Total enolase activity Enol ase is enzymes (‘4 total activity)

U/g of wet tissrie’ U/mg of protein” an’ ny yy’

Astrocytomas t2345

96—“

108—“

ND

1019102

ND

0.30.50.40.2ND

7829646459

1838253024

433II6

17Mean ± SEM 10.2 ± 3.5 0.3 ±0.06 58.8 ±8.1 27 ± 3.3 14.2 ±5.2

Anaplastic astrocytomas 1 20 11 0.8 45 27 2823456

2348566936

2011

88

12

0.30.30.20.20.6

5578ND3441

2716

ND3232

186

ND3427

Mean ±SEM 42 ±7.8 11.6 ± 1.8 0.4 ± (1.1 50.6 ± 7.6 26.8 N 2.9 22.6 ± 4.8

Glioblastomas I234567

91312480

11

181929

533

826

0.41.11.10.21.30.50.6

60585178545862

27222319293430

132026

31788

Mean N SEM 9.6 N 1.1 19.7 ± 3.9 t).74 ± 0.1 60.1 ± 3.3 26.3 ± 1.9 13.6 ± 3.0

Meningiornas 1 —“ 12 0.5 68 22 102345

—“

—“

—“

—“

1569

13

0.50.20.30.9

66797778

171917IS

17264

Mean ± SEM II N 1.6 0.48 N 0.1 73.6 N 2.7 18.6 N 0.9 7.8 N 2.6

Control tissue”Mean N SEM 20.6 ± 1.1 1.89 ± 0.1 45.5 N 1.4 29.2 N 0.8 25.4 ± 0.9

ND, not determined.“Control tissue: 20 specimens from 41—68 years normal brain.“After 10 years follow-up, still survives and no recurrence has been registered.The comparisons are as follows:’ Control vs. astrocytomas,p < t).05; control vs. anaplastic astrocytomas,p <0.05; control vs. glioblastomas,

not significant; control vs. meningiomas, p < 0.05. “Control vs. astrocytomas, p < 0.001; control vs. anaplastic astrocytonias, p < 0.001;control vs. glioblastomas, p < 0.001; control vs. meningiomas, p < 0.001. ‘Control vs. astrocytomas, not significant; control vs. anaplasticastrocytomas. not significant; control vs. glioblastomas, p < 0.05; control vs. meningiomas, p < 0.001; meningiomas vs. anaplastic astrocytomas,p < 0.01. ‘Control vs. astrocytomas,p <0.05; control vs. anaplastic astrocytomas, not significant; control vs. gliohlastomas.p < 0.01; controlvs. meningiomas, p < 0.001; meningiomas vs. anaplastic astrocytomas, p < 0.05.

1982), the presence of both a- and y-enolase subunitshas been shown by immunohistochemistry and by im-munoassay in several types of brain tumors: ganghio-gliomas, astrocytomas, glioblastomas, oligodendro-gliomas, ependymomas, meningiomas, neuroblasto-mas, ganglioneuroblastomas, ganghioneuromas, andmedulloblastomas (Bonnin ci al., 1984; Nakajima cial., 1984, 1985; Vinores et al., 1984; Tsuchida et al.,1985; Cras et al., 1986, 1988; Kuramitsu et al., 1986;Royds et al., 1986; Van den Dod et al., 1986; Zeltzerci al., 1986; Kamitani et al., 1988). In contrast, the /3-enolase subunit has not been found in any of the tumorsinvestigated, except for a single medulloblastoma(Royds et al., 1982; Nakajima ci al., 1984).

Using enzyme immunoassay, the y-enolase contentin astrocytomas, glioblasiomas, oligodendroghiomas,ependymomas, and meningiomas has been shown to

be lower than in normal brain (Kuramitsu ci al., 1986).Using elcctrophoretic analysis of astrocytomas withdifferent degrees of malignancy, oligodendrogliomas,and ependymomas, the proportion of aa-enolase hasbeen shown to be higher and the proportion of yy-enolase lower than in normal brain (Beemer ci al.,1984; Van den Dod et al., 1986). The relative propor-tions of enolase isoenzyrnes in neuroblastomas and inmedulloblastomas, determined by electrophoretic anal-ysis, did not differ significantly from normal brain(Beemer et al., 1984). However, the relative levels ofNSE in neuroblastomas, ganglioncuroblastomas, andganglioneuromas, calculated from the determination ofenolase isoenzymes by ion-exchange chromatography,were found to be lower than in human fetal brain(Odelstad et al., 1981, 1982). Determined by immuno-assay, act- and ay-enolase levels in neuroblastomas,

I. Neuro,/,e,n.. Vol. 66, No. 6, 1996

ENOLASE ISOENZYMES IN HUMAN BRA/N 2489

expression of y-enolase, it has been shown in the ratby immunohistochemistry that the levels of NSE inneurons depend on their metabolic activity (Silverman,1992).

Acknowledgment: This work was supported by FISSgrant 93/0573, by BIOMED-l project (PL930354), and byHospital Clinic grant 1993.

REFERENCES

FIG. 2. Electrophoretograms of enolase isoenzymes in extractsof human brain tumors. Astrocytomas are shown in A (lanes 5,6, and 7) and C (lanes 1 and 2). Anaplastic astrocytomas areshown in A (lane 4), B (lane 6), and C (lanes 3, 4, 7, and 8).Glioblastomas are shown in A (lanes 1, 2, and 3), B (lanes 7and 8), and C (lanes 5 and 6). Meningiomas are shown in B(lanes 1, 2, 3, 4, and 5).

ganglioneuroblastomas, and ganglioneuromas werehigh, similar to levels of yy-cnolase (Ishiguro et al.,1983).

The differences in the relativeproportions of enolaseisoenzymes in brain tumors compared with normalbrain tissue could be explained by the different expres-sion of enolase subunits in thevarious cell populations.As already discussed, in normal brain, y-enolase sub-unit is expressed only in neurons, whereas a-cnolasesubunit is expressed in glial and in neuronal cells. Ingangliogliomas and in ganglioncuromas, y-enolasewas localized in the ganglion cell population whereascr-enolase was found in astrocytic cells as well as inoccasional ganglion cells. In astrocytomas, a signifi-cant proportion of astrocytes stained for y- and for a-enolase (Royds et al., 1986).

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