9
50 Biochimica et Biophysica Acta 841 (1985) 50-58 Elsevier BBA 22090 Tissue distribution, developmental profiles and effect of denervation of enolase isozymes in rat muscles Kanefusa Kato "~*, Atsuko Shimizu a, Reiji Semba b and Toyohiko Satoh ~ Departments of" Biochemistry and i, Perinatology, Institute for Dec,elopmental Research, A ichi Prefectural Col.~ 1', Kamiva, Kasugai, Aichi 480-03, and ' Department of Pt~vsiology, School of Dentistry, Aichi-Gakuin Unwersi(v, ( 'a-ku, Nag(~va 464 (Japan) Received February 12th, 1985) Key words: Enolase isozyme; Denervation; lmmunoassay; (Rat muscle) The tissue distribution of muscle-type a/8 and /8/8 enolases in rats were determined with the sandwich-type enzyme immunoassay method which utilized the purified antibodies specific to the a and to the/8 subunit of enolase, and/8-D-galactosidase from Escherichia coli as label. All the tissues examined contained detectable levels of both a/8 and /sfl enolases. The/8/8 enolase was found at high levels in the skeletal muscle tissues (tongue, esophagus, diaphragm and leg muscles) and in the cartilages (xipoid process and auricular cartilage). The a/8 enolase was distributed at a relatively high concentration in the heart and in the above-mentioned tissues. The /8/8 enolase in the leg muscles, diaphragm and tongue was present on the day of birth at a concentration higher than that of the aa and aft enolases, and its concentration further increased in a manner apparently related to the functional state of each tissue. Denervation of the leg muscles by cutting the sciatic nerve in adult rats resulted in a drastic change in the isozymes profile. The concentration of/8/8 enolase in the tibialis anterior gastrocnemius lateralis and extensor digitorum Iongus (about 800 pmoi/mg protein) decreased to about a half in a few weeks after denervation. In contrast, the concentrations of aa (2 pmol/mg) and a/8 (80 pmoi/mg) usually showed a slight increase by the treatment (aa, 7 pmol/mg; a/8, 100 pmol/mg after 2 weeks). As compared with these three muscles, the soleus had normally a low enolase level and the effect of denervation was less drastic. These results seem to suggest that the concentration of B/8 enolase is closely correlated with the functional state of the muscle tissue. Introduction Molecules of the glycolytic enzyme enolase (EC 4.2.1.11) have, in mammalian tissues, a dimeric structure composed of three distinct subunits a, fl and ~,. Five forms (aa, BB, "~7, aB and a~) of enolase have been found in rat tissues. It is known that some of these forms have a characteristic distribution in specific tissues; aft enolase in the heart, fib enolase in the skeletal muscles, and ay * To whom correspondence should be addressed. and ~'3' enolases in the nervous tissue [1,2]. It has been reported that enolase molecules in the fetal skeletal muscle are mostly of aa form, and the tiff form increases in parallel with the postnatal maturation [3]. However, precise determination of each form of enolase has not been done. The present paper describes a sensitive enzyme immunoassay system for aB and BB enolases, the tissue distribution of these enolases in adult rats, and the developmental profiles of aa, aB and Bfl enolases in the leg muscles, tongue, diaphragm and heart, together with the effect of denervation of four leg muscles (tibialis anterior, gastrocnemium 0304-4165/85/$03.30 ":~ 1985 Elsevier Science Publishers B.V. (Biomedical Division)

Tissue distribution, developmental profiles and effect of denervation of enolase isozymes in rat muscles

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50 Biochimica et Biophysica Acta 841 (1985) 50-58

Elsevier

BBA 22090

T i s s u e d i s t r i b u t i o n , d e v e l o p m e n t a l p r o f i l e s an d e f f e c t o f d e n e r v a t i o n o f e n o l a s e

i s o z y m e s in r a t m u s c l e s

K a n e f u s a K a t o "~*, A t s u k o S h i m i z u a, Re i j i S e m b a b a n d T o y o h i k o S a t o h ~

Departments of" Biochemistry and i, Perinatology, Institute for Dec, elopmental Research, A ichi Prefectural Col.~ 1', Kamiva, Kasugai, Aichi 480-03, and ' Department of Pt~vsiology, School of Dentistry, Aichi-Gakuin Unwersi(v, ( 'a-ku,

Nag(~va 464 (Japan)

Received February 12th, 1985)

Key words: Enolase isozyme; Denervation; lmmunoassay; (Rat muscle)

The tissue distribution of muscle-type a/8 and /8/8 enolases in rats were determined with the sandwich-type enzyme immunoassay method which utilized the purified antibodies specific to the a and to the/8 subunit of enolase, and/8-D-galactosidase from Escherichia coli as label. All the tissues examined contained detectable levels of both a/8 and /sfl enolases. The/8/8 enolase was found at high levels in the skeletal muscle tissues (tongue, esophagus, diaphragm and leg muscles) and in the cartilages (xipoid process and auricular cartilage). The a/8 enolase was distributed at a relatively high concentration in the heart and in the above-mentioned tissues. The /8/8 enolase in the leg muscles, diaphragm and tongue was present on the day of birth at a concentration higher than that of the aa and aft enolases, and its concentration further increased in a manner apparently related to the functional state of each tissue. Denervation of the leg muscles by cutting the sciatic nerve in adult rats resulted in a drastic change in the isozymes profile. The concentration of/8/8 enolase in the tibialis anterior gastrocnemius lateralis and extensor digitorum Iongus (about 800 pmoi /mg protein) decreased to about a half in a few weeks after denervation. In contrast, the concentrations of aa (2 pmol /mg) and a/8 (80 pmoi /mg) usually showed a slight increase by the treatment (aa, 7 pmol /mg; a/8, 100 p m o l / m g after 2 weeks). As compared with these three muscles, the soleus had normally a low enolase level and the effect of denervation was less drastic. These results seem to suggest that the concentration of B/8 enolase is closely correlated with the functional state of the muscle tissue.

Introduction

Molecules of the glycolytic enzyme enolase (EC 4.2.1.11) have, in mammalian tissues, a dimeric structure composed of three distinct subunits a, fl and ~,. Five forms (aa, BB, "~7, aB and a~) of enolase have been found in rat tissues. It is known that some of these forms have a characteristic distribution in specific tissues; aft enolase in the heart, fib enolase in the skeletal muscles, and ay

* To whom correspondence should be addressed.

and ~'3' enolases in the nervous tissue [1,2]. It has been reported that enolase molecules in the fetal skeletal muscle are mostly of aa form, and the tiff form increases in parallel with the postnatal maturation [3]. However, precise determination of each form of enolase has not been done.

The present paper describes a sensitive enzyme immunoassay system for aB and BB enolases, the tissue distribution of these enolases in adult rats, and the developmental profiles of aa, aB and Bfl enolases in the leg muscles, tongue, diaphragm and heart, together with the effect of denervation of four leg muscles (tibialis anterior, gastrocnemium

0304-4165/85/$03.30 ":~ 1985 Elsevier Science Publishers B.V. (Biomedical Division)

lateralis, extensor digitorum longus and soleus) of adult rats.

Materials and Methods

Enolase isozymes Rat aa and 77 enolases were purified to homo-

geneity from brains as described previously [4]. Rat tiff enolase was purified from leg muscles following the method of Fletcher et al. [2] with modifications as described for the purification of human tiff enolase [5]. About 100 mg purified enzyme were obtained from 100 g muscle. When examined by sodium dodecyl sulfate-acrylamide gel electrophoresis, the purified tiff enolase pre- paration showed a single band with a relative mobility corresponding to a molecular weight of 44000. The amino acid composition of the puri- fied rat tiff enolase is shown in Table I. The composition is slightly different from those of rat aa and ~,y enolases, but very similar to that of

human tiff enolase.

TABLE I

AMINO ACID COMPOSITION OF RAT tiff ENOLASE: COMPARISON WITH THE COMPOSITIONS OF RAT aa AND ~,~' and HUMAN tiff ENOLASES

Values are expressed as mol% and are the means of three determinations. Variations in the assay were within the range 90-110% that of the mean values.

Amino acid Rat enolase Human enolase

Aspartic acid 11.2 13.0 11.5 12.1 Threonine 4.2 3.5 3.8 4.0 Serine 6.8 6.2 5.5 5.3 Glutamic acid 10.3 11.9 9.9 9.1 Proline 2.7 2.5 3.4 3.4 Glycine 8.8 10.1 9.8 10.0 Alanine 11.3 12.1 11.4 11.3 Valine 6.6 5.3 7.1 7.1 Methionine 2.0 2.2 1.9 1.9 Isoleucine 5.7 3.8 5.1 6.0 Leucine 8.0 9.3 9.2 9.0 Tyrosine 2.7 2.6 2.5 2.2 Phenylalanine 4.2 4.7 4.0 4.0 Lysine 8.0 5.8 9.0 8.5 Histidine 2.7 2.9 2.0 2.3 Arginine 4.0 4.2 4.0 3.9

a Data taken from Ref. 4. b Data from Ref. 6.

51

Rat aft enolase, used as standard for the im- munoassay, was prepared from the purified rat aa and tiff enolases as described for the preparation of human hybrid enolases [6]. A mixture (0.8 ml) of aa (0.6 mg) and tiff (1.2 mg) enolases was dissociated in buffer containing 1 M KBr without Mg z+, and when the activity of the mixture was reduced to about 30%, reactivation back to 85% was performed by reassociation of the subunits in buffer containing Mg 2+ without KBr. The mixture (0.8 ml) was applied to a column (1.2 × 27 cm) of QAE-Sephadex, equilibrated with 10 mM Tris-HC! buffer (pH 8.5) containing 4 mM MgCI 2. The column was washed with the same buffer (200 ml) and then with buffer containing a linear gradient (0-50 mM) of NaC1. Three peaks of enolase activ- ity were eluted from the column. They were identi- fied as/3fl, aft and aa, respectively, by the order of elution, and also by the reactivity with antibod- ies to the a or fl subunit of enolase [6]. The location of aft enolase was detected by the specific immunoassay system (see below). Fractions con- taining aft enolase were pooled, concentrated, and stored at - 2 0 ° C in 2.5 mM sodium phosphate buffer (pH 6.8) containing 2.5 mM MgSO 4 and 50% glycerol. Under this condition, the hybrid enolase was stable for at least 3 months and showed a specific activity (about 70 uni ts /mg) similar to that of the parental homodimeric enolases. The antiserum specific either to a or fl subunit of enolase precipitated more than 90% of the enzyme activity, indicating that the prepara- tion was not seriously contaminated with aa and tiff enolases.

Antibodies to enolase isozymes Antisera specific to the a subunit of rat enolase

were raised in New Zealand white rabbits, and the specific antibody immunoglobulin G (IgG) was purified by means of immunoaffinity chromatog- raphy as described previously [7]. Antisera to the/3 subunit of rat enolase were raised by the same procedures in rabbits by serial injection of the purified fifl enolase (1 mg/rabbi t ) emulsified with Freund's complete adjuvant. Antibodies to enolase /3 subunit were purified with a column (0.9 × 5 cm) of purified tiff enolase- (5 mg) coupled Sep- harose 4B as described for the purification of antibodies to the a and ~, subunits of enolase [7].

52

About 20 mg antibody IgG were obtained by the above procedures from 300 ml pooled antisera. Upon double-immunodiffusion testing, the puri- fied antibodies produced a precipitin arc not only with rat /3/3 enolase but also with human /3/3 enolase. However, the antibodies did not form any visible precipitin lines with rat aa and 3'7 enolases (data not shown). The antibody IgG was digested with pepsin to obtain the F(ab')2 fragments for the preparation of the immunoassay system [8].

Antibody Fab' fragments labeled with fl-v-galac- tosidase

The F(ab')~ fragments of antibodies were reduced with 2-mercaptoethylamine, and resultant Fab' fragments were coupled with fl-D-galactosi- dase from Escherichia coli (Boehringer Mannheim, Mannheim) by using N,N'-o-phenylenedimalei- mide as described previously [8,9]. Amounts of the labeled antibodies were expressed as units of galactosidase activity (1 unit = 1 /~mol p roduc t / min at 30°C).

Antibody F(ab')_,-immobilized polystyrene balls The F(ab')2 fragments were immobilized non-

covalently on polystyrene balls (3.18 mm in diam- eter, Precision Plastic Ball Co., Chicago) as de- scribed previously [10], and the balls were stored in 0.01 M sodium phosphate buffer (pH 7.0) con- taining 0.1 M NaC1, 1 mM MgCI 2, 0.1% bovine serum albumin (fraction V, Armour Pharmaceuti- cal Co., Chicago) and 0.1% NaN~ (buffer A) at 4°C for at least 2 days to stabilize the immunoas- say.

lmmunoassay procedures The assay of aa, aft or fl/3 enolase was carried

out with the procedures already described in the assay of other enolase isozymes [7], but with some modifications. In brief, a single polystyrene ball with immobilized anti-a (for aa and aft assays) or anti-fl (for tiff assay) was incubated with known amounts of standard enolase or the sample in duplicate at 30°C for 5 h under continuous shak- ing in a final volume of 0.5 ml with 0.01 M sodium phosphate buffer (pH 7.0) containing 0.3 M NaCk 1 mM MgCl 2, 0.1% bovine serum albumin, 0.5% proteinase-treated gelatin [11] and 0.1% NaN s (buffer G). After washing in chilled buffer A, the

polystyrene ball was incubated at 4°C overnight in 0.2 ml buffer A containing 1 munit of anti-a (for aa assay) or anti-fl (for aft and tiff assays) labeled with galactosidase. The galactosidase activity bound on the ball was assayed with 4-methyl- umbelliferyl-fl-D-galactoside as substrate [7].

Sensitivity and cross-reactivity of the immunoassay Standard curves for the assay of aft (A) and fl/3

(B) enolases are shown in Fig. 1. Both assay sys- tems had a sufficient sensitivity to obtain a dose- response curve of the activity of bound galactosi- dase ranging between 0.1 and 100 fmol for each form of enolases. The aft assay system was specific to aft, showing no cross-reactivity to aa, tiff and 77 enolases, and was not influenced by the coexis- tence of up to 30 fmol aa and 100 fmol /3fl enolases (data not shown). The tiff assay system did not cross-react with aa and Y7 enolases, but did so with aft enolase containing fl subunit in the molecule (about 7%, when the cross-reactivity was assessed by comparing the amounts of aft and tiff enolases required to display a fluorescence inten- sity of 50 of the bound galactosidase activity in Fig. 1). Since the a/3 assay system is specific and there is no evidence for the presence of f17 enolase

'°°°f A I B I

ili 0 0.1 1 10 100 0 0.1 1 10 103

Enolase isozymes ( fmo[ )

Fig. 1. Standard curves in the assay of rat aft (A) and tiff (B) enolases, and cross-reactivities with other enolase isozymes. The indicated amounts of rat aa (O), aft (O) and tiff (O) or Vy (A) enolase were incubated with a polystyrene ball with immobilized anti-a (A) or anti-fl (B). Then the ball was in- cubated with the anti-fl labeled with galactosidase. The activity of fl-D-galactosidase bound on the ball was expressed as the fluorescence intensity of 4-methylumbelliferone (100 = 1.10 v M) produced in a 20 min reaction with 0.1 mM 4-methylumbel- liferyl-fl-D-galactoside.

in rat tissues, the amount of 1313 enolase could be calculated by subtracting the cross-reacted value of a/3 enolase in each sample. The precision of the two assay systems was tested by assaying three samples of leg muscle extract ten times in one assay. The coefficients of variation in the a/3 and 13/3 assays were less than 7%.

The aa enolase was assayed with the same procedures as those for a/3 and /3/3 enolases using reagents prepared with antibodies specific to en- olase a subunit [7]. The measurable range of aa enolase under the conditions was between 0.03 and 100 fmol. The assay did not cross-react with /3/3 and "/3' enolases, but did so by about 20% with a/3 and a3' enolases (data not shown). Since a3' enolase concentrations are low in the heart, and skeletal muscles [12], aa enolase concentrations in the extracts of these tissues were estimated by simply subtracting the cross-reacted a/3 enolase. Therefore, act values in the samples with high a/3 concentrations might be inaccurate.

Tissue samples and preparation of the extract Various tissues were sampled from male Wistar

rats of 3 months old immediately after decapita- tion. The rats of both sexes were used in the developmental studies. Male rats of 6 months old were employed in the denervation experiments. The unilateral sciatic nerve trunk was severed at the midpoint between the caudal gluteal nerve and the fibular nerve as described previously [13]. The incised skin was sutured, and the rat was kept for 2 or 4 weeks after which it was killed by decapita- tion. Four hind limb muscles, tibialis anterior, gastrocnemius lateralis, extensor digitorum longus and soleus, were dissected bilaterally at 0°C. The homonymous muscles from the contralateral un- treated hind limb were used as control. All tissue samples were stored at - 2 0 ° C for a few days before analysis.

The frozen tissue was homogenized with a glass homogenizer in 9 vol. of 0.05 M sodium phosphate buffer (pH 7.0) containing 5 mM MgSO 4. The homogenate was centrifuged at 4°C for 20 min at 20 000 × g, and the supernatant was used for the assay of enolase isozymes. Protein concentration of the extracts was estimated with Bio-Rad Protein Assay (Bio-Rad Laboratories, Richmond), using bovine serum albumin as standard.

53

Immunohistochemical staining Tissues were fixed in 10% formalin. Sections of

3 ~m thick were immunostained by the indirect peroxidase-labeled antibody method [14]. The purified antibody (anti-/3) solution (4 ~tg/ml) was used as the primary antibody, and the peroxidase- labeled goat (anti-rabbit IgG) IgG (Miles Labora- tories, Elkhart), diluted 100-fold, was employed as the second antibody [7].

Other methods Enolase activity was measured spectrophoto-

metrically at 340 nm, and polyacrylamide gel elec- trophoresis and amino acid analysis were per- formed as described previously [4]. Amounts in micrograms of the standard enolases were esti- mated with Bio-Rad Protein Assay, and amounts in fmoles were calculated based on the molecular weight of each enolase (aa, 98 000; aft, 93 000; tiff 88 000).

TABLE II

DISTRIBUTION OF aft AND fir ENOLASES IN RAT VARIOUS TISSUES

Values are the means+ S.D. of four rats.

Tissue Enolase isozymes (pmol /mg protein)

Brain 0.44 4- 0.06 Spleen 0.14 4- 0.02 Lung 0.20 4- 0.04 Liver 0.13 + 0.03 Adrenal 0.30 4- 0.07 Thymus 0.20 4- 0.07 Adipose tissue 0.45 + 0.11 Bladder 0.53 4- 0.07 Small intestine 0.23 + 0.04 Colon 0.32 + 0.06 Stomach 0.22 4- 0.02 Xiphoid process 2.82 4- 0.39 Heart 18.5 4-1.9 Tongue 23.5 4- 2.9 Esophagus 16.1 + 3.6 Diaphragm 23.2 + 6.0 Leg muscle 25.4 4-1.6 Serum 0.06 + 0.03 a

0.07 + 0.10 0.03 + 0.01 0.06 + 0.02 0.002 4- 0.002 0.47 _ 0.27 0.55 + 0.21 0.48 + 0.18 0.78 + 0.21 0.03 4- 0.02 0.45 4- 0.14 0.68 4- 0.60

10.6 + 5.5 2.20 4- 0.27

144 4-12 247 + 4 320 + 38 582 + 58

0.011 + 0.009 ~

a pmol/ml .

54

Results

Distribution of the a/3 and /3fl enolases in various tissues

In order to de termine the distr ibution of muscle- type a/3 and/3/3 enolases in the tissue, the soluble extract of various tissues of adult male rats were prepared. The tissue extract was diluted 50-50000- fo ld with buffer A, and 100-/~1 aliquots of the diluted sample were subjected in duplicate to the immunoassay . As shown in Table II, muscle- type fl-enolase was detected in all the tis- sues examined, and the serum was the lowest in the level of both a/3 and /313 enolases. The di- aphragm, tongue, esophagus and leg muscles, which in the rat are all composed of striated

muscle fibers, contained more than 100 pmol (or 10 ~g) tiff enolase per mg soluble protein. The aft enolase was found also in those muscles at a relatively high concentra t ion (about 20 p m o l / m g protein). In the heart the dominan t form was the aft type, conf i rming the previous report by Rider and Taylor [1 ], and its concentra t ion was about 20 p m o l / m g protein. The stomach, gut and bladder, which are smooth muscle organs, contained aft and /3fl enolases at less than 1 p m o l / m g protein. In the lung, spleen, liver, brain, thymus and adrenal their level was also low. However, the xiphoid process, which is totally a cart i laginous tissue, conta ined a relatively high level of/3-enolase. Simi- larly, the auricular cartilage of adult male rats (n = 3) contained a/3 and fl/3 enolases at a level of

Fig. 2. Immunohistochemical staining of fl-enolase in sections of tongue (A), esophagus (B), diaphragm (C) and xiphoid process (D), photographed under Nomarski interference microscope. Some straited muscle fibers were stained intensely or moderately, but the others were entirely pale (A, B and C). The cytoplasm of chondrocytes was stained intensely (D). Control staining of each adjacent section with anti-fl-depleted IgG fractions of antisera remained pale (not shown). Scale bar, 50/~m.

55

6.20 + 2.66 and 99.0 + 77.2 pmol/mg protein, re- spectively.

Immunohistochemical demonstration of the localiza- tion of fl-enolase

Fig. 2 shows the immunohistochemical staining of /3 antigens in the sections of the tongue (A), esophagus (B), diaphragm (C) and xiphoid process (D), as visualized under the Nomarksi interference microscope. The skeletal muscle fibers were stained with various intensities, but in each fiber the inten- sity was apparently homogeneous (A, B and C). The intensity seemed to be divided into three categories: high, moderate and low.

The chondrocytes of the xiphoid process were also stained with antibodies to/3 subunit of en- olase (D), and the staining was found to be limited to the cytoplasm.

Developmental profiles of enolase isozymes in rat leg muscles, diaphragm, tongue and heart

Changes in the concentration of aa, aft and tiff enolases in rat skeletal muscle tissues and heart were determined during 3 months after birth, and the results were expressed in pmol of each en- olase/mg soluble protein. As shown in Fig. 3A, the concentration of /3fl form increased sharply

A , B -c- -o-

600 80

400 1

200

0 0

ul 400

200

2OO 100

0 0 0 20 40 ~ 0

Fig. 3. D e v e l o p m e n t a l prof i les o f a a ( O ) , aft ( ~ ) a n d / 3 f l (O)" enolases in the rat leg muscles (A), diaphragm (B), tongue (C) and heart (D). Each point represents the mean+ S.D. of 4-10 animals.

during 1 month after birth and reached a plateau (about 600 pmol/mg protein), while the con- centration of aa form decreased progressively to a level less than 1 pmol/mg protein in the same period. The aft enolase was present with a rela- tively constant level during the observation period.

The developmental profile of enolase isozymes in the diaphragm was similar to that in the leg muscles (Fig. 3B). After birth, the concentration of /3/3 form increased gradually to reach the adult level. On the other hand, concentrations of aa and aft enolases decreased rapidly during the first month after birth, and then gradually to the adult level.

In the tongue (Fig. 3C), the amount of enzyme in /3/3 form increased sharply just after birth and reached a maximal level (more than 300 pmol/mg protein) within 10 or 20 days, and then decreased gradually to the adult level (about 150 pmol/mg protein). The an and tx/3 forms were at a relatively constant level.

In the heart the distribution pattern was unique (Fig. 3D). The dominant form in the newborn was aa (about 270 pmol/mg protein), which after birth decreased rapidly at first, but later decreased gradually. However, its concentration was always maintained above that of aft. The fl/3 form showed an increase after birth, but always remained at a low level ( < 3 pmol/mg protein).

Effect of denervation on the three enolase isozymes in leg muscles

Changes in the concentration of aa, aft and tiff enolases in the four hind timb muscles were de- termined in adult male rats which were subjected to unilateral complete severance of the sciatic nerve. The results are summarized in Table III.

The concentration of/3fl enolase in the control samples obtained from the contralateral untreated hind limb was about 5-fold higher in the tibialis anterior, gastrocnemius lateralis and extensor dig- itorum longus than in the soleus. However, the concentrations of aa and a/3 enolases in these four muscle tissues were similar.

The denervation brought about a drastic change in 1313 enolase concentrations in these muscles. In the three muscles other than soleus the/3/3 enolase concentration decreased by about 40 and 60%, in 2 and 4 weeks after the teatment, respectively. In

56

TABLE III

EFFECT OF DENERVATION ON THE C O N C E N T R A T I O N OF ENOLASE ISOZYMES IN F O U R LEG MUSCLES

Values are the means ± S.D.

Muscles Weeks of No. of denervation samples

Enolase isozymes (pmol / mg protein)

Tibialis anterior

Gastrocnemius lateralis

Extensor digitorum longus

Soleus

0 12 2.4_+1.4 71.4_+ 8.4 782 _+88 2 6 7.0_+1.9 97.4+ 9.0 486 _+62 4 6 8.7_+3.9 71.4_+10.9 318 _+33 0 12 2.8_+1.3 49.7+ 6.9 860 _+92 2 6 8.0_+1.7 82.0_+ 7.5 510 _+26 4 6 8.5_+1.9 74.8± 4.4 383 +36 0 12 2.6-+2.2 72.2±16.1 856 +90 2 6 6.2-+2.1 104 ±21 431 -+33 4 6 6.3_+2.4 85.3±23.9 293 +43 0 12 6.2_+3.1 74.7-+15.4 142 -+18 2 6 17.4_+2.6 105 _+18 107 -+11 4 6 19.8 -+ 3.1 83.9 _+ 14.1 83.6 _+ 22.8

contrast, the aa and a/3 levels in these three muscles appeared to be slightly increased when expressed relative to the total soluble protein con- tent. The decrease in the /3/3 enolase and the increase in the a s and a/3 enolases were also observed in the soleus muscle. However, the reduc- tion in/3/3 enolase was less striking in this muscle; 25 and 40% in 2 and 4 weeks, respectively.

Extensor digitorum longus, which was de- nervated for 17 days, was subjected to immuno- histochemical staining of/3 antigens in order to be compared with the contralateral innervated con- trol (Fig. 4). When visualized under the Nomarski interference microscope, the denervated muscle fibers were very much reduced in their diameter, and the number of intensely stained fibers ap- peared to be much smaller than that of weakly stained or pale fibers. In contrast, in the in- nervated normal muscle almost all fibers appeared dark with scattered, less intensely stained fibers. Similar results were also obtained in soleus.

Fig. 4. Immunohistochemical staining of fl-enolase in sections of the denervated (A) and control (B) extensor digitorum longus muscles, photographed under Nomarski interference microscope. Muscle samples were obtained 17 days after sever- ing the unilateral sciatic nerve. Control samples were taken

from the contralateral untreated hind limb. The ratio of the intensely stained fibers to the weakly stained or pale fibers is apparently reversed in the denervated muscle. Scale bar, 100 p.m.

Discussion

Determination of aft and fir enolases in vari- ous tissues of the rat has revealed that both forms of enolase are present in detectable amount in all the tissues examined, but they are highly con- centrated in the skeletal muscles and heart, and also in the cartilaginous tissues, such as xiphoid process and auricular cartilage. The skeletal muscles and xiphoid cartilage originate from the mesoderm, while the auricular cartilage is consid- ered to be of neuroectodermal origin. Such a dis- tribution over cytogenically distant cell groups is also seen in the T-enolases (aT and TT); they are found mainly in neurons and neuroendocrine cells of neuroectodermal origin, but also at a relatively high concentration in the blood platelets and lymphocytes of mesodermal origin [12,15,16]. It is of interest how the gene expression of enolase isozymes is controlled in the differentiated cells.

The developmental profiles of aa, aft and fir enolases in the rat muscle tissues described in this paper are largely consistent with those obtained in the mouse [3]. Until day 15 prenatal, aa is the dominant form of enolase isozymes in the muscle tissues of the mouse embryo. Thereafter the con- centrations of aft and fir enolases begin to in- crease gradually. In the newborn rat, the level of fir enolase was highest in the tongue, and it fur- ther increased to reach a maximal level at 2-3 weeks postanal, the period where milk sucking would be most active. After this period the fir concentration gradually decreased with the begin- ning of the weaning period. These results seem to suggest that the concentration of fir enolase may have an important relation with the functional state of the muscle tissue.

Concentrations of fir enolase in the rat ex- tensor digitorum were much higher than those in the soleus. It is reported that the skeletal muscle fibers can be divided into two types on the basis of the difference in myosin ATPase activity [17]. The type I fibers are highly oxidative and lowly glyco- lytic, while the type II fibers are lowly oxidative and highly glycolytic [18]. Extensor digitorum longus and soleus of the rat are typical fast- and slow-twitch muscle, respectively [19]. Immunohis- tochemical study with antibodies to the fast and

57

slow myosins has shown that 97% of the muscle fibers in the rat extensor digitorum longus are fast-twitch and glycolytic and 3% are slow-twitch and oxidative, while in the rat soleus almost all fibers are slow-twitch and oxidative [20]. The im- munohistochemical staining of sections with the anti-fl has demonstrated a differential distribution of fl antigens among muscle fibers as shown in Fig. 2. Taking into consideration all of these find- ings and a recent report [21] that in human skeletal muscles a high immunochemical reaction to fl-en- olase is found in type II fibers, it may be con- cluded that the main source of fir enolase in skeletal muscles is the type II fibers.

The expression of glycolytic enzymes in differ- ent muscle fibers is considered to be under neural control, because experimental cross-innervation of fast-twitch and slow-twitch muscles can reverse the twitch properties of these muscles [22,23]. After denervation of the rat leg muscles the fir enolase decreased drastically, and the reduction was more striking in extensor digitorum longus, gastro- cnemius lateralis and tibialis anterior which con- tain a great proportion of type II muscle fibers. These results are in line with the observation [24] that within 2 weeks after denervation of the chicken breast fast-twitch muscles the concentrations of several glycolytic enzymes became only 50% of normal. In soleus the decrease of the fir enolase concentration was relatively small. This might be due to later development of post-denervation atrophy in the type I fibers as compared to the type II fibers [25]. However, lower fir level in the normal soleus might also have any significance.

The concentrations of aa and aft in the leg muscles usually showed a slight increase after de- nervation. It has been suggested that the isozyme patterns in the extant muscles of the patient suffer- ing from neuromuscular disorders might be pro- duced by reversion of gene expression to the fetal pattern [26]. Such an interpretation might be ap- plicable also to the denervated muscles which are liberated from the regulation by the central nervous system, the circumstance of the fetal muscles with poor or practically no central control.

It remains to be determined which extrinsic factor(s) is the major determinant of the fib en- olase level.

58

Acknowledgement

This work was supported in part by a Grant-in Aid for Cancer Research (58-2) from the Ministry of Health and Welfare of Japan, and by a research grant from the JAMW Ogyaa Donation Founda- tion.

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