Neural Regulation of Cholinesterase in Newt Skeletal Muscle I DISTRIBUTION AND CHARACTERIZATION OF THE ENZYME SPECIES

MICHEL P. RATHBONE,' JOHN D. VICKERS, ' MAILVAGANAM GANAGARAJAH, JUDY A. BROWN AND DAVID M. LOGAN 'Department of Neurosciences, McMaster University Medical Centre, 1200 Main Street West, Hamilton, Ontario, Canada L8S 459 and 'Department of Biology, York University, 4700 Keele Street, Downsview, Ontario, Canada M3J l P 3

ABSTRACT The molecular forms of acetylcholinesterase (AChE) in triceps muscles of newts (Notophthalmus viridescensi were identified and their dis- tribution in the muscle determined. Six forms of AChE were separated from crude muscle homogenates using velocity sedimentation on sucrose gradients. Their sedimentation coefficients were 16.5S, 1226, 9.8S, 7S, 5.5S, and 3.7s. They were identified as acetylcholinesterases by their ability to hydrolyze acetyl-/3-methylcholine, but not butyrylthiocholine. The 7 s form, however, has not been as thoroughly characterized as i t can only be consistently identified in sucrose gradients of Tris-Triton X-100-NaC1 extracts of muscle previously ex- tracted with Tris-Triton X-100. The Tris-Triton X-100 extraction removes the 5.55 and 9.8s AChE forms which mask the 7 s form in sucrose gradients of unfractionated material. The region of muscle containing the motor endplates was enriched in the 16.5s form, whereas the musculotendinous junction con- tained a high proportion of the 12.8s form. Contamination by AChE present in blood and nerve, did not account for the distribution of the 12.8s species within the muscle. The 3.7s and 5.5s forms were soluble in Tris buffer and are proba- bly located in the cytoplasm. The 9.8s forms required 1 M NaCl for solubiliza- tion, indicating that i t is associated with an osmotic shock-resistant intracellu- lar compartment, possibly the sarcotubular system. Triton X-100 was required to solubilize the 16.5S, 12.8s and 7 s forms indicating that they may be mem- brane bound. Crude muscle homogenates and each of the forms of AChE iso- lated on sucrose gradients yielded nine bands of AChE activity when subjected to polyacrylamide gel electrophoresis (PAGE). The bands of AChE were ap- parently monomers and multimers from which the larger forms of AChE were assembled.

Acetylcholinesterase (E.C. 3.1.1.7) (AChE) hydrolyzes acetylcholine (AChl a t vertebrate neuromuscular junctions, thereby terminat- ing its action (Eccles e t al., '42; Fatt and Katz, '51; Thesleff and Katz, '57), and liberating choline that is used for resynthesis of ACh in presynaptic nerve terminals (Potter, '70). Al- though AChE is concentrated a t the neu- romuscular junction (Couteaux, '55; Salpeter, '67) AChE activity is also found in regions of muscle distant from the motor endplates (Guth et al., '64) such as the myotendinous junctions (Couteaux, '63; Lubinska and Zelena, '67). The physiological role of AChE in these latter locations is unknown.

J . EXP. ZOOL. (1979) 210: 435-450

Three major molecular forms of AChE are found in rat skeletal muscles (Hall, '73; Vigny et al., '76). The form with the greatest sedi- mentation coefficient, 16S, is specifically as- sociated with the motor endplate (Hall, '73; Reiger and Vigny, '76). The physiological sig- nificance of the two more slowly sedimenting forms is unknown. Moreover, i t is not known whether the forms undergo metabolic inter- conversion under physiological conditions or whether they are multimeric structures hav- ing common subunits. Although a structural

JTo whom correspondence should be addressed. Post-Doctoral Fellow of the Muscular Dyetrophy Association of

Canada.

435

436 RATHBONE, VICKERS, GANAGARAJAH, BROWN AND LOGAN

Abbreviations

ACh, Acetylcholine AcThCh, Acetylthiocholine MeCh, Acetyl-P-methylcholine BuThCh, Butyrylthiocholine AChI, Acetylcholine iodide Che, Acetylcholine iodide

model has been proposed tha t interrelates the various molecular forms of AChE in the elec- tric organs of electric eels (Cartaud et al., '751, i t does not appear to be directly applicable to AChE from rat muscles (Reiger and Vigny, '76).

The activity of cholinesterase (ChE) in mus- cles is regulated by the "trophic" influence of the motor nerves and is reduced by as much as 60% after denervation (Guth et al., '64). The synthesis of ChE is more rapid in innervated than in denervated muscles (Filogamo and Gabella, '66; Rose and Glow, '67) and reinner- vation of denervated muscles increases the rate of ChE synthesis in the muscles (Guth et al., '66). The AChE activities a t both the musculotendinous junctions (Beckett and Bourne, '58; Mumenthaler and Engel, '61; Lubinska and Zelena, '67) and at the motor endplates (Guth et al., '64; Lentz, '71, '72, '74) of a variety of vertebrate species are under neural regulation. Moreover, after denerva- tion of rat muscles there is a preferential loss of the 16s form of AChE which is associated with the motor endplate (Hall, '73; Vigny e t al., '76), indicating that the activity of the various forms may be regulated independent- ly. The details of this regulation require fur- ther elucidation.

We are investigating the nature and mecha- nism of the neurotrophic regulation of AChE in muscles of newts fNotophthaZmus uirides- censi. Since the properties of newt muscle ChE have not been reported previously, i t was nec- essary first to define some of the biochemical properties of cholinesterase from newt mus- cles. In this paper we are reporting the proper- ties and distribution within newt muscles of six forms of AChE.

MATERIALS AND METHODS

Unless otherwise stated, all reagents and chemicals used in these investigations were of reagent grade and were obtained either from Fisher Chemical Co., Canlab, or British Drug Houses (Toronto, Ontario).

Source and maintenance of newts Newts (Notophthalmus uiridescens) were

obtained from M. Tolley, Nashville, Tennes- see. They were kept in aquaria a t 22°C and fed fresh lean ground meat at weekly intervals.

Preparation of tissue homogenates Newts were anaesthetized in 1: 1,000 (w/v)

solution of MS 222 (Sandoz, Canada, Ltd., Dor- Val, Quebec) in tap water. All operative pro- cedures were done with the aid of a dissecting microscope.

The triceps muscles were dissected from each forelimb and the nerve cut as i t entered the muscle. The muscles from 1 to 2 newts (4-8 mg wet weight) were weighed rapidly on a Cahn electrobalance, Model G 1501 (Cahn Division, Ventron Instrument Corp., Para- mount, California) and homogenized in 200 p1 of either Tris-Triton-NaC1 buffer (1 M NaCl, 0.5% (v/v) Triton X-100, 50 mM Tris-HC1, 0.2 mM EDTA, pH 7.3) or Tris buffer solution (50 mM Tris-HC1,0.2 mM EDTA, pH 7.3) at 0°C in a glass homogenizer (Micrometric Instru- ments, Inc., Cleveland, Ohio). Homogenates used in the polyacrylamide gel electrophoresis (PAGE) study were frozen and thawed 3 times. This improved the resolution of the spe- cies on the gels without affecting the profiles obtained on sucrose gradient fractionation.

Peripheral nerves were dissected from both forelimbs of newts from the level of the distal humerus to the level of the intervertebral foramen. The nerves were carefully stripped of adherent adventitia, pooled (2 newts yielded 5-8 mg wet weight of tissue) and homogenized in 200 p1 of Tris-Triton-NaC1 buffer in a glass homogenizer.

Blood (100 ~ 1 ) obtained by cardiac punc- ture, was drawn into a heparinized (10,000 unitdml, M.T.C. Pharmaceuticals, Hamilton, Ontario) 1-ml syringe, transferred to a micro- centrifuge tube, and centrifuged to separate the plasma and RBCs (red blood cells). The RBC pellet was initially washed with 0.9 mg/ ml of NaC1, adjusted to 224 milliosmoles, re- suspended and centrifuged again. The result- ing pellet was homogenized in 300 pl of Tris- Triton-NaC1 buffer.

Newts were exsanguinated and perfused by removing the heart and catheterizing the aor- ta with a fine polyethylene cannula attached to a syringe. The newts were then perfused over a 5-minute period with 1 ml of solution containing 1 mg/ml of bovine albumin (SchwartdMann, Orangeburg, New York) in amphibian Ringer's and both triceps muscles were removed.

CHOLINESTERASE IN NEWT MUSCLE 437

Fractionation of AChE by velocity sedimentation i n sucrose gradients

Tissue homogenates prepared in Tris- Triton-NaC1 buffer were centrifuged a t 3,000 x g for 10 minutes. Portions of the supernates (0.1 ml) were layered onto 3.8 ml linear su- crose gradients (5-20X w/v) prepared in Tris- Triton-NaC1 buffer. Velocity sedimentation was done by a modification (Hall, '73) of the method of Martin and Ames ('61). Sedimenta- tion coefficients were determined as described by Hall ('73).

The sedimentation coefficients of the various molecular forms of AChE were deter- mined by comparison with those of P-galac- tosidase (16s), catalase (11.3s) and bacterial alkaline phosphatase (6.13 as sedimentation markers. These enzymes were added to muscle homogenate prior to velocity sedimentation in a sucrose density gradient. P-Galactosidase activity was measured by the method of Lederberg ('50), catalase activity by the meth- od of Beers and Sizer ('521, and alkaline phos- phatase as described in the Sigma Technical Bulletin No. 104, using glycine buffer.

The gradients were centrifuged a t 4°C in an IEC 488 rotor (2,912 adaptors) in an IEC B-60 ultracentrifuge. Centrifugation was carried out so that the product of gmaxx hours was 2.45 X lofi.

Fractionation ofAChE by polyacrylamide gel electrophoresis (PAGE)

Chemicals for PAGE were obtained from Eastman Chemical Co., Rochester, New York. Electrophoresis gels consisted of an 8-cm layer of 6% acrylamide overlaid by a 1-cm layer of 4.5% acrylamide. They were cast in 0.5 x 10 cm tubes. The casting mixtures contained either 6% acrylamide (w/v) of which 2.6% (wlw) was bisacrylamide or 4.5% (wlv) acrylamide of which 2.6% (w/w) was bisacrylamide and 0.375 M Tris-sulphate buffer pH 9.0, 0.045% (v/v) N, N, N', "-tetra- methylethylenediamine and 0.0525% (w/v) ammonium persulphate. Before electrophore- sis, samples were mixed with 0.25 volumes of a solution that contained 50% (w/v) sucrose and 0.075 M Tris-sulphate buffer, pH 9.0 (at 7°C) and 0.004% (w/v) bromophenol blue. The tank electrolyte was 0.065 M Tris-borate buffer pH 9.3. Samples were layered onto the top of the gels under the electrolyte buffer and elec- trophoresed a t 2.5 mA/tube for approximately 2 hours after which the tracking dye was

within 1 cm of the bottom of the gel. Gels were removed from the tubes and soaked for 1.25 hours in 0.5 M boric acid (pH 4.1) to lower the pH of the gels minimizing alteration of the in- cubation mixture pH when they were assayed for AChE activity. Gels were stored a t 4°C until they were sliced into 1.6-mm pieces with a wire gel slicer.

Esterase assays Radiometric assays of AChE in tissue ho-

mogenates and sucrose gradient fractions were done by a modification of the method of Potter ('67). Portions of the solution to be as- sayed for AChE activity were made up to 80 ,LLI by the addition of deionized water. Twenty microliters of concentrated buffer-substrate solution were then added so that the final reaction mixture contained 0.1 M sodium phosphate, and either 2 mM lacetyl-3Hl AChI (Specif ic r a d i o a c t i v i t y 1 mCi /mmole) (NEN Canada, Ltd., Lachine, Quebec) or 2 mM [acetyl-l-I4C1 acetyl-P-methylcholine (Specific radioactivity 0.1 mCi/mmole) (NEN Canada, Ltd.), pH 8.0. Incubations were done in 5-ml Mini-vials (Canatech Inc., Toronto, Ontario) a t 30°C for periods from 5 to 180 minutes depending on the activity of the en- zyme. In no case was more than 25% of the substrate converted. Under these conditions the rate of conversion was directly propor- tional to the enzyme activity present. The spontaneous hydrolysis of ACh under the reac- tion conditions was determined from incuba- tions in which the enzyme was replaced by buffer. The reactions were stopped by cooling to 0°C and adding 100 p1 of 1 N monochlo- roacetic acid, pH 3.0. Four milliliters of to1uene:isoamyl alcohol (9: 1 v/v) that con- tained 2 g of 2, 5-diphenyloxazole (PPO) and 0.1 g of p-bis-2-(5-phenyloxazoyI)-benzene (POPOP) per litre were added to each Mini- vial. The acetate was extracted into the organic phase by vortexing the vials for 5 sec- onds each. The vials were placed in Mini-vial adaptors (Canatech Inc., Toronto, Ontario) and the radioactivity determined by liquid scintillation counting. To determine the effi- ciency of the extraction and the radioactivity determinations, control reaction mixtures were hydrolyzed with 2.5 units of purified electric eel AChE (Sigma Chemical Co., St. Louis, Missouri) for 1 hour a t 30°C which was sufficient to convert the ACh quantitatively to acetate and choline. Activity is expressed as the mean k standard error of the mean.

438 RATHBONE, VICKERS, GANAGARAJAH, BROWN AND LOGAN

Colorimetric determinations of AChE and butyrylcholinesterase (BuChE) activity were done on muscle homogenates by the method of Ellman et al. ('61) using either acetylthiocho- line (AcThCh) (Sigma Chemical Co.) or butyrylthiocholine (BuThCh) (Sigma Chemi- cal Co.) as substrates. In some experiments physostigmine (British Drug House, Toronto, Ontario) was added to the muscle homoge- nates.

The AChE and BuChE activities of the PAGE gels were determined by a modification of this technique. Slices of gels 1.6 mm thick were each placed into individual 10 x 75 mm test tubes that contained 1.5 ml of 0.1 M sodium phosphate buffer pH 8.0, 10 p l of 0.075 M AcThCh and 50 pl of 5, 5-dithiobis-2- nitrobenzoic acid DTNB (Sigma Chemical Co.) solution (39.6 mg DTNB and 15 mg NaHC03 in 10 ml of 0.1 M sodium phosphate buffer pH 7.0) and the mixture incubated for 40 minutes a t 30°C. The reaction was terminated by the addition of 100 pl of 1 N acetic acid and by cooling to 4°C. The tubes were stored a t 4°C for 16 hours to allow any remaining chro- mophore to diffuse out of the gel slices. The optical density a t 412 nm of the solution in each tube was then determined. The optical density of a blank consisting of reaction mix- ture and a slice of gel, from the lower end of the sliced gel that contained no cholinesterase activity, was subtracted from each experimen- tal value. Pseudocholinesterase activity was determined by substi tuting BuThCh for AcThCh in the above assay a t the same con- centration.

Distribution of esterase in gels was deter- mined by incubating whole gels in 3 ml of a solution of 0.1% (w/v) Fast Blue RR Salt (Sig- ma Chemical Co., St. Louis, Missouri) to which were added 100 p1 of 0.02% (w/v) P-naphthyl- acetate (in 0.1 M sodium phosphate buffer, pH 7.6) a t 30°C for 1 to 2 hours until the bands of esterase activity were clearly visible. The reaction was terminated by washing the gels in 7.5% (v/v) acetic acid, in which solution the gels were stored. The distribution and relative intensity of staining of the gels was deter- mined by scanning them in a Clifford model 345 densitometer.

Histochemical demonstration o f endplates Triceps muscles were dissected and fixed for

1 hour in 4% buffered formalin, pH 7.3 (0.05 M sodium phosphate, 4% formalin vlv) at 4"C, washed with distilled water a t 4"C, oriented in

gelatin and post-fixed in buffered formalin a t 4°C. Sections 40 pm thick were cut with a cytostat and stained for cholinesterase using the technique of Karnovsky and Roots ('64).

Calculation and graphical representation of AChE activity i n gradient fractions

The radioactivity of the 13H1 acetate re- leased from the substrate was determined by liquid scintillation counting. The 1 "HI acetate produced by non-enzymatic spontaneous hy- drolysis of the substrate under the incubation conditions and the [3Hl acetate produced by total hydrolysis of the substrate were simi- larly determined. A program designed by Judy Leon of the Department of Neurosciences, McMaster University, for a Hewlett-Packard desk calculator, Model 9810A, interfaced with an X-Y plotter, Model 9862A, enabled us: (i) to calculate and substract the background due to the non-specific hydrolysis of the substrate; (ii) to plot (a), the activity of each fraction, and (b), the activity of each fraction expressed as a percentage of the total activity on the gradient; and (iii) to calculate the proportion of the total activity in each of the forms sepa- rated an the gradients.

RESULTS

Characteristics o f cholinesterase activity in unfractionated muscle homogenates

Unfractionated homogenates of newt tri- ceps muscles hydrolyzed I 3H1 ACh. The muscle homogenates also hydrolyzed AcThCh a t about 40% of the rate a t which ACh was hydrolyzed (table 1). This hydrolysis showed substrate inhibition a t concentrations above 2.5 mM a feature that is characteristic of acetyl-, but not pseudo-cholinesterase (Silver, '74) (fig. 11. Acetyl-P-methylcholine, a specific substrate of AChE (Silver, '741, was hydro- lyzed a t approximately 20% of the rate of ACh. In contrast, BuThCh, a substrate of pseudo- cholinesterase, was hydrolyzed a t less than 5% of the rate of AcThCh (table 1). Hydrolysis of AcThCh was inhibited by physostigmine in a dose-dependent fashion. A concentration of 10 pM physostigmine was sufficient to reduce the rate of hydrolysis by 50% (fig. 2).

Fractionation of cholinesterase species f rom whole muscles by velocity sedimentation

on sucrose density gradients Velocity sedimentation of the newt triceps

homogenates resulted in separation of five fractions containing AChE activity (fig. 3).

CHOLINESTERASE IN NEWT MUSCLE 439

TABLE 1

F P 4 1

x

0 0 4 8

SUBSTRATE CONCENTRATION (mM) Fig. 1 The effect of the concentration of AcThCh on

the rate of hydrolysis of AcThCh by a crude homogenate of newt triceps muscle. Incubation conditions are de- scribed in the text.

loo t

~~ - 0 50 100 150 INHIBITOR CONCENTRATION (M x I$)

Fig. 2 The inhibition by increasing concentrations of physostigmine of AcThCh hydrolysis by a crude homoge- nate of newt triceps muscle. Incubation conditions are de- scribed in the text.

The principal activity sedimented with the value of 5.5s. Other activities had sedimenta- tion coefficients of 3.7S, 9.8S, 12.8s and 16.5S5 The 3.7s material always separated as a shoulder on the 5.5s peak. However, re-

Hydrolysis of Ach, MeCh, AChThCh and BuThCh by ho- mogenates o f newt triceps muscles. In each case the re- sults are the mean and standard error of Foe separate ex- periments done at 30°C and pH 8.0

Cholinesterase activity

nmoleslminlmg nmoleslminlmg wet weight protein

Substrate ’

ACh 13.3 -t 1.4 57.2 2 10.4 16.0-t 4.6 MeCh 3.7-t l . l

AcThCh 5.420.6 23.22 2.1 BuThCh 0.620.2 2.6& 1.3

’ ACh, Acetylcholine; AcThCh. Acetylthiocholine: MeCh, Acetyl-8-methylcholine; BuThCh, Butyrylthiocholine.

centrifugation of the 3.7s material (see below) demonstrated that i t was, in fact, a different form from the 5.5s AChE activity. All five fractions hydrolyzed acetyl-/3 -met hylc hol i ne a t 10-2096 of the rate of ACh (fig. 41, but none of the fractions hydrolyzed BuThCh a t a sig- nificant rate. This indicated that the ChE ac- tivity of all five fractions was AChE rather than BuChE (Mendel et al., ’43; Augustinsson, ’49).

To determine whether the fractions were stable multimeric structures or were capable of interconversion during the isolation pro- cedure, each of the peaks of AChE activity ob- tained by velocity centrifugation was re- covered from t h e gradient and dialyzed against 0.2 M NaC1, 50 mM Tris-HC1, 0.2 mM EDTA, pH 7.3 for 12 hours a t 4°C. Portions of the fractions (200 p.1) were recentrifuged on sucrose gradients. Recentrifugation of the five fractions each resulted in a single peak of AChE activity with the same sedimentation coefficient as the original fraction (fig. 5) .

AChE forms in endplate-rich, endplate-poor and rnusculotendinous junction regions

Histochemical staining of whole triceps muscles for AChE revealed that the endplates were located in a transverse band, approx- imately 2 mm wide, midway between the ori- gin and insertion of the muscles (fig. 6). “Endplate-rich” tissue was obtained by excis- ing a 3-mm-long segment that contained the band of endplates. The “endplate-poor’’ tissue was the 2-mm segment of muscle immediately distal to the “endplate-rich’’ segment. Tissue from the musculotendinous junction region

Although the true sedimentatlon coefficients of these forms are 12.85 and 16.55, we frequently refer to them as the 12s and 16s forms. The abbreviation to 16s of the AChE forms that sediment in the 16s region is commonly accepted in the literature in the field.

440 RATHBONE, VICKERS, GANAGARAJAH, BROWN AND LOGAN

U D > r z 0 a

n c 0 .-

i rt \ t

\ E v) Q)

0 - E n

800

400

0

I6 S L

11.3 s *L

6.1 S 3.

10 20 FRACTIONS

Fig. 3 ACh hydrolysis by fractions of an homogenale of newt triceps muscle separated by sedimentation on a sucrose density gradient. Arrows indicate t h e positions of t he marker enzymes in the gradients: 6-galactosidase, 16s; catalase. 11.3s; alkaline phosphatase, 6.1s. The sedimentation coefficient of each form of AChE was determined by comparison of i ts mobility with the mobility of the marker enzymes activity in the same gradient. Since the sedimentation coefficients of t he forms of AChE were consistent in all experiments, indications of t he marker enzymes are omitted from some of the figures.

was comprised of the muscle tissue distal to the “endplate-poor” segment (about 1 mm) and the tendon up to its insertion into the radius bone. These three regions of the mus- cles contained different proportions of the various AChE forms. In homogenates of whole muscle, the 16s form accounted for 10-121%, of the total AChE activity (fig. 3 ) . In contrast, the endplate-rich segments had a higher pro- portion of the 16s form-15-2O‘X of the total activity (fig. 7A) -while in endplate-poor seg- ments the 16s form comprised only 2.5% (fig. 7B) and this form was barely detectable in the musculotendinous junction segment (fig. 7 0 .

The distribution of the 12s form was the reverse of that of the 16s form. Although it was a major activity in the musculotendinous junction segment (fig. 7C), it was present as a

much lower proportion of the total activity in the endplate region (fig. 7A). The 9S, 5.5s and 3 .7s forms were present in tissue samples from each of the three parts of t he muscle. The 9s fraction, however, was present in only very small quantities in the musculotendinous junction region (fig. 7C). Another feature peculiar to AChE forms from this region of the muscle was the relatively large proportion of 3 .7s form compared to the 5.5s one.

Soluhilization ofAChE by Triton X - 1 0 0 and NaCl

After homogenization in Tris buffer solu- tion and using AcThCh as a substrate, the AChE activity of newt triceps muscles at pH 8.0 and 30°C was 5.4 -+ 0.6 nmoleslminimg wet weight muscle (23.2 * 2.1 nmoles/min/

CHOLINESTERASE IN NEWT MUSCLE 44 1

80

n cn 5 >- J 0 a 0 >- I

I

09 I 0 a

C 0

-- 40 E \ m Q) - E n U

0 10 20 FRACTIONS

Fig. 4 Acetyl-P-methylcholine hydrolysis by forms of AChE separated from homogenates of newt triceps muscles fractionated by velocity sedimentation on sucrose gradients.

mg protein) respectively (table 1). After cen- trifugation of this homogenate for 30 minutes a t 10,000 x g, 50% of the AChE activity re- mained in the supernate. The pellet from the first centrifugation was homogenized in 150 pl of 50 mM Tris-HC1,O.Z mM EDTA, 0.5% (v/ v) Triton X-100, pH 7.3 and recentrifuged a t 10,000 X g for 30 minutes. A further 20% of the total AChE activity was solubilized by this procedure and remained in the 10,000 x g su- pernate. The Tris-Triton insoluble pellet was rehomogenized in 150 p1 of Tris-Triton-NaC1 buffer and recentrifuged at 10,000 x g for 30 minutes. A further 15% of the total AChE ac- tivity was solubilized by this treatment and remained in the 10,000 x g supernate after centrifugation. Since the final pellet did not contain detectable AChE activity i t is ap- parent that the repeated homogenization and recentrifugation of the muscle homogenates results in some small (about 15%) loss of AChE activity. A similar small loss of activity has been reported by others who have undertaken

similar determinations in other systems (Gisiger et al., '78).

The fractions of AChE solubilized by homog- enization in the Tris, Tris-Triton and Tris- Triton-NaC1 buffers were each further frac- tionated by centrifugation on sucrose density gradients. The Tris-soluble material con- tained a large proportion of 3.7s and 5.5s forms of AChE and a smaller amount of the 9.8s form (fig. 8A). In contrast to homoge- nates of whole muscles in which the 5.5s form predominates (fig. 3), the proportion of the 3.7s form was almost double that of the 5.5s form. Negligible quantities of the 12s and 16s forms were extracted when the muscles were homogenized in Tris buffer. Extraction of the 10,000 x g pellet with Tris-Triton buffer ex- tracted the same three forms as did Tris alone. However, in contrast to the Tris soluble mate- rial in the Tris-Triton extracts, the amount of 5.5s AChE was much greater than that of the 3.7s form (fig. 8B). Re-extraction of the 10,000 x g pellet of the Tris-Triton homoge-

442 RATHBONE, VICKERS, GANAGARAJAH, BROWN AND LOGAN

A

0 1 2ool h

0 \ 0 10 20

FRACTIONS

Fig. 5 Re-sedimentation of isolated AChE species. Each form was isolated by velocity centrifugation, dia- lyzed and re-centrifuged as described in the text. A, 3.7s form; B, 5.5s form; C, 9.8s form; D, 12.8s form; E, 16.5s form.

nate with Tris-Triton-NaC1 buffer resulted in the solubilization of three AChE forms tha t the previous treatments had failed to solubil- ize. These forms had sedimentation coeffi- cients of 16.5S, 12.8s and 7S , (fig. 8C). In addi- tion, lesser amounts of t he 9.8S, 5.5s and 3.7s forms were present. The amounts of t he 9.8S, 5.5s and 3.75 forms relative to one another were approximately the same as in the Tris- Triton-soluble material. The 7s form was ap- parently not an artefact produced by repeated homogenization, since it was seen in a small proportion (approximately 15%) of gradients of whole muscle homogenates and likely was

not apparent in other gradients because i t was hidden by the 5 . 5 s and the 9.8s peaks. More- over, the centrifugation conditions used in these studies were optimized for t he separa- tion of the five major AChE forms. Although under these conditions the 7s form failed to separate consistently from the abundant 5 . 5 s and 9.8s forms tha t sedimented on either side of i t , we were able to separate these forms in whole muscle homogenates by more extensive centrifugation. However, the studies reported- here deal predominantly with the five forms reproducibly separated on sucrose gradients designed to separate these forms optimally.

Fractionation ofAChE from blood and peripheral nerves

The muscle samples were necessarily con- taminated with small amounts of both blood and intramuscular branches of peripheral nerves. Both of these have AChE activity. Therefore, i t was necessary to determine whether regional differences in the content of t he nerve tissues or blood within the muscle could account for the distribution of t he various AChE species in the endplate and musculotendinous junction regions of the muscles.

The principal AChE species in nerves a re the lower molecular weight forms that sedi- ment at approximately 3.7S, 5 . 5 s and 9.8s re- spectively. The more rapidly sedimenting 12s and 16s forms are present in nerves in very low amounts (fig. 9A). Thus, regional dif- ferences in the content of nerves within the muscles a re unlikely to account for the dif- ferent proportions of the 12s and 16s forms of AChE in the endplate-rich and musculoten- dinous junction regions of t he muscles.

Blood erythrocytes contained forms of AChE which sedimented a t approximately 3.7S, 5.5s and 75 (fig. 9B). Blood plasma (fig. 9C), however, contained AChE which sedi- mented between 5.5s and approximately 10.5s. It was therefore likely tha t regional dif- ferences in muscle blood supply could account for the different proportions of 12s and 16s forms of AChE observed in the muscles. To verify this, the sedimentation profiles of AChE species from triceps muscles removed from newts by our usual procedure were com- pared with those from muscles removed from newts tha t had been exsanguinated and per- fused with amphibian Ringer’s solution con- taining 5 mg/ml of bovine albumin. The sedi-

CHOLINESTERASE IN NEWT MUSCLE 443

Fig. 6 Longitudinal section of a triceps muscle stained by the method of Karnofsky and Roots ('64) dem- onstrating AChE activity a t the endplates. The endplate hand extends diagonally from the bottom left to the top right of the picture. Lines indicate the limit of the endplate region.

mentation profiles of each were the same. This demonstrates t ha t the blood content of newt triceps muscles does not make a detectable contribution to the sedimentation profile of AChE species from the muscles under the con- ditions of our experiments.

Fractionation of individual AChE species by PAGE

Both crude muscle homogenates and the AChE species separated by velocity gradient centrifugation were further fractionated by electrophoresis on polyacrylamide gels. The fractions from the sucrose gradients were sep- arated into three groups. One group contained the AChE species tha t had sedimentation coefficients of 16.5s and 12.8S, one the 9.8s species and one the 5.5s and 3.7s species.

Upon electrophoresis of the unfractionated muscle homogenates a total of 12 bands stained for esterase activity when stained using 0-napthylacetate and Fast Blue RR. All t h ree groups of samples from gradients showed essentially the same pattern of bands as the crude homogenate except t he bands with higher mobility ( > 0.70 R,) were vari- ably present. However, when the distribution of ChE species on gels was determined using AcThCh as the substrate, they were also ab- sent, indicating they represent non-specific

esterase. Storage of the muscle homogenates a t 4°C for periods of up to one week did not al- ter the pattern of bands on the gels.

Using AcThCh, the effect of substrate con- centration on the AChE activity of bands 1-9 in gels of muscle homogenate was determined. Bands 1 through 7 showed maximum activity between 2.5 and 3.75 mM above which activity dropped (fig. 10). In several cases (bands 5 , 6 and 7) a n increase was observed a t 8 mM AcThCh which may be due to non-specific ChE. In contrast, bands 8 and 9 showed no sub- strate inhibition, but rather their activities were maximal at AcThCh concentrations of 1.8 mM and above. Physostigmine, at a con- centration of 1 x M inhibited the hydrolysis of bands 1-9 by more than 85%. Bands 1 to 9, hydrolyzed BuThCh a t less than 5% of the rate of AcThCh (fig. 11).

DISCUSSION

In unfractionated homogenates of newt t r i - ceps muscles we have demonstrated tha t t he majority of the esterase activity is AChE, both on the basis of substrate specificity (Mendel et al., '43; Augustinsson, '49) and its inhibition by high substrate concentrations (Augustins- son, '49) or by low concentrations of physostig- mine (Easson and Stedman, '37; Mendel et al., '43). The AChE activity was separated into six

444

8

4

t z w a $ 0 0 C

RATHBONE, VICKERS, GANAGARAJAH, BROWN AND LOGAN

16s 12s 9.8s 5.5S3.7S L 1 3 . 4 1

A

d a

0 20 40 FRACTIONS

Fig. 7 Activity of forms of AChE separated by velocity centrifugation of homogenates of various regions of newt triceps muscles.

A Endplate rich region. B Endplate poor region. C Musculotendinous junction.

The activity in each fraction was expressed as a percen- tage of the total activity on each gradient. The numbers and arrows indicate sedimentation coefficients of the forms of AChE. The position of the sedimentation stan- dard marker enzymes is omitted for clarity.

major fractions on sucrose density gradients. Five of these a re apparent in sucrose gra- dients of homogenates while the sixth is infre- quently seen in homogenate gradients and consistently seen after differential solubiliza-

tion of AChE. This number of fractions is con- siderably greater than the three major frac- tions of AChE separated from ra t muscles by this technique (Hall, '73; Reiger and Vigny, '761, and is more like the number of forms ob- tained from the electric organs of the phyloge- netically less advanced ray (Torpedo marmo- rata) and the electric eel (Electrophorus elec- tricus). The various forms of AChE showed major differences in their solubility in various extraction media. The 3.7s form was readily solubilized in Tris buffer, and therefore was probably cytoplasmic. The 5.5s and 9s forms were partially solubilized in Tris buffer, but a substantial further proportion of these forms was released by extraction in Tris-Triton buffer. This indicates tha t these forms are partially localized within a compartment re- sistant to osmotic shock during aqueous ho- mogenization. A likely candidate for such a compartment is the sarcotubular system of the muscles. Histochemical studies of mam- malian muscles have revealed the presence of membrane-associated AChE in this location (Tennyson et al., '77). Moreover, Gisiger et al. ('78) have evidence tha t the 10s form of AChE in sympathetic neurons is also located in the endoplasmic reticulum. Three forms of AChE, with sedimentation coefficients of 16S, 12s and 7S, were solubilized only in the Tris- Triton-NaC1 medium. It is likely tha t these forms are each bound to membranes by similar mechanisms. I t has been implied in some ear- lier reports t ha t soluble forms of AChE may be derived artefactually from membrane bound forms (for example, see Hollunger and Nik- lasson, '67; Whittaker and Dowdall, '72). Al- ternatively, i t might be argued tha t the various forms of AChE separated by velocity sedimentation arose due to artefactual aggre- gation of subunits during the experiments. These explanations are unlikely since: (i) two of the forms had specific anatomical distribu- tions in the muscles; (ii) there were tissue- specific differences in sedimentation pat- terns; and (iii) t he various forms retain their initial sedimentation characteristics when they are isolated and recentrifuged, without any evidence of interconversion. We conclude, therefore, tha t the six forms observed in these experiments represent naturally-occurring entities and are unlikely to be experimental artefacts.

The biological roles of t he multiple forms of AChE in newt muscles a re enigmatic. In stud- ies of mammalian muscles Hall ('73) and Vig-

CHOLINESTERASE IN NEWT MUSCLE 445

NEWT TRICEPS (homogenized in Tris-Triton-NaCI+ 100% Activity) MUSCLE Supernates Fractionated on

Sucrose Gradients I Homogenized in Tris buffer

Centrifuged 1

Supernate ( 5 0 ~ ~ of act.) ’

+ Pellet

(50% of activity)

I Rehomogenued in T ris -Triton h f f er

Centrifuged I

(20% of act) I Pellet

(30% of activity)

Rehomogenized in Tris-Triton,-NoCI buffer

Centrifuged

Supernate (15% of act.) t-

Pellet (0 activity)

C 0

> > I- 0

k

a

-

J

I- 0 I-

0

a

u-

* e

c 0 a

0 15 30 F RACTiONS

Fig. 8 Fractionation on sucrose gradients of forms of AChE obtained by sequential extraction of the mus- cles and pellets in (A) Tris buffer, (B) Tris-Triton buffer and (C) Tris-Triton-NaCI buffer.

446 RATHBONE, VICKERS, GANAGARAJAH, BROWN AND LOGAN

I6 S 11.3s 6.1s the motor endplate region of the muscles with AChE from nerves or blood was eliminated by the observation tha t the 16s form was not present in blood and represented only a very small proportion of the AChE activity in the nerves. Additional evidence tha t the 16s frac- tion is the endplate form in newt muscles is presented in an accompanying paper (Rath- bone et al., '791, in which i t is reported tha t the proportion of the 16s form is specifically reduced following denervation of muscles. The

L A

I suggestion has been made recently that the

c

0 15 30 FRACTIONS

Fig 9 Forms of AChE separated by velocity gradient centrifugation from ( A ) peripheral nerve, (BJ blood erythrocytes and (C) blood plasma of newts

ny et al. ('76) found tha t the 1 6 s form of AChE is the endplate form. A similar conclusion can be made about the 16s form in newt muscles since regions of muscle containing the motor endplates contain proportionally more of this form of AChE. The possibility t ha t this obser- vation could be attributed to contamination of

16s-form of AChE at the neuromuscular junc- tion may be neuronal in origin (Fernandez and Festoff, '78; Festoff and Fernandez, '78). This species is transported bidirectionally along motor nerve axons and may be released a t the neuromuscular junctions and become a t - tached postsynaptically to the surface of the muscle membrane. These data do not permit us to determine whether the 16s form at the newt neuromuscular junction has a neuronal or muscular origin, since we also find a small proportion of the 16s form in newt peripheral nerves. The very high proportion of the 12s form of AChE in regions of muscle containing the musculotendinous junctions raises the possibility tha t this species is actually associ- ated with these junctions. Cholinesterase ac- tivity a t this site has been well described (Couteaux, '53; Gerebtzoff, '54), characterized as an acetylcholinesterase (Schwarzacher, '60a) and shown to be located in the infoldings of the surface membrane of the muscle fibres a t the points of attachment of the collagenous fibres of the tendons (Couteaux, '58, '59; Schwarzarcher, '60b). However, its function is obscure since cholinergic nerve endings have not been found in this region of muscles. Ap- parently, the 125 form does not have a counter-part in mammalian muscles. In ra t muscles, only three forms have been found which have sedimentation coefficients of 16S, 10s and 4s (Hall, '73; Vigny et al., '76) and there is no evidence tha t any of these forms are present in increased amounts at the musculotendinous junctions. The identifica- tion of a specific molecular form of AChE that is more abundant at the musculotendinous junction may provide the basis for further in- vestigations of t he role of AChE at this loca- tion, and of i ts relationship to other forms of AChE located elsewhere in the muscles.

The various forms of AChE obtained by cen- trifugation on sucrose density gradients could

1 PEAK 0.6 -

0.4 - c

\

.- E E 0.2 N t 0 s o > > 0

w

!I

j~ 0.6

a

5 0.4

0.2

0

CHOLINESTERASE IN NEWT MUSCLE

PEAK 2 [ PEAK 3

I PEAKS 7 Ef8(0)

44 7

PEAK 4

- 0 4 8 0 4 8 0 4 8 0 4 8

SUBSTRATE CONCENTRATION (mM) Fig. 10 Hydrolysis of AcThCh in response to increasing substrate concentrations by the fractions of AChE separated

by PAGE from crude muscle homogenates.

be fralctionated further by PAGE into multi- ple bands of AChE activity. Each of the frac- tions from the sucrose gradients and the crude muscle homogenates gave similar banding patterns when fractionated by this procedure. These patterns remained unaltered even when the crude homogenate was stored a t 4°C for up to one week before they were fractionated. Therefore, i t is unlikely that these bands re- sulted from proteolytic degradation of larger molecules since such a process would be ex- pected to be time dependent. It is more likely that forms separated on the sucrose density gradients represent multimeric complexes. The large number of bands on the PAGE gels is difficult to explain. It is possible that in the presence of Triton, the stability of the multi- meric complexes may be reduced, and an equi- librium established between dissociated sub- units and the intact multimeric complexes. Since migration in the sucrose gradient is

slow, the individual subunits are not dissoci- ated from the multimeric structures for a suf- ficient time to be physically separated from them. Thus, the multimeric complexes are ef- fectively stable in the sucrose gradients. How- ever, under the influence of the electric field of the electrophoretic system the individual components may undergo a more rapid physi- cal separation which is maintained once they enter the gel, resulting in monomers, dimers, trimers, etc. being present. Furthermore, Dudai et al. ('72) and Massoulie et al. ('71) have shown that the large multimeric AChE structures from electric eels cannot enter polyacrylamide gels and thus only the smaller multimeric structures and monomers pro- duced by dissociation of the multimers would be found in the gels.

Of the 9 bands of AChE activity separated on PAGE the two most rapidly migrating ones did not exhibit substrate inhibition. Although

448

1.0

- C

\

.- E I 8 0.6 0

> W

k f 0 4

9 0 0.2

0

RATHBONE, VICKERS, GANAGARAJAH, BROWN AND LOGAN

R 6

4 i

0 20 40 GEL SLICE NUMBER

Fig. 11 Profile of AcThCh (0-0) and BuThCh (0-0) hydrolysis in fractions of PAGE gels of crude muscle homogenate. Assays were done using the colorimetric technique described in the text.

substrate inhibition is characteristic of AChE (Silver, ’74), Hall (’73) isolated a low molecu- lar weight form of rat muscle AChE that also failed to show a substrate inhibition. Coopera- tive behaviour is found usually in enzymes that are a t least dimers, and frequently more complex multimeric structures (Koshland and Meet, ’68). A lack of cooperative behaviour might therefore be expected of monomer sub- units of AChE.

The finding that the several forms of AChE separable by sedimentation on sucrose density gradients each yield similar AChE activities when subfractionated on PAGE provides the basis for a hypothesis that describes the mo- lecular structure of the various forms of AChE in newt muscles. We propose that the forms of AChE from newt muscles are analogous to those reported in Electrophorus electricus by Dudai et al. (’73) and Bon et al. (’73). Thus the

16S, 1 2 s and 7 s like the 19S, 1 4 s and 8s forms of the electric eel consist of a multimeric “head” of enzymatically active subunits and an elongated, enzymatically inactive “tail,” and the 9.8S, 5.5s and 3.7s forms are respec- tively tetramers, dimers, and monomers of the catalytic subunit(s1. The smaller forms of AChE are readily soluble in aqueous buffers containing low concentrations of salt. This raises the possibility that they are cytoplas- mic and thus, perhaps, are near their site of synthesis. The insolubility of the larger forms in aqueous buffers indicates that they are pro- gressively more firmly bound to membranes. The smaller, soluble, cytoplasmic forms of AChE may be precursors of the larger, insolu- ble, membrane-bound forms, a possibility that we are currently investigating.

The elucidation of the molecular structures, possible metabolic interrelationships, bio-

CHOLINESTERASE IN NEWT MUSCLE 449

chemical characteristics and ultrastructural localization of specific forms of AChE from newt muscles may provide the basis for a bet- ter understanding of the regulation of this en- zyme activity by various neural and myogenic factors.

ACKNOWLEDGMENTS

This work was supported through grants-in- aid of research from the M.R.C. of Canada (Grant MA 6326) and the Muscular Dystrophy Association of Canada to M.P.R. and from the N.R.C. of Canada (Grant A49141 to D.M.L.

We thank Carmella Yacoob and Patricia Martin for expert technical assistance, Drs. G. M. Brown, R. Carlone and E. S. Werstiuk of the Department of Neurosciences, McMaster University for their helpful advice during preparation of this manuscript, and Ms. Carol Habinski for typing the manuscript.

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