The collagen-like subunits of acetylcholinesterase from the eel electrophorus electricus

Neurochemistry International Vol.2, pp.135-147. Pergamon Press Ltd. 1980. Printed in Great Britain.

THE COLLAGEN-LIKE SUBUNITS OF ACETYLCHOLINESTERASE FROM THE EEL ELECTROPHORUS ELECTRICUS Terrone L. Rosenberry, Philip Barnett, and Carol Mays The Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106 and the Department of Neurology, Columbia University, New York, New York 10032

ABSTRACT Pepsin-resistant fragments of the t a i l subunits of 14S and 18S acetylcholinesterase from eel electric organ have been isolated and characterized. The native fragments are composed of three 24,000 molecular weight polypeptides linked by intersubunit disulfide bonds in a collagen-like t r i p l e helix. Intact t a i l subunits have also been isolated. These subunits appear to contain both the triple-helical domain and a noncollagenous domain that is linked to catalytic subunits by disulfide bonds. KEYWORDS Acetylcholinesterase; collagen-like subunits; pepsin-resistant fragments; basement membrane; neuromuscular junction INTROOUCTION In 1937, David Nachmansohn observed that acetylcholinesterase activity in muscle appeared at least three to six times higher in endplate regions than in extrajunctional regions (Marnay and Nachmansohn, 1937). This observation led Nachmansohn to investigate the enzyme activity in e l e c t r i c organs of e l e c t r i c f i s h , tissues known to be rich in synaptic innervation. Extraordinary concentrations were found, and the f i r s t s o l u b l e extract of acetylcholinesterase from the electric organ of the ray Torpedo marmorata was obtained in 1938 (Nachmansohn and Lederer, 1939). A 16S form of acetylcholinesterase was l a t e r shown to be s p e c i f i c a l l y associated with rat diaphragm endplates (Hall, 1973) after Massoulie and Rieger (1969) had demonstrated the u t i l i t y of sucrose gradient sedimentation in characterizing the distribution of acetylcholinesterase forms in eel electric organ extracts. 14S and 18S Eel forms of the enzyme appear to be closely analogous to the 16S rat form, and the protein structure of these 14S and 18S forms has been studied in some detail (Rosenberry and Richardson, 1977). Collagen-like subunits have been identified that appear to anchor these enzyme forms in the extracellular basement membrane at peripheral cholinergic synapses (McMahan and colleagues, 1978). Because basement membrane components may serve important regulatory roles in developing synapses (Sanes and colleagues,1978), we have characterized in some detail the very unusual collagen-like t a i l subunits of 14S and 18S eel acetylcholinesterase. ISOLATION OF PEPSIN-RESISTANT FRAGMENTS OF ACETYLCHOLINESTERASE The digestion of tissue or tissue extracts with pepsin is frequently employed to solubilize collagen-like proteins or to selectively degrade noBcollagen-like regions in these proteins. The technique was introduced by Rubin and colleagues (1963), who showed that pepsin digestion of acid-soluble c a l f skin collagen at low pH released small peptides to the dialysate with amino acid compositions not typical of triple-helical collagen while retaining most of the protein in native triple-helical form. The collagen-like regions that resist degradation by pepsin can be fractionated into various collagen types by d i f f e r e n t i a l salt precipitation (Orkin and colleagues, 1977; Sage and Bornstein, 1979). In preliminary experiments we established that salt precipitation was not required for the isolation of pepsin-resistant fragments of 18S and 14S eel acetylcholinesterase and that gel exclusion chromatography in a nondenaturing solvent was more direct and provided a better yield. The isolation procedure is ]35

136

T.L.

I

Rosenberry,

f

l

I

P. Barnett and C. Mays

I

A

I

B

~,

70,000

O.

30,000

CPM

A280nn

0

40

80 VOLUME, mL

120

160

Fig. 1. Gel exclusion chromatography of a pepsin digest of 14S and 18S acetylcholinesterase. The acetylcholinesikerase had been prelabelled with a substoichiometric amount of [~H]diisopropylfluorophosphate to indicate peptides containing the enzyme catalytic site. Digestion of 10 mg of labelled enzyme (6 mL) with pepsin (100 ug/mL) in 0.5 M acetic acid for 6 hrs at 15°C was followed by neutralization to pH 8 with Tris and application to a Sepharose CL-6B column equilibrated with buffered 1.0 M sodium chloride. The absorbance )(A~n"~) (2~ v~ (0-0) and cpm (10 uL aliquot) ( ~ of each column fraction are indicated. Elution volumes of the following standard proteins were determined from parallel chromatographic runs in the same buffer: A: 18S eel acetylcholinesterase (MW 1,100,000); B: 11S eel a c e t y l cholinesterase (MW 320,000); C: soybean trypsin i n h i b i t o r (MW 22,000). shown in Fig. 1. Gel exclusion chromatography resulted in fractions that can be grouped somewhat a r b i t r a r i l y into five pools. Pool I eluted between 44 and 60 mL and corresponded to the column void volume. Protein in this pool was aggregated, as suggested by the low absorbance r a t i o ApRN/A~(I of about 0.8 for the fractions in this pool. In contrast, fractions that elute-d--aft~-Pool I were characterized by A280/A250 values of greater than 2 that are t y p i c a l of undigested acetylcholinesterase. Gel electrophoresis in sodium dodecylsulfate of samples from selected fractions in Fig. 1 gave banding patterns shown in Fig. 2. Pepsin-digested fragments large enough to be retained by dialysis tubing are shown in the gel corresponding to the column onput (0). The largest discrete polypeptide fragment bands are denoted F3 and F2 and are the only major polypeptides in the digest larger than pepsin (P). F3 is a single band while F2 is a closely spaced doublet. After column fractionation, F3 and F2 are the only major polypeptide components found in Pool I and Pool I I . A faint diffuse band just below pepsin at the position labelled F1 is observed in Pools I and I I in some pepsin digests. Pool I contains aggregated material in the column void volume, and Pool I I is comprised of apparently soluble protein in the included volume of the column with a separate peak of F2 and F3 distinct from that in Pool I. Thus F3 and F2 are obtained both in large aggregates and as discrete "dissociated" complexes prior to denaturation and separation into individual polypeptides in sodium dodecyl sulfate. The elution volume of the dissociated complex can be estimated from the staining intensities of F3 and F2 on sodium dodecyl sulfate

Collagen-Like Subunits of Acetylcholinesterase

137

gels that we routinely use to characterize fractions from column fractionations similar to that in Fig. 1. For twelve such fractionations, this estimate is 78 ± 1 mL and corresponds to a Stokes radius between that of 18S and of 11S acetylcholinesterase (Fig. 1). Pool I l l is a mixture that contains most of the polypeptides in the column onput, and Pool IV includes only pepsin and smaller polypeptides. Pool V (115-160 mL) contains nearly 70% of the total A2R0 onput, but v i r t u a l l y everything is dialyzable; thus l i t t l e gel staining is observed under The conditions in Fig. 2. The chromatographic procedure in Fig. 1 is intended to isolate collagen-like regions of the acetylcholinesterase t a i l subunits that are resistant to pepsin, and several criteria were examined to demonstrate that F3 and F2 were collagen-like fragments that were essentially free of other polypeptide contaminants. Contamination could arise either from small amo~,ts of residual undigested acetylcholinesterase or from noncollagen-like fragments. The H-cpm p r o f i l e in Fig. 1 indicates that very l i t t l e 14S and 18S ~cetylcholinesterase survived the digestion intact. Although more than one-half of the H label remains attached to the c a t a l y t i c sites under the digestion condition~ in Fig. 1 in the absence of pepsin (Mays and Rosenberry, 1980), less than 1.5% of the total °H cpm eluted in Pools I-IV after the pepsin digestion in Fig. 1. V i r t u a l l y all the label in these pools appeared at or near the elution volume of 11S acetylcholinesterase, and a negligible amount appeared at the earlier elution volumes that correspond to 14S and 18S acetylcholinesterase. These percentages are considerably less than the corresponding A2BCOrecoveries, but i t is reasonable that pepsin should p r e f e r e n t i a l l y degrade the relative-Fy exposed region of the enzyme c a t a l y t i c site. Since collagen-like t a i l subunits are associated with 14S and 18S acetylcholinesterase but not with 11S acetylcholinesterase, pepsin digestion of the 11S enzyme should provide an excellent control for collagen-like fragments arising from the catalytic subunits. A gel profile (C) corresponding to this control is included in Fig. 2, and i t is apparent that polypeptide bands including F3 and F2 above pepsin are v i r t u a l l y nonexistant, while bands below pepsin roughly correspond to the 14S and 18S enzyme digest. Thus F3 ~nd F2 appear to be excellent candidates for pepsin-resistant t a i l fragments. To insure that H-labelled noncollagen fragments do not coincidentally electrophorese with F3 and F2, several~of the gels in Fig. 2 were sliced and counted (Mays and Rosenberry, 1980). Four d i s t i n c t °H-labelled peaks were apparent, all corresponding to polypeptides with apparent molecular weights equal to or less than pepsin. The same four peaks characterized gel slice profiles for gels I, I l l and C in Fig. 2 with only minor variations. Virtually no JH label corresponded to intact catalytic subunit monomers or oligomers. Thus the light bands above F3 in gel I are probably oligomers of F3 and/or F2 and are not undigested acetylcholinesterase. To further support the collagen-like nature of F3 and F2 as well as to quantitate the recovery

of total hydroxyproline and hydroxylysine from the column chromatography procedure, column pool samples were hydrolyzed and subjected to amino acid analysis. The sample digestion and column fractionation procedures employed and the elution volumes pooled in this experiment were s i m i l a r to those in Figs. 1 and 2, but the pepsin appeared s l i g h t l y less active. This lower a c t i v i t y had three pronounced effects (Mays and Rosenberry, 1980): 1) The A280 recoveries were about twice, in Pool I , and four times, in Pool I I , the correspondlng recoveries in Fig. 1. 2) The amount of F3 r e l a t i v e to F2 in gels appeared to be about twice that shown in Fig. 2 for a l l fractions. 3) Pool I I gels contained polypeptide bands below pepsin s i m i l a r to those in the Pool I l l gel in Fig. 2. Pool I gels remained free of any polypeptide bands below pepsin. The quantitative amino acid recoveries in the five pools were determined, and a summary of these recoveries is presented in Table 1. Pool I and thus i t s major components F3 and F2, with nearly 30% glycine and over 5%each of hydroxyproline and hydroxylysine, is predominantly collagen-like in composition. Pool II has only one-half the mole percents of hydroxyproline and hydroxylysine found in Pool I and appears to consist of about equal amounts of the F3-F2 mixture and of noncollagen-like fragments. Pool I l l seems to be at most 20% collagen-like, and Pools IV and V are devoid of any fragments derived from collagen-like domains. The overall amino acid recovery was quantitative, although the total recoveries of hydroxyproline and hydroxylysine were 81% and 79% respectively. Of the recovered hydroxyproline and hydroxylysine, 66% was obtained in Pool I. Thus Pool I appears to represent a r e l a t i v e l y contaminant-free isolation of over one-half of the collagen-like regions of the 14S and 18S acetylcholinesterase t a i l subunits.

]38

T.L.

Rosenberry,

P. Barnett and C. Mays

CH~ACTERIZATION OF PEPSIN-RESISTANTFRAGMENTSOF ACETYLCHOLINESTERASE Pool I contains predominantly F3 and F2. Several observations suggest that F2 is derived from F3 by further pepsin degradation. The increased ratio of F3 to F2 under conditions of lower pepsin a c t i v i t y was noted above, and we have confirmed that this ratio is also increased when the time of exposure to pepsin is decreased. I f F3 and F2 represent c o l l a g e n - l i k e t r i p l e h e l i c a l domains, as anticipated f o r the enzyme t a i l s t r u c t u r e , then increasing the pepsin digestion temperature above the helix melting point should result in rapid degradation of F3 and F2. To t e s t t h i s expectation, pepsin digestion of 2 mg of 14S and 18S a c e t y l c h o l i n esterase was conducted in two stages (Mays and Rosenberry, 1980): in the f i r s t stage, digestion was conducted at 15°C for 6 hrs to give a fragment population in which the ratio of F3 to F2 was later shown to be about 5; immediatiately following the f i r s t stage, the sample was divided into seven equal portions, each of which was incubated one additional hour at a fixed temperature that ranged from 0° to 45 °C. The samples were dialyzed and subjected to gel el ectrophoresis as in Fig. 2. For samples that had second stage incubations below 25°C, the F3 to F2 ratio remained at about 5 and the total quantity of F3 and F2 remained constant. Between 25° and 35°C, a sharp transition occurred in which the F3 to F2 ratio dropped to below 0.4 and the total quantity of F3 and F2 was about 60% of that recovered at lower temperatures. Thus one region of F3 appears to undergo a temperature melt and consequent pepsin cleavage between 25 ° and 35°C, but the remaining structure associated with F2 remains r e s i s t a n t to pepsin up to 45°C. Molecular Weight Estimates of Fragments The isolated F3 and F2 fragments retain endogenous interpolypeptide disulfide linkages, and exposure to d i s u l f i d e reducing agents converts them to smaller species on sodium dodecylsulfate gels. A pepsin digest that had been fractionated as in Fig. 1 was selected for analysis in Fig. 3 because Pool I was composed almost e x c l u s i v e l y of F3. Exposure to d i t h i o t h r e i t o l in the sodium dodecylsulfate sample buffer resulted in dissociation to a rather broad primary band FI. Molecular weights of the pepsin-resistant fragments were estimated

TABLE 1 Recovery of Total Amino Acids and of Three Amino Acids C h a r a c t e r i s t i c of C o l l a g e n - l i k e Polypeptides from the Column Chromatographic Fractionation of the Pepsin Digest. Fraction

Column onput Pool Pool Pool Pool Pool Total

I II Ill IV V recovery

Total Amino Acids* mg

Mole Percent HYP GLY HYL

10.53

0.6

10.9

0.7

0.59 0.41 0.76 0.73 8.37

5.2 2.3 0.9 0.0 0.0

27.9 18.2 12.8 6.1 8.9

5.6 2.5 2.5 0.0 0.0

10.86

0.5

10.3

0.5

*Pool samples were not dialyzed to avoid the loss of small peptide fragments, and residual sodium chloride and buffer from the column solvent were carried through the i n i t i a l dehydration, acid hydrolysis, and amino acid analysis. Amino acid standards were prepared with an equivalent amount of salt and carried through the hydrolysis procedure in parallel with the samples (Mays and Rosenberry, 1980). Total amino acids are expressed in mg mean residue weights.

Collagen-Like Subunits of Acetylcholinesterase

F3 F2 P

i

]39

0

~

~,

I

4

++

II

III

FI

0

I

IV

C

Fig 2. Gel electrophoresis of pepsin digests and of selected fractions in Fig I in 1% sodium dodecylsulfate. Polyacrylamide gels (5.8%) were stained with Coomassie B r i l l i a n t Blue R. Samples were dialyzed overnight against water, lyophilized, dissolved in 25 uL of 1% sodium dodecylsulfate sample buffer in the absence of disulfide reducing agents, and run as outlined previously (Rosenberry and Richardson, 1977). (0) 0.16 mL of the column onput in Fig. 1; (1) 0.7 mL of fraction at 52 mL that represents Pool I (44-60 mL); ( I I ) 0.9 mL of fraction at 77 mL that represents Pool II (60-80 mL); ( I I I ) O~ mL of fraction at 91 mL that represents Pool I l l (80-95 mL); (IV) 0.8 mL of fraction at 100 mL that rep~resents Pool IV (95-115 mL). (C) 0.2 mL pepsin digest of 0.2 mg of [ H]diisopropylfluorophosphatelabelled 11S acetylcholinesterase prepared as in Fig. 1.

]40

T.L.

Rosenberry,

P. Barnett and C. Mays

T

60

F2 O 29 25 1

F1

18 ~

A

B

C

Fig. 3. Molecular weight determinations of polypeptides arising from Pool I by gel electrophoresis in 1% sodium dodecylsulfate. Electrophoresis conditions are s i m i l a r to those in Fig. 2. (B, C) Samples correspond to the peak fraction in Pool I; (B) 14 ~g protein prepared without d i s u l f i d e reduction; (C) 20 ~g protein subjected to reduction w i t h I00 mM d i t h i o t h r e i t o l at 50°C f o r 30 min in the 1% sodium dodecylsulfa~e sample solution prior to electrophoresis. (A) Cyanogen bromide fragments of ~ i ( I ) and e2(1) acid soluble calf skin collagen run in parallel with samples B and C. Indicated molecular weights in thousands (from Fietzek and Kuhn, 1976, assuming mean amino acid r e s i due weights of 91.0) correspond r e s p e c t i v e l y to ~1,e2: 94; ~2CB3,5: 60; ~2CB4: 29; ~ICB8: 25; ~ICB6: 18.

Collagen-Like Subunits of Acetylcholinesterase

i

,5| •27 .

| I

A

B

C

Fig. 6. Polypeptide components in fractions obtained during isolation of intact t a i l subunits displayed by gel electrophoresis in 1% sodium dodecylsulfate. Electrophoresis conditions are similar to those in Fig. 2. (A) 14S and 18S enzvme sample that had been completely reduced and alkylated with [T4C]N-ethylmaleimide in 6 M guanidine hydrochloride. Indicated molecular weights in thousands correspond to c a t a l y t i c subunit monomers and c a t a l y t i c subunit fragments (Rosenberry and colleagues, 1974). (B) Fraction not retained by a DEAE cellulose column after dissolution of the sample in 10 M urea buffered with 5 mM sodium acetate at pH 6.5 and chromatography ~ this solvent. (C) Peak fraction obtained from gel exclusion chromatography on Sepharose CL-6B in 6 M guanidine hydrochloride after d i a l y s i s , l y o p h i l i z a t i o n and redissolution of fraction B in this solvent. To each gel was applied 40 ~g protein.

14]

Collagen-Like Subunits of Acetylcholinesterase

143

from electrophoresis standards composed of the well-characterized cyanogen bromide fragments of the al(1) and a2(1) chains of acid-soluble Type I collagen. Fl corresponds to about 24,000 molecular weight. The reduced fragments s t i l l associate rather strongly even in 1% sodium dodecylsulfate, as additional bands with decreasing intensities correspond to 47,000, 74,000 and 98,000 molecular weights. These relative molecular weights are consistent with oligomers of F1, and thus the designation F2 and F3 in Fig. 3 indicate dimers and trimers of Ft. Although the electrophoretic mobilities of F2 and F3 are somewhat greater prior to disulfide reduction, use of these designations i nterchangeably between the corresponding nonreduced and reduced gel bands seems a relatively safe assumption. Pool I samples that are predominantly F3 and those that are largely F2 give v i r t u a l l y identical polypeptide banding patterns when run under the completely reduced conditions corresponding to gel C in Fig. 3. I t should be noted, however, that F1 in this gel may be quite heterogeneous, as suggested by i t s broad band. Circular Dichroism Spectra To confirm that the pepsin-resistant fragments obtained in Pool I indeed have a collagen-like t r i p l e helical secondary structure, c.d. spectra of this pool were compared with those of acid-soluble calf skin collagen and with 11S acetylcholinesterase in Fig. 4. Triple helical collagen is characterized by [e]MR values of 5000-6000 at 220-223 nm and -50,000 at 198 nm (Kefalides, 1968; Brodsky-Doyle an~-colleagues, 1976). The c.d. spectra for collagen and for the pepsin-resistant fragments in several solvents are virtually identical and consistent with these literature values. In contrast, the c.d. spectrum of 11S acetylcholinesterase appears qualitatively similar to that for an a-helical conformation (see Brodsky-Doyle and colleagues, 1976), although the [e]MR values for the 11S form are only about 20% of those for a homogeneous e-helix and suggest the presence of considerable amounts of alternative secondary structures. The c.d. spectrum of 14S and 18S acetylcholinesterase was very similar to that of the 11S form in Fig. 4. A difference spectrum for these two acetylcholinesterase samples

I

I

I

I

I

5

0

2 X

-5

-10

-15 200

I

i

I

I

~

210

220

230

240

250

nm Fig. 4. C.~ spect~ of llS aceVlcholinesterase, collagen, and pepsinr e s i s t a n t fragments of 14S and 18S acetylcholinesterase at 25°C. The mean residue e l l i p t i c l t y , [O]MR, is expressed .in units (deg)(cmZ)/dmOlMg with mean residue weights o f I 1 2 g/mol for 11S acetylcholinesterase an~ 100 g/mol for collagen and pepsin-reslsta~ f r a ~ e n t s . 0 11S A c e ~ l cholinesterase in 20 mM phosphate buffer. (O) Acid-soluble c a l f skin collagen (Sigma) in either 10 mR acetic acid or lOOmM sodium chloride and lOmR acetic acid. (0) P e p ~ n - r e s i s t a n t fragme~s of 14S and 18S acetylch-olinesterase in phosphate-buffered 1.0 or 2.0 M sodium chloride.

144

T.L.

Rosenberry,

P. Barnett and C. Mays

could not be obtained with s u f f i c i e n t accuracy to c l e a r l y demonstrate the presence of the t r i p l e helical structure in the 14S and 18S sample. We have shown that the major covalently linked polypeptides F3 and F2 in the pepsin-resistant fragment pool are primarily converted to FI on disulfide reduction in sodium dodecyl sulfate. Furthermore, F3 appears to be r e a d i l y converted to F2 by pepsin at temperatures of 25 ° to 35°C. F i n a l l y , the Pool I f r a c t i o n p r i o r to denaturation has a c.d. spectrum t y p i c a l of t r i p l e h e l i c a l collagen. These observations suggest the following structural relationships among the pepsin-resistant fragments of 14S and 18S acetylcholinesterase. The largest

t/-~.,s.Sj~ ,.- ,\,

.-~ s s4

[i.sLj'

\j

TRYPSIN

i

is.s~j~

i

ENDOGENOUS\\ PROTEASE \

S ~ s s S ~, ~ )- ~ '

C)

I

/

YPSIN

,j.

/PEPSIN i

?

,s /

Fig. 5. Schematic representation of 18S eel acetylcholinesterase and the patterns of degradation shown by three different proteases. The central structure represents an 18S molecule, in which twelve catalyt i c subunits ( c i r c l e s ) are arranged as three tetrameric groups. Within each tetramer, two catalytic subunits are directly disulfide linked while the remaining two are each covalently attached by a single disulfide bond to one t a i l subunit (Rosenberry and Richardson, 1977). Pepsin degrades the c a t a l y t i c subunits and the noncollagenlike domains of the t a i l subunits that are adjacent to the catalytic subunits to give t r i p l e - h e l i c a l t a i l subunit fragments. An endogenous protease in stored eel e l e c t r i c organ tissue can cleave the t a i l subunits at a point that releases an 11S enzyme tetramer which s t i l l retains an 8000 molecular weight residual t a i l subunit (Barnett and Rosenberry, 1978). Exposure of e i t h e r t h i s 11S form or the 18S form to t r y p s i n generates an 11S tetramer in which the residual t a i l subunit is cleaved between its disulfide linkages to the two catalytic subunits (Rosenber~ and colleagues, unpublished observations).

Collagen-Like Subunlts of Acetylchollnesterase

145

fragment with i n t a c t polypeptide chains is F3, a d i s u l f i d e - l i n k e d complex of three F1 polypeptides each of approximately 24,000 molecular weight. The d i s u l f i d e linkage from at least one of these FI units to the other two is associated with a t r i p l e - h e l i c a l region that melts between 25° and 35°C and can then be degraded by pepsin. The remaining t r i p l e - h e l i c a l structure, consisting of a disulfide-linked F2 and a noncovalently linked residual FI, is only slowly susceptible to further pepsin degradation below 45°C. This residual FI component does not stain as intensively as F2 on sodium dodecylsulfate gels prior to disulfide reduction, but i t can be detected in Pool I samples in which F3 has been largely converted to F2. The question of whether the amino acid sequences of the FI polypeptides are i d e n t i c a l remains open. ISOLATION OF "INTACT" TAIL SUBUNITS OF ACETYLCHOLINESTERASE Our approach to a structural characterization of 14S and 18S eel acetylcholinesterase can be summarized in Fig. 5. Tail subunits can be fragmented by pepsin to permit i s o l a t i o n of polypeptides corresponding to t h e i r t r i p l e - h e l i c a l domain or by an endogenous protease to permit isolation of a noncollagen-like domain that is disulfide-linked to catalytic subunits. We have recently developed an isolation procedure for "intact" t a i l subunits associated with 14S and 18S acetylcholinesterase (P. Barnett and L L. Rosenberry, unpublished observations). The word "intact" is used advisedly because the 14S acetylcholinesterase i t s e l f appears to be derived from the 18S form by the endogenous protease cleavage of the t a i l subunit to release one 11S tetramer (McCann and Rosenberry, 1977). Dissociation of the intact t a i l subunits from catalytic subunits requires both reduction of the intersubunit disulfide bonds and exposure to a denaturing solvent. The subsequent f r a c t i o n a t i o n procedure is summarized in Fig 6.

TABLE 2 Comparison of the Amino Acid Mole Percentages of Intact and Fragmented Tail Subunits of 14S and 18S Acetylcholinesterase. Amino Acid HYP ASP THR SER GLU PRO GLY ALA VAL MET ILE LEU TYR PHE HYL LYS HIS ARG hal f-CYS

Collagen-like Domain 5.0 5.0 2.3 5.9 9.1 8.7 27.2 3.4 4.3 2.8 2.2 5.2 1.7 1.2 5.3 2.3 2.2 4. I

Noncollagenous Domain 0.0 11.4 4.2 4.5 11.9 13.8 6.0 5.7 6.9 2.6 4.3 9.9 1.8 4.0 0.0 4.5 1.1 5.0 2.2

Intact Subunits observed calculated 4.2 6.7 2.4 5.2 9.8 11.0 23.3 3.5 4.7 2.0 2.4 6.5 1.5 2.0 4.6 2.6 1.8 4.8 1.4

3.8 6.5 2.7 5.6 9.7 9.9 22.3 3.9 4.9 2.8 2.7 6.3 1.7 1.9 4.1 2.8 1.9 4.3

*Uncorrected 20 hr hydrolyses at 110°C in 6 N hydrochloric acid containing 80mM mercaptoethanol under argon. The samples corresponding to each domain are described in the t e x t . The t h e o r e t i c a l amino acid composition was calculated from the data in columns 2 and 3 assuming a 32,000 dalton intact subunit, composed of a 24,000 dalton c o l l a g e n - l i k e domain (100 mean residue weight) and an 8000 dalton noncollagenous domain (110 mean residue weight).

]46

T.L.

Rosenberry,

P. Barnett and C. Mays

Labelling the reduced sulfhydryl groups with [14C]N-ethylmaleimide permitted rapid monitoring of the t a i l subunits. At the pH used in the DEAE-cellulose ion exchange chromatography, the catalytic subunit monomers and the 50,000 molecular weight catalytic subunit fragments were completely adsorbed to the column while the t a i l subunits and some of the 24-27,000 molecular weight catalytic subunit fragments were not adsorbed. These latter components were readily separated by the f i n a l gel exclusion chromatography step. The overall recovery of hydroxylysine in the i s o l a t i o n procedure was 40%, and most of the loss occurred in the dialysis steps. The absence ok catalytic subunits in the isolated fraction was confirmed by conducting the procedure with [~H]diisopropylfluorophosphate-labelled enzyme. The isolated fraction C in Fig. 6 gave a major polypeptide band with a closely spaced minor band on either side. The molecular weight of the major band was estimated by the noncollagen standards in Fig. 6 to be 40,000 but by the collagen standards in Fig. 3 to be 30,000. Since the t a i l subunits are a mixture of collagen-like and noncollagenous domains, a precise molecular weight is d i f f i c u l t to assign. Basic amino acid hydrolysates (Butler and colleagues, 1977) revealed that most of the hydroxylysine residues were linked to 2-O-:-D-glucopyranosyl-O-B-Dgalactopyranose. In Table 2, the amino acid compositions of the pepsin-resistant Pool I fraction, referred to as the collagen-like domain, and the 8000 molecular weight residual t a i l subunit fragment associated with 11S acetylcholinesterase, referred to as the noncollagenous domain, are compared with the composition of the intact t a i l subunit fraction. The amino acid composition for intact subunits can be estimated as an appropriate weighted average of the compositions of the two isolated domains. This estimate is in good agreement with the observed composition, suggesting that no distinctive regions of the intact subunits are omitted in the two domains. This suggestion is supported by the satisfactory agreement of the molecular weight estimates of the intact subunits with the sum of the estimated molecular weights of these two domains. REFERENCES Barnett, P., and T. L. Rosenberry (1978). A residual subunit fragment in the conversion of 18S to 11S acetylcholinesterase. Fed. Proc., 36, 485. Brodsky-Doyle, B., K. R. Leonard, and K. B. M. Re~(1976). Circular-dichroiSm and electronmicroscopy studies of human subcomponent Clq before and after limited proteolysis by pepsin. Biochem. J__~., 159, 279-286. Butler, W. T., J. E. Finch Jr., and E. J. M i l l e r (1977) Covalent structure of cartilage collagen. Amino acid sequence of residues 363-551 of bovine e l ( I I ) chains. Biochemistry, 1__6, 4981-4990. Fietzek, P. P., and K. KUhn (1976). The primary structure of collagen. Int. Rev. Conn. Tiss. Res., 2, 1-60. Hall, Z. W. (1973). Multiple forms of acetylcholinesterase and their distribution in endplate and non-endplate regions of rat diaphragm muscle. J__~.Neurobiol., 4, 343-361. Kefalides, N. A. (1968). I s o l a t i o n and characterization of the collagen from glomerular basement membrane. Biochemistry, 7, 3103-3112. Marnay, A., and D. Nachmansohn (f93~. Sur la r~partition de la cholinest~rase dans le muscle couturier de la grenouille. ~ R e n d . Soc. Biol., 125, 41. Massouli~, J., and F. Rieger (1969) L'ac~tylcholinest~rase des organes ~lectriques de poissons ( t o r p i l l e et gymnote); complexes membranaires. Europ. J. Biochem., 1__1, 441-455. Mays, C., and T. L. Rosenberry,(1980). Characterization of pepsin-resistant collagen-like t a i l subunit fragments of 14S and 18S acetylcholinesterase from Electrophorus electricus. Biochemistry, in press. McCann, W. F. X., and T. L. Rosenberry (1977). I d e n t i f i c a t i o n of discrete d i s u l f i d e - l i n k e d oligomers which distinguish 18S from 14S acetylcholinesterase. Arch. Biochem. Biophys., 183, 347-352. McMahan, U. J., J. R. Sanes, and L. M. Marshall (1978). Cholinesterase is associated with the basal lamina at the neuromuscular junction. Nature, 271, 172-174. Nachmansohn, D., and E. Lederer (1939). Sur la biochemie de la cholinesterase. Bull. Soc. Chim. b i o l . Paris, 21, 797-808. Orkin, R. W., P. Gehron, E. B. McGoodwin, G. R. Martin, T. Valentine, and R. Swarm (1977). A murine tumor producing a matrix of basement membrane. J_~.Exp. Med., 145, 204-220. Rosenberry, T. L., and J. M. Richardson (1977). Structure of 18S and 14S acetylcholinesterase. I d e n t i f i c a t i o n of collagen-like subunits that are linked by d i s u l f i d e bonds to catalytic subunits. Biochemistry, 16, 3550-3558. Rosenberry, T. L., Y. T. Chen, and E. Bock (1974). Structure of 11S acetylcholinesterase. Subunit composition. Biochemistry, i__3, 3068-3079.

Collagen-Like Suhunits of Acetylcholinesterase

]47

Rubin, A. L., D. P f a h l , P. T. Speakman, P. F. Davison, and F. O. Schmidt (1963). Tropocollagen: significance of protease-induced interactions. Science, 139, 37-38. Sage, H., and P. Bornstein (1979). Characterization of a novel collagen chain in human placenta and its relation to AB collagen. Biochemistry, 18, 3815-3822. Sanes, J. R., L. M. Marshall, and U. J. McMahan (1978). Reinnervation of muscle f i b e r basal lamina after removal of myofibers. J. Cell Biol.~ 78, 176-198. This research was supported, in part, by U.S. National Institutes of Health Grant NS-03304, by U.S. National Science Foundation Grant PCM77-09383, by the Muscular Dystrophy Association of America, and by the New York Heart Association.