©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Activity of an Endoplasmic Reticulum-localized Pool of Acetylcholinesterase Is Modulated by Heat Shock (*)

(Received for publication, July 11, 1994; and in revised form, October 27, 1994)

Jerry Eichler (§) Israel Silman (¶)

From the Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Primary cultures prepared from embryonic chick pectoral muscle were subjected to heat shock, and the effect on acetylcholinesterase activity in the cultures was examined. A rapid recovery in enzyme activity was observed soon after an initial heat shock-induced drop and was shown to be independent of de novo synthesis of protein, since it could occur in the presence of an inhibitor of protein synthesis. Lectin binding and sucrose gradient centrifugation studies suggested that molecular monomers and dimers found in the endoplasmic reticulum are involved in the observed recovery of acetylcholinesterase activity. Enhanced activation of a pre-existing pool of inactive enzyme was clearly not the main agent of the recovery in enzymic activity. Recovery relied principally on restoration of the activity of previously active, heat-denatured acetylcholinesterase molecules found in the endoplasmic reticulum. Possible agents involved in the recovery of enzymatic activity might be heat shock proteins acting as molecular chaperones.


INTRODUCTION

Acetylcholinesterase (EC. 3.1.1.7, AChE) (^1)occurs in a variety of synapses, where its main role is to terminate impulse transmission by rapid hydrolysis of the neurotransmitter acetylcholine. AChE exists in multiple molecular forms, conveniently classified as either asymmetric (A) or globular (G)(1) . Much information is available concerning the molecular biology, structure, and distribution of these forms of AChE, but little is known regarding the biosynthetic steps involved in their folding and assembly. Most research on AChE assembly has involved use of primary muscle cultures or cell lines(2, 3, 4, 5) . Studies involving recovery of AChE activity after application of irreversible AChE inhibitors or in the presence of heavy-atom precursors(2, 6, 7, 8) suggest that complex forms of AChE are derived from catalytically active simpler forms. Whereas globular dimers and tetramers of AChE are assembled in the endoplasmic reticulum, A forms of the enzyme are assembled in the Golgi apparatus(3, 5) . Not all catalytically active subunits are destined for incorporation into the more complex oligomeric forms; substantial amounts of catalytically active AChE are degraded(4, 5) . The simpler active forms of AChE are, in turn, derived from a pool of inactive precursors. The existence of a large, catalytically inactive pool of subunit monomers and dimers has been demonstrated in cultured chick muscle (4, 9, 10) as well as in other systems(11, 12) . The inactive molecules are, however, organized as monomers and dimers which closely resemble their active counterparts in physicochemical parameters(13) . It has been proposed that such inactive pools become activated by an as yet unidentified mechanism or that much of their content is destined for destruction(2, 4, 5) . It is possible that subpopulations exist within the inactive pool(s) and that both pathways are followed(2) .

The reason for the lack of catalytic activity of the inactive pool of AChE is not known. Although it does not require any covalent modification or processing, AChE does not acquire its catalytic activity immediately after biosynthesis(2, 14) , possibly because it is a large polypeptide, and its active conformation, with its correct arrangement of disulfide bonds, is not immediately realized. Thus, incomplete or incorrect folding may be a possible explanation for the presence of inactive enzyme.

Although the three-dimensional structures of many proteins, including AChE(15) , have been solved, only limited information is available regarding the pathways used by these polypeptides to achieve their native configurations in situ. For many years, it was widely accepted that the information required for the realization of a protein's final structure is contained in its primary structure(16) . However, when one takes into the account the efficiency and time required for protein folding in vitro, it is clear that protein folding and related processes must be directed processes within the cell(17) . Accordingly, the concept of self-folding and assembly has been challenged recently by the identification of molecular chaperones. These are a group of proteins which aid in protein assembly by mediating the folding of polypeptides and, in some cases, assist their assembly into oligomers, yet are not part of the final structure (cf. 18, 19). Chaperones may enhance proper folding, protect against undesired modes of folding, or interact with existing protein aggregates(19, 20) . Among the elements shown to act as chaperones are several heat shock proteins (HSPs), highly conserved proteins expressed by organisms ranging from Escherichia coli to man, in response to heat or other forms of stress(21) . Cells constitutively express proteins highly homologous to the HSPs, and it is assumed that these proteins fulfil chaperone functions under normal growth conditions(22, 23) .

In order to investigate a possible role for HSPs, acting as molecular chaperones, in the folding and assembly of AChE, we examined the effect of heat shock on AChE activity in chick muscle primary cultures. Chick muscle cultures represent the model of choice for such studies since the kinetics of AChE synthesis, degradation, oligomerization and transport are well documented in this system(4, 7, 8) . An initial examination of the effects of heat shock on AChE activity in chick myocultures has already been reported(24) . In the present article, characterization of those AChE molecules affected by heat shock is presented.


EXPERIMENTAL PROCEDURES

Materials

12-day-old, fertilized white Leghorn chick eggs were obtained from Kfar Bilu Hatcheries (Kfar Bilu, Israel). Horse serum and penicillin/streptomycin/neomycin were supplied by Biotek Industries (Jerusalem, Israel) and Biological Industries (Kibbutz Beit Haemek, Israel), respectively, while gelatin was from Difco Laboratories (Detroit, MI). Anti-proteolytic agents (aprotinin, bacitracin, benzamidine, EDTA, leupeptin, pepstatin A), bovine serum albumin, catalase, cycloheximide, diisopropylfluorophosphate (DFP) and 5,5`-dithiobis-(2-nitrobenzoic acid) were obtained from Sigma. [^3H]Acetylcholine (ACh) iodide, [^3H]DFP, and Na[I] came from Amersham (Buckingham, United Kingdom). Concanavalin A (ConA)-Sepharose, ConA-agarose, and underivatized Sepharose beads were from Pharmacia (Uppsala, Sweden), while wheat germ agglutinin (WGA)-agarose and ricin I agglutinin (RCA)-agarose came from BioMakor (Rehovot, Israel). All other reagents used were of analytical grade.

Preparation, Maintenance, and Heat Shock of Chick Muscle Cultures

Primary pectoral muscle cultures were prepared from 12-day-old chick embryos as described previously(24) . In some cases, cultures were subjected to a 45 °C heat shock by replacing the culture medium with Dulbecco's modified Eagle's medium containing 10% horse serum, preheated to 45 °C, followed by transfer to an incubator set at that temperature. 45 min later, culture medium was replaced with 37 °C medium containing 10% horse serum and 100 µg/ml of cycloheximide, an inhibitor of protein synthesis(7) , and the cultures were returned to a 37 °C incubator.

Determination of AChE Activity in Myocultures

Cultures were washed with 3 times 5 ml of cold Hank's balanced salt solution (HBSS), on ice, to reduce AChE secretion. Cells were scraped, collected, and pelleted. The pellet was homogenized in a glass homogenizer in 100 µl of AChE extraction buffer (1 M NaCl, 1% Triton X-100, 10 mM EDTA, 0.1 mg/ml bacitracin, 1 mM benzamidine, 1.1 unit/ml aprotinin, 10 mM Tris, pH 7.0). The homogenate was centrifuged (100,000 times g, 10 min) in a Beckman airfuge. AChE activity in the supernatant was determined radiometrically (see below). Inhibition of myoculture AChE was achieved by first washing the cultures with 3 times 5 ml of HBSS on ice and incubating with 10M DFP in HBSS, at room temperature, for 20 min. Under such conditions >95% inhibition of AChE activity is achieved(6, 7) . The cultures were then rinsed with 4 times 10 ml of HBSS to remove free DFP. Medium was reapplied to the cultures, which were then returned to the incubator.

Lectin-binding Studies

6-day myocultures were subjected to a 45-min heat shock, returned to 37 °C in the presence of 100 µg/ml cycloheximide, and then extracted at 5-10-min intervals in a volume of 400 µl of borate extraction buffer (1 M NaCl, 1 mg/ml bovine serum albumin, 1% Triton X-100, 2 mM benzamidine, 1 mg/ml bacitracin, 1.1 unit/ml aprotinin, 5 mMN-ethylmaleimide, 20 mM sodium borate, pH 9.0)(3) . For lectin binding studies, 100-µl aliquots were incubated with 40 µl of ConA, WGA, or RCA conjugated to either Sepharose or agarose beads or with underivatized Sepharose beads, all previously washed in AChE extraction buffer, together with 1 mM CaCl(2) and 1 mM MnCl(2), in a final volume of 200 µl. The mixture was rocked gently overnight at 4 °C, following which the beads were precipitated and AChE activity remaining in the supernatant was determined radiometrically.

Detection of Inactive AChE

The presence of inactive AChE molecules was detected using an immunoradiometric assay, as described previously(13) . Briefly, purified mAbs (10 mg/ml) were nonspecifically adsorbed to the walls of plastic tubes (Immuno Maxisorp Startube, Nunc) by incubation in 50 mM potassium phosphate, pH 7.0, for 5 h at room temperature or overnight at 4 °C (final volume 200 µl). The immobilized antibody was either mAb C-131, which retains only molecules containing active AChE subunits, or mAb C-6, which retains all AChE molecules, including those lacking catalytic activity(10) . Nonspecific binding sites were saturated by 1 ml of coating buffer (0.1% bovine serum albumin, 0.4 M NaCl, 0.1% Triton X-100, 0.01% NaN(3), 50 mM potassium phosphate, pH 7.0). Before use, the tubes were rinsed with 1 ml of coating buffer. The tubes were then incubated with 100 µl of the analyzed fraction, together with 100 µl of a saturating concentration of a second I-labeled purified monoclonal antibody, one which recognizes both active and inactive AChE species, in coating buffer, for 6 h at RT or overnight at 4 °C. Next, the tubes were washed twice with 1 ml of rinse buffer (0.05% Tween 20, 50 mM potassium phosphate, pH 7.0) and radiometrically counted to determine the level of AChE protein present. Solid-phase bound AChE activity was revealed by addition of 1 ml of Ellman's reagent to the tubes(25) , which were then vigorously agitated. AChE activity was then determined as described below.

Determination of AChE Activity

AChE activity was determined either colorimetrically on ATCh (25) or radiometrically on [^3H]ACh(26) . The reaction mixture for the Ellman determination contained 1 mM 5,5`-dithiobis-(2-nitrobenzoic acid), 0.01% gelatin, 100 mM Tris-HCl, pH 7.6 and 0.5 mM ATCh. The reaction mixture for the radiometric assay contained 3 mM [^3H]ACh, 1 mM MgCl(2)/100 mM NaCl, 50 mM Tris-HCl, pH 7.4.

Sedimentation Analysis of AChE Molecular Forms

Cell extracts (100 µl) were deposited onto 5-20% (w/v) sucrose gradients prepared in 1 M NaCl, 50 mM MgCl(2), 1% Triton X-100, 10 mM Tris-HCl, pH 7.0 and centrifuged using a SW41 rotor in a Beckman L8-70 ultracentrifuge (38,000-40,000 revolutions/min, 21 h, 4 °C)(27) . Sedimentation coefficients were determined by comparison with beef liver catalase (11.4 S), included in the gradients as an internal standard. 200-µl fractions were collected and tested for enzymic activity colorimetrically (see above).


RESULTS

Recovery of AChE Activity Lost in Heat Shock Does Not Require De Novo Protein Synthesis

Results presented in an earlier study showed that both the transient reappearance of AChE activity during prolonged heat shock, and the permanent recovery subsequent to transient heat shock, occurred faster than might be expected from the rates of de novo synthesis(24) . These experiments could not, however, exclude some contribution of de novo synthesis to the reappearance. To examine this possibility, 6-day-old chick pectoral muscle cultures were subjected to a 45 °C heat shock for 45 min, and then returned to 37 °C, in the presence of 100 µg/ml cycloheximide, an inhibitor of protein synthesis. At 5-min intervals, plates were extracted and their AChE content determined radiometrically. As shown in Fig. 1, over 30% of the activity found in control cultures was lost as a result of heat stress. Activity remained at this reduced level for 10 min, after the cultures had been returned to 37 °C. Within the next 5 min, a rapid recovery in AChE activity was observed. In fact, immediately after reversal of the loss in AChE activity induced by heat shock, the level of activity transiently exceeded, by 10%, that found in control cultures not exposed to heat stress. Cultures experiencing a constant heat stress of 45 °C in the presence of cycloheximide were able to maintain their AChE activity levels for the first 20 min, after which a rapid loss of activity occurred (not shown).


Figure 1: The AChE activity of chick myocultures can recover from heat shock in the absence of de novo protein synthesis. 6-day-old chick myocultures were incubated for 45 min at 45 °C, transferred to a 37 °C environment in the presence of 100 µg/ml cycloheximide, and then extracted at 5-10-min intervals. The AChE activity at each time point was determined radiometrically and expressed as a percentage of the AChE activity of control cultures. Each point represents the average of three to seven plates ± S.E., collected in two to three experiments.



Lectin-binding Properties of AChE in Heat-shocked Myocultures

In order to characterize the population of AChE molecules involved in the rapid recovery of activity observed subsequent to a 45-min heat shock, the lectin binding ability of the AChE content of the cultures was examined at 5-min intervals. It had previously been shown that in myocultures, glycosylation of AChE molecules and, consequently, recognition by the appropriate lectins, occurs in a stepwise manner, reflecting transit of the enzyme through the secretory machinery(3) . Recognition by ConA is achieved in the endoplasmic reticulum (ER), while WGA only binds to AChE that has passed into the cis-Golgi, and RCA only recognizes enzyme which has passed into the trans-Golgi(3) . Accordingly, these lectins were used to determine the extent of processing and hence the location, within the secretory pathway, of the AChE molecules involved in the observed recovery from heat shock. The results of these experiments are presented in Fig. 2A. At all time points tested, ConA-Sepharose was able to precipitate over 80% of the AChE activity present. Within the first 15 min after removal of heat stress, however, both WGA and RCA were able to precipitate a higher percentage of AChE activity than under control conditions (control levels corresponding on average to 35% in the case of WGA and 20% in the case of RCA). After 15 min, corresponding to the period of time needed for recovery of AChE activity, binding by these lectins returned to or dropped slightly below control levels. Thus, during the period of elevated binding of AChE activity by WGA and RCA, corresponding to the period of heat shock-induced loss of AChE activity, enzyme that had passed into the Golgi apparatus and had acquired complex glycosylation accounted for a larger proportion of the AChE content of the cultures than is the case in control cultures. Recovery of AChE activity was accompanied by a restoration of binding of WGA and RCA to control levels. This return of WGA and RCA binding to normal levels is due to the appearance of a pool of AChE molecules which have yet to undergo complex glycosylation, and would not, therefore, be recognized by either WGA or RCA. This suggests that the AChE molecules affected by heat shock and subsequently responsible for the observed recovery of activity are found in the ER. Indeed, if the levels of activity precipitated by the various lectins immediately after heat shock and after return of AChE activity to control levels (15 min later) are expressed in terms of units of activity rather than as percentages of control levels, the increased contribution of ConA-binding, ER-localized AChE molecules to the total enzymatic activity of the cultures can be seen clearly (Fig. 2B).


Figure 2: Lectin-binding capacity of AChE in heat-shocked chick myocultures. A, 6-day-old myocultures were subjected to a 45-min heat shock, returned to 37 °C in the presence of 100 µg/ml cycloheximide, and then extracted at 5-10-min intervals. Aliquots were incubated with ConA, WGA, or RCA conjugated to either Sepharose or agarose beads, or with underivatized Sepharose beads as described under ``Experimental Procedures.'' After overnight incubation, the beads were precipitated, and the AChE activity remaining in the supernatant was determined radiometrically. The bound activity in the lectin-exposed samples is expressed as a percentage of the activity found in the supernatants of samples incubated with underivatized Sepharose beads, and represents the average of three to six samples ± S.E. In addition, although not included in the figure, the AChE activity found in the cultures at each time point was also determined. The arrows on the right correspond to the percentage of soluble activity remaining after precipitation by WGA (upper arrow) and RCA (lower arrow). B, the AChE activities bound by ConA, WGA, and RCA after a 45-min heat shock and 15 min later, upon recovery of AChE activity to control levels, were determined by subtracting the activity remaining in the soluble fraction of lectin-exposed samples from the activity found in the supernatant of samples exposed to underivatized Sepharose beads at each time point. Values represent the average of three cultures.



To further support the hypothesis that the pool of AChE involved in the recovery from heat shock had yet to transit the Golgi complex, cultures were first treated with DFP, an organophosphate which irreversibly binds to the enzyme's active site, thereby irreversibly inhibiting almost all (>95%) pre-existing AChE activity(6, 7) . Since de novo recovery of AChE activity following DFP inhibition occurred at a rate of 30%/h, such an approach permitted monitoring of movement through the secretory pathway of AChE synthesized de novo(4, 24) . After removal of excess DFP, cultures were allowed to recover for 30 min and subjected to heat shock for 35 min. After return to a 37 °C environment, in the presence of cycloheximide, the activity and lectin-binding properties of the AChE in the cultures were determined at 5-min intervals. It had been previously shown that, from the onset of their biogenesis, AChE molecules in chick muscle cultures require 90 min for transport to the trans-Golgi, where they acquire glycosylation that can be recognized by RCA(4) . Therefore, during the 65 min from the onset of de novo synthesis until extraction of enzyme was performed, the newly synthesized AChE molecules had yet to reach the trans-Golgi. This was confirmed by lectin precipitation experiments, which showed that RCA binding only occurred 25 min later or 90 min after the onset of de novo AChE synthesis. ConA was able to recognize AChE at all time points after heat shock. Determination of the enzymic activity of the cultures subsequent to heat stress showed the characteristic recovery of activity soon after their return to 37 °C (Fig. 3). This experiment supports the thesis that the AChE molecules responsible for recovery of activity after heat shock have yet to transit the Golgi or to achieve their final glycosylation. Again, this supports our contention that these molecules are found in the ER.


Figure 3: Lectin-binding behavior of AChE in DFP-treated, heat-shocked chick myocultures. The AChE activity of 6-day-old chick myocultures was blocked by incubation with 10M DFP. After washing away unbound DFP, the cultures were allowed to synthesize new AChE molecules for 30 min at 37 °C. They were then subjected to 45 °C heat shock for 35 min, followed by transfer to 37 °C medium containing 100 µg/ml cycloheximide. The cultures were then extracted and processed for lectin binding and determination of activity as described in the legend to Fig. 2. AChE activity (open circles) is expressed as a percentage of the activity associated with cultures which had been exposed to DFP and then returned to a 37 °C environment. The bound AChE activities in the samples exposed to ConA (closed circles) and RCA (closed squares) are expressed as percentages of the activity found in samples incubated with underivatized Sepharose beads and represent the averages for three samples ± S.E.



Molecular Forms of AChE Affected by Heat Shock

Sucrose density gradient centrifugation was employed to determine which molecular forms of AChE in chick muscle cultures were affected by heat shock (Fig. 4). Fig. 4, A and B, shows profiles of AChE activity after heat shock and after recovery from heat shock, respectively. Heat shock leads to a decrease in the activity of the G(1) and G(2) molecular forms, whereas G(4)-AChE is not substantially affected by heat shock (control not shown). Recovery from heat shock leads to an increase in the activities of the monomeric and dimeric forms of the enzyme (compare Fig. 4A and B). Indeed, integration of the areas under the peaks corresponding to the G(1) and G(2) molecular forms from both heat-shocked and recovered cultures shows that the activities associated with these molecular forms had increased 30 and 45%, respectively, whereas the G(4) peak had increased only 6%. Furthermore, the profile of enzyme activity observed after recovery from heat shock resembled that of control cultures (not shown), showing that no redistribution of molecular forms of AChE had occurred as a result of heat shock.


Figure 4: G(2) and G(1)-AChE are affected by heat shock. 6-day-old chick myocultures were subjected to heat shock, after which the cultures were either directly extracted, or transferred to 37 °C in the presence of 100 µg/ml cycloheximide, and extracted after AChE activity had returned to control levels. The extracts were separated on 5-20% sucrose gradients, and the AChE activity of 20 µl aliquots was determined colorimetrically. A, AChE activity profile of a culture extracted after heat shock. B, AChE activity profile of a culture extracted after recovery from heat shock. The position of catalase (11.4 S), included as an internal marker, is shown by an arrow.



Effect of Heat Shock on Inactive AChE Molecules

The recovery in AChE activity observed subsequent to heat shock could represent recovery of the activity of mature existing enzyme lost as a result of the heat stress; alternatively, this recovery might reflect heat shock-induced activation of a pool of inactive AChE molecules, since, as shown in Fig. 1, de novo synthesis is not involved in the restoration of AChE activity. Pools of inactive AChE molecules have been described in various systems, including chick myocultures, where they were localized to the ER(4, 13) . To determine whether activation of inactive AChE was responsible for the observed recovery from heat shock-induced loss of AChE activity, the following experiment was performed. Cultures were heat-shocked in the presence of DFP and allowed to recover at 37 °C in the presence of cycloheximide. In this protocol, >95% of pre-existing AChE activity was inhibited, and de novo synthesis of new AChE molecules subsequent to heat shock should be prevented. Thus, any AChE activity detected should be due to the activation of pre-existing inactive AChE molecules. Determination of AChE activity subsequent to removal of the heat stress revealed a slight increase in enzymatic activity, corresponding to 10% of that present in normal cultures (not shown). Heat shockinduced activation of pre-existing inactive AChE molecules thus appeared to be insufficient to account for the recovery of enzymatic activity observed following removal of heat stress. These results were confirmed in a second experiment in which cultures were again subjected to heat stress in the presence of DFP. This time, however, the cultures were allowed to recover at 37 °C in the presence of cycloheximide together with [^3H]DFP. In this way, any newly activated AChE would incorporate the radiolabeled DFP. The level of radioactivity associated with cultures which had experienced heat shock was not significantly different from that of the appropriate control (not shown).

These experiments cannot, however, discount the possibility that inactive as well as active AChE molecules bind DFP. If this were the case, the full extent of heat stress-induced activation of inactive AChE would be masked, due to binding of DFP to inactive AChE. Accordingly, a second approach was employed to examine the response of inactive AChE to heat shock: use of an immunoradiometric assay capable of distinguishing between active AChE molecules and total AChE protein (13) . The relative contributions of inactive AChE to the total enzyme pool were compared in control cultures which had never experienced heat shock and in cultures which had been allowed to recover from heat shock in the presence of cycloheximide (Fig. 5). In this way, the possible involvement of inactive AChE molecules in the observed recovery of activity subsequent to heat shock was examined, independent of inhibitor binding properties. Recovery from heat shock was accompanied by 10% decrease in the relative amount of inactive AChE, presumably due to activation of AChE in the inactive pool. The magnitude of this presumed activation appears, however, to be insufficient to account for the post-heat shock recovery in AChE activity.


Figure 5: The effect of heat shock on the proportion of inactive AChE in chick myocultures which have recovered from heat shock. Extracts obtained from 6-day-old myocultures grown either under control conditions, or subsequent to a recovery from a 45 min heat stress in the presence of cycloheximide, were analyzed on 5-20% sucrose gradients, as described above. In each fraction, the level of AChE activity was determined colorimetrically, while the amount of bound radiolabeled antibody was used to quantitate total AChE protein levels, as described under ``Experimental Procedures.'' Shown are the profiles obtained with mAb C-6, able to recognize both active and inactive chick AChE species. Closed circles, AChE activity; open circles, AChE immunoreactivity. A, control cultures; B, cultures which had recovered from heat shock. The position of catalase (11.4 S), included as an internal marker, is shown by an arrow.



A Priming Heat Shock Delays Loss of AChE Activity in Response to a Subsequent Prolonged Heat Shock

The effect was examined of a priming heat shock, applied 24 h before a maintained heat stress, on the AChE activity of chick myocultures. A priming heat shock is an exposure to elevated temperatures for a period of time sufficient to elicit the production of heat shock proteins, yet insufficient to cause heat shock(28) . Cultures were subjected to a 45 °C heat shock for 30 min, and then returned to a 37 °C environment. 24 h later, the cultures were challenged with a maintained 45 °C heat stress. As shown in Fig. 6, the characteristic loss of AChE activity observed upon prolonged heat stress could be delayed for 60 min.


Figure 6: Loss of AChE activity is delayed in thermotolerant chick myocultures. 5-day-old chick myocultures were subjected to a 30 min, 45 °C heat stress and then returned to a 37 °C environment. 24 h later, these cultures were exposed to a prolonged 45 °C heat shock, as were non-thermotolerant cultures. AChE activity of the cultures was assayed at 20-min intervals and is expressed as a percentage of the activity of control cultures, taken as 100%. Each point represents the average of 9-12 cultures ± S.E., collected over three to six experiments.




DISCUSSION

For many years, it was widely accepted that the information required for realization of a protein's final structure is contained in its primary structure(16) . However, this concept of self-folding and assembly has recently been challenged by the identification of molecular chaperones which aid in protein assembly by mediating the folding of polypeptides and, in some cases, assist their assembly into oligomers(17, 18, 19) . Members of the chaperone family include heat shock proteins, a group of proteins expressed in response to various stress stimuli(21, 29) . Due to its large subunit size, different activation states, and complex oligomeric diversity, it is reasonable to assume that AChE might enlist the aid of chaperones at various stages of its folding and/or assembly. The effect of heat shock on AChE activity in chick muscle cultures was, therefore, investigated. Previous work showed that when the AChE activity of chick muscle cultures was examined under conditions of maintained or transient heat shock, activity decreased initially, but the cultures subsequently recovered a substantial percentage, if not all, of the AChE activity lost. This recovery could not be accounted for solely by synthesis of new AChE molecules(24) . The present report confirms that recovery occurred even in the absence of protein synthesis and describes the population of enzyme molecules involved in the post-heat shock recovery of activity.

Lectin-binding studies were undertaken to define the intracellular localization of the enzyme molecules involved in the recovery of activity following heat shock. Such studies take advantage of the fact that nascent glycoproteins, such as AChE, are processed in an orderly fashion, beginning with cotranslational addition of high-mannose asparagine-linked oligosaccharides in the ER, followed by a step-wise modification of the oligosaccharide chains in the Golgi apparatus(3, 30) . The presence of the various sugars, as detected using the appropriate lectins, reflects the intracellular localization of a given pool of AChE molecules. When such an approach was employed here, it was shown that the AChE molecules affected by heat shock are found in the ER or possibly in the early Golgi complex. Previous studies have shown that the ER and early Golgi represent those compartments where most of the decisions are made concerning the fate of a given AChE molecule, in terms of activation, addition of non-catalytic subunits, oligomerization, and degradation(4, 9, 10, 31) . Sucrose density gradient experiments showed that the molecules responsible for the observed recovery in AChE activity exist as catalytic subunit monomers and dimers.

The mechanism underlying the recovery of AChE activity is unknown. Recovery depends on the presence of intact intracellular structural elements, as clearly demonstrated by the fact that if extracts of cultures are exposed to elevated temperatures, AChE activity falls rapidly and no recovery is observed (not shown). Furthermore, purified chick AChE heated to 45 °C loses its catalytic activity rapidly and irreversibly (not shown). Thus, the trivial explanation of spontaneous refolding and reactivation of heat-denatured AChE seems unlikely.

The reappearance of AChE activity might be due to a heat shock-enhanced activation of the large pool of catalytically inactive AChE molecules previously described in chick myocultures(4, 13) . It has been suggested that inactive AChE arises as the result of an alternate folding pathway(10) . This has been indeed shown to be the case for human insulin-like growth factor 1, where two thermodynamic ground-state in vitro folding products can appear(32) . Thus, it is possible that heat shock-induced denaturation of AChE may offer both denatured, previously native and previously inactive enzyme species an opportunity of being refolded into the active conformation. The effect of heat shock on catalytically inactive AChE molecules was examined by two approaches. In the first, the existing population of active AChE was irreversibly inhibited by DFP treatment. Thus, upon recovery from heat shock in the presence of a blocker of protein synthesis, the contribution of newly activated pre-existing AChE could be detected. While some increase in activity was observed, it was inadequate to account for the recovery of AChE activity observed subsequent to heat shock. This experimental approach is based on the assumption that only active AChE subunits bind DFP. This may not, however, be the case. Indeed, it was reported that both active and inactive chicken AChE catalytic subunits bind the organophosphate active site inhibitor, MPT(10) , and Stieger et al.(11) reported earlier that inactive Torpedo AChE could bind [^3H]DFP, but not edrophonium. Such observations could be explained by assuming that putative misfolded or immature AChE molecules are not totally inactive, but rather possess a very low catalytic activity and are capable of carrying out the phosphorylation reaction involved in covalent binding of the organophosphate inhibitors (10) . Accordingly, a second approach was employed to examine the response of inactive AChE to heat shock. The relative contribution of inactive AChE to the total enzyme pool was examined in control cultures and in cultures which had recovered from heat shock in the presence of cycloheximide. Recovery from heat shock was accompanied by a modest decrease in the relative amount of inactive AChE, presumably due to activation of part of the inactive pool. Possibly due to differences in myoculture preparation, the proportion of inactive AChE protein in control cultures was somewhat higher in the present experiments than was previously reported(13) . Nonetheless, the magnitude of this activation could not account for the post-heat shock recovery of AChE activity. Thus, two different experimental approaches show that heat shock leads to only a minor enhancement of the activation of inactive AChE. This small degree of activation might, however, account for the transient increase of 10-20% above control levels of AChE activity observed either upon transfer of the chick myocultures to 45 °C (24) , or during recovery at 37 °C in the presence of cycloheximide. It might also explain the slightly enhanced rate of reappearance of AChE activity observed at 45 °C subsequent to DFP treatment(24) . Walker and Wilson (14) reported a similar increase in AChE activity in chick myocultures treated with cycloheximide 4 h after DFP inhibition and attributed this increase to an activation step. Thus, it appears that during restoration of AChE activity subsequent to reduction in activity by various treatments, chick muscle cultures overcompensate; as a result, higher than normal levels of AChE activity appear transiently. Nevertheless, heat shock-enhanced activation of pre-existing inactive enzyme is clearly not the sole or principal route by which the observed recovery in AChE activity is achieved.

An alternate explanation for the recovery of activity might be the rescue of heat-denatured, inactivated AChE molecules. The obvious candidates to serve as agents in this rescue would be HSPs, acting as molecular chaperones. Indeed, HSPs have been previously shown to protect or rescue proteins from thermal inactivation(33, 34) . Several observations offer indirect support for an involvement of HSPs in the rapid recovery of AChE activity observed subsequent to heat shock. It was observed that de novo protein synthesis during the period of exposure to elevated temperature is essential for recovery of activity and that the observed recovery of enzymatic activity does not occur in heat-shocked culture lysates. Furthermore, the time required for enhanced HSP expression in response to heat stress corresponds to the period of time required for recovery of AChE activity(24) . Finally, the notion that heat-shocked cultures are able to modulate the activity of their existing pool of active AChE molecules through changes brought about by heat shock is supported by the observation that thermotolerant cultures are able to delay the heat shock-mediated loss in AChE activity. HSPs are believed to serve as the mediators of thermotolerance(28) .

It remains to be demonstrated that HSPs recognize heat-denatured AChE or are involved in the restoration of enzymatic activity subsequent to heat shock. Recovery of enzymic activity does not seem to involve either de novo AChE synthesis or spontaneous, unassisted refolding of heat-denatured AChE. While enhanced to some degree, activation of pre-existing catalytically inactive AChE does not seem to make a major contribution to the recovery in activity observed. Thus, refolding assisted by a molecular chaperone-like protein such as a HSP remains an attractive possibility.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry, Dartmouth Medical School, Hanover NH 03755-3844.

To whom correspondence and reprint requests should be addressed: Dept. of Neurobiology, Weizmann Institute of Science, Rehovot, Israel. Tel.: +972-8-343649; Fax: +972-8-344131.

(^1)
The abbreviations used are: AChE, acetylcholinesterase; ACh, acetylcholine; ATCh, acetylthiocholine; ConA, concanavalin A; DFP, diisopropylfluorophosphate; ER, endoplasmic reticulum; G-AChE, globular acetylcholinesterase; HBSS, Hank's buffered salt solution; HSP, heat shock protein; RCA, ricin I agglutinin; WGA, wheat germ agglutinin; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We thank Dr. Richard Rotundo for valuable discussions and for critical reading of the manuscript and Drs. Jean Massoulié and Jean-Marc Chatel for their generous gift of monoclonal antibodies. The technical assistance of Lilly Toker is gratefully acknowledged.


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