(Received for publication, July 11, 1994; and in revised form, October 27, 1994)
From the
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.
Acetylcholinesterase (EC. 3.1.1.7, AChE) ()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.
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.
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.
Figure 4:
G and G
-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.
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.
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.
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 [H]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.