(Received for publication, January 30, 1997, and in revised form, April 15, 1997)
From the Research Laboratory of Resources Utilization, R-1, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226, Japan
Chaperonin from Thermus thermophilus (Tcpn6014· Tcpn107) splits at the plane between two Tcpn607 rings into two parts in a solution containing ATP and K+ (Ishii, N., Taguchi, H., Sasabe, H., and Yoshida, M. (1995) FEBS Lett. 362, 121-125). When Escherichia coli GroEL14 was additionally included in the solution described above, hybrid chaperonins GroEL7·Tcpn607 and GroEL7· Tcpn607·Tcpn107 were formed rapidly (<20 s) at 37 °C. The hybrid was also formed from Tcpn6014 and GroEL14 but not from a mutant GroEL14 lacking ATPase activity. The hybrid formation was saturated at ~300 µM ATP and ~300 mM K+. These results imply that GroEL14 also splits and undergoes a heptamer exchange reaction with Thermus chaperonin under nearly physiological conditions. Similar to parent chaperonins, the isolated hybrid chaperonins exhibited ATPase activity that was susceptible to inhibition by Tcpn107 or GroES7 and mediated folding of other proteins. Once formed, the hybrid chaperonins were stable, and the parent chaperonins were not regenerated from the isolated hybrids under the same conditions in which the hybrids had been formed. Only under conditions in which GroEL in the hybrids was selectively destroyed, such as incubation at 70 °C, Thermus chaperonin, but not GroEL14, was regenerated from the hybrid. Therefore, the split reaction may not be an obligatory event repeated in each turnover of the chaperonin functional cycles but an event that occurs only when chaperonin is first exposed to ATP/K+.
GroEL14 is an Escherichia coli chaperonin that facilitates the proper folding of proteins in an ATP-dependent manner (1-3). As determined by x-ray crystallography, GroEL14 seems to be a hollow cylinder of 14 identical 57-kDa subunits consisting of 2 heptamer rings stacked back-to-back with a dyad symmetry (GroEL7·GroEL7) (4). GroEL14 functionally cooperates with co-chaperonin GroES7 (5), a single heptameric ring of 10-kDa subunits that binds one end, or two ends in some cases, of the GroEL14 cylinder (6-8). From a thermophilic bacterium, Thermus thermophilus, GroEL homolog Tcpn601 (Thermus chaperonin 60) is purified as a complex with GroES homolog Tcpn10. The complex, termed Thermus holo-chaperonin (T.holo-cpn), is composed of two heptameric rings of Tcpn60 and a single ring of Tcpn107 (9-12). In contrast to chaperonins from E. coli and T. thermophilus, several members of the chaperonins, including those from Thermoanaerobacter brockii (13, 14) and mitochondria (15, 16), are purified as a single heptameric ring. In addition, purified chaperonin from Paracoccus denitrificans is a mixture of a large number of tetradecamers and a small number of heptamers (10). The physiological meaning of such a divergence in the quarternary structure of chaperonin has not been understood.
Recently, we found that when T.holo-cpn is incubated with ATP and K+, it splits into two parts at the equator plane between the two rings of Tcpn607, producing cone-shaped particles (Tcpn607·Tcpn107) and ring-shaped particles probably corresponding to Tcpn607 (17). Then we observed that the products of the split reaction can reassociate to form T.holo-cpn under the appropriate conditions (18). In contrast to the split reaction, Todd et al. (14) reported that the single-ring T. brockii cpn607 dimerizes to form a double-ring structure in the presence of T. brockii cpn107 and ATP. These results raise the question of whether GroEL14 also undergoes the tetradecamer-heptamer transition.
Here, we report that when GroEL14 and T.holo-cpn are incubated with ATP and KCl, hybrid chaperonins such as GroEL7·Tcpn607·Tcpn107 are formed as a result of the heptamer exchange reaction. This suggests that ATP/K+-dependent transient dissociation of a tetradecamer into heptamers occurs not only in Thermus chaperonin but also in GroEL.
Isopropylmalate dehydrogenase (IPMDH) from T. thermophilus strain HB8 was a kind gift from Dr. T. Oshima and his colleagues (Tokyo University of Pharmacy and Life Science, Hachioji, Japan) (19). (2R*,3S*)-3-Isopropylmalic acid, a substrate of IPMDH, was purchased from Wako Pure Chemical Corp. T.holo-cpn was purified as described previously (9, 20). Tcpn6014 expressed in E. coli was purified using modifications of procedures described previously (12). The lysate of E. coli cells containing expressed Tcpn6014 was heated at 70 °C for 20 min. The supernatant containing Tcpn6014 was recovered by centrifugation and applied to a hydrophobic interaction chromatography (Butyl-Toyopearl). The fractions containing Tcpn6014 were pooled and concentrated by ammonium sulfate precipitation. The concentrated protein solution was applied to a gel permeation HPLC column (G3000SWXL; Tosoh) equilibrated with 25 mM Tris-HCl, pH 7.0, 100 mM Na2SO4, and 20% (v/v) methanol. The fractions containing Tcpn6014 were further purified by a DEAE-5PW HPLC column. Tcpn107 expressed in E. coli was purified as described previously (12). GroEL14 and GroES7 were purified as described previously (21) from the lysate of E. coli cells bearing the multicopy plasmid pACYC 184 carrying groES-groEL genes, which was a kind gift from Dr. K. Ito (Kyoto University, Kyoto, Japan) (22). A mutant GroELAEX14 (C138S, C458S, C519S, D83C, and K327C) was purified as described previously (21). Purified chaperonins were stored as a suspension in 65% ammonium sulfate at 4 °C.
Formation of Hybrid ChaperoninT.holo-cpn or Tcpn6014 (5 µg) was mixed (final volume, 10 µl) with GroEL14 (5 µg) and incubated at 37 °C for 10 min in Buffer A (25 mM Tris-HCl, pH 7.5, 300 mM KCl, and 5 mM MgCl2) containing 1 mM ATP, unless otherwise indicated. The sample solutions were applied to nondenaturing polyacrylamide gel electrophoresis (6% acrylamide), and electrophoresis was continued for about double the duration of the period required for the leading dye (bromphenol blue) to reach the front of the gel. The protein bands were stained by Coomassie Brilliant Blue. Only the regions of the chaperonin protein bands are shown in the figures.
Isolation of Hybrid ChaperoninThe ammonium sulfate precipitate of the mixture of parent and hybrid chaperonins was solubilized in a minimum volume of Buffer B (25 mM Tris-HCl, pH 7.5, and 5 mM MgCl2) and applied to a Sephadex G-25 (Pharmacia) column to remove the excessive salts. The eluted protein solution was applied to a DEAE-5PW (Tosoh) column equilibrated with Buffer B and eluted with a 0-1.0 M NaCl gradient at 1 ml/min. The chromatography was monitored by absorbance at 280 nm.
ATPase AssayATPase activities were assayed by measuring the amount of produced inorganic phosphate (23). Typically, the reaction was started by the addition of ATP (final concentration, 1 mM) to Buffer A containing 0.88 µM2 GroEL14 or Tcpn6014 and, when indicated, 1.3 µM GroES7 or Tcpn107. The assay solution was preincubated for 10 min at 37 °C before the addition of ATP. The reactions were terminated by the addition of perchloric acid after incubations at 37 °C for 5, 10, 15, and 20 min. The solution was treated with a malachite green reagent, and the absorbance at 630 nm was measured. One unit of activity is defined as the activity that hydrolyzes 1 µmol of ATP/min.
Chaperonin-promoted IPMDH FoldingIPMDH (16.2 µM) denatured in 6.4 M guanidine HCl was diluted 25-fold at 37 °C in Buffer A containing the components indicated in the figure legends. After a 20-min incubation at 37 °C, an aliquot was withdrawn, and the reactivated IPMDH activity was determined as described previously (9).
Other MethodsProtein concentration was determined by the method of Bradford with bovine serum albumin as a standard (24). Proteins were analyzed by polyacrylamide gel electrophoresis either on a 10% polyacrylamide gel in the presence of SDS (SDS-PAGE) or on 6% polyacrylamide gels without SDS (native PAGE) (25). To obtain higher resolution on the native PAGE, electrophoresis was continued for about double the duration of the period required for the leading dye (bromphenol blue) to reach the front of the gel. Gels were stained by Coomassie Brilliant Blue R-250.
After we found the T.holo-cpn split (17), we attempted
to identify the heptameric state of GroEL under various conditions but
had no success. If GroEL14 splits only transiently in its ATPase cycle, we could not detect the heptamer GroEL by the usual methods. Then we used Thermus chaperonin as a trap for
GroEL7, that is, we incubated GroEL14 with
ATP/K+ in the presence of T.holo-cpn and examined whether
the hybrid chaperonin containing GroEL7 and
Tcpn607 was formed. We took advantage of the different
electrophoretic mobility of GroEL14 and T.holo-cpn in
native PAGE (Fig. 1A, lanes 1-5)
to identify the hybrid. As shown in Fig. 1A, lane
6, two closely moving bands appeared between T.holo-cpn and
GroEL14 when they were incubated with ATP/K+.
The NH2-terminal amino acid sequences of the two bands
confirmed that the upper band contained GroEL, Tcpn60, and Tcpn10 and
that the lower band contained GroEL and Tcpn60 (data not shown). Yields of phenylthiohydantoin derivatives from GroEL, Tcpn60, and Tcpn10 (upper band) were generally close to each other, indicating that the
upper and lower bands corresponded to
GroEL7·Tcpn607·Tcpn107 and
GroEL7·Tcpn607, respectively. As
described later, an analysis of isolated hybrid chaperonins supported
the structures described above. For these hybrids to be formed,
heptamer exchange reactions should occur, that is, both T.holo-cpn and
GroEL14 should split into heptamers that then rebind each
other with a random combination into tetradecamers. Hereafter, we term
the hybrid chaperonins as follows:
GroEL7·Tcpn607·Tcpn107, Hybrid
(EL-60-10); and GroEL7·Tcpn607, Hybrid
(EL-60).3
The conditions required for the formation of hybrid chaperonins were
the same as those required for the split reaction of T.holo-cpn (17);
formation was absolutely dependent on ATP and K+, and other
combinations such as adenosine 5-(
,
-imino) triphosphate + K+ (Fig. 1A, lane 7), adenosine
5
-O-(thiotriphosphate) + K+ (data not shown),
ADP + K+ (lane 8), and ATP + Na+
(lane 10) were not effective in generating hybrids. When
free Mg2+ was removed by
trans-1,2-diaminocyclohexanetetraacetic acid, no hybrid was formed
(lane 9). The relatively high concentrations of ATP and
K+ were necessary. The hybrid formation was half-maximal at
~50 µM ATP and ~50 mM K+ and
saturated at ~300 µM ATP and ~300 mM
K+ (Fig. 1, B and C). The addition of
excess Tcpn107 to the reaction mixture resulted in an
increased yield of hybrid chaperonins with a simultaneous decrease of
T.holo-cpn (Fig. 1D, lane 2), but the addition of
GroES7 had only little, if any (lane 3), effect.
The hybrids were formed rapidly. In 1 min (Fig. 1E,
lane 5), hybrids with an amount similar to that formed in 10 min (lane 6) were detected, and a significant amount of
hybrids was formed even in 20 s (lane 4). That is as
fast as a single ATPase turnover in the GroEL catalytic cycle (6).
The ATPase activity of Thermus chaperonin at 37 °C, the
temperature at which hybrid formation was observed, is very low (see Fig. 5); hydrolysis of a single ATP molecule by Tcpn6014
takes ~15 s. Nevertheless, hybrid chaperonin was formed within
20 s. This result means that only the hydrolysis of a single ATP
by Tcpn6014 is sufficient to form the hybrid chaperonins.
This rapid formation of hybrid might be related to the observation that
the formation of a tetradecamer from T. brockii
cpn607 is also very rapid, occurring before all the cpn60
subunits could hydrolyze ATP (14). Although the real reason why the
hybrid was formed in such a short period is not known, one of the
possible explanations is that the initial single turnover by one cpn60
in the tetradecamer might induce a quarternary structural change in the
double ring of cpn607 in the presence of a high
concentration of K+.
Cpn10 Is Not Required for Hybrid Formation
To know whether
cpn10 is required for hybrid formation or not, next we used
Tcpn6014 instead of T.holo-cpn as one of the parent chaperonins. Tcpn6014 was isolated from recombinant
E. coli (12), and we confirmed that Tcpn6014
also split in the presence of
ATP/K+.4 The hybrid chaperonin
was formed between Tcpn6014 and GroEL14 in the
presence of ATP/K+ (Fig. 2, lane
6). Unlike the experiment using T.holo-cpn, only a single band
appeared between the parent chaperonins. As expected, this band was
indeed Hybrid (EL-60), because NH2-terminal amino acid
sequencing showed that the band contained an almost equal amount of
Tcpn60 and GroEL. It is likely that in the experiments described in
Fig. 1, Hybrid (EL-60) was formed at first, and Hybrid (EL-60-10) was
generated next by attaching Tcpn107 to Hybrid (EL-60).
ATP Hydrolysis by GroEL14 Is Essential for Hybrid Formation
In spite of the fact that the condition required for
hybrid formation as described above was the same condition required for the split reaction of Thermus chaperonin (17, 18), there was no direct evidence of a requirement for ATP hydrolysis by
GroEL14 for hybrid formation. To address the question, we
used a GroEL mutant called GroELAEX instead of the wild-type GroEL as a
parent chaperonin (21). GroELAEX is a mutant in which apical and
equatorial domains in the same GroEL subunit can be cross-linked in a
reversible manner (apical-equatorial cross
(X)-link) (21). In the presence of a reducing reagent,
GroELAEX14 retains normal functional activity as a
chaperonin. In contrast, oxidized GroELAEX14, which is
locked in a "closed" conformation by an interdomain disulfide bond,
can bind but not hydrolyze ATP (21). Therefore, the requirement for ATP
hydrolysis of GroEL14 in the hybrid formation would be tested in the presence or absence of a reducing reagent. Note that the
Thermus chaperonins have no cysteine residue (12). Just like
wild-type GroEL14, the hybrid chaperonin was formed from
GroELAEX14 and Tcpn6014 in an
ATP/K+-dependent manner under reducing
conditions (Fig. 3, lane 6). However, the
hybrid was not formed under the oxidizing condition (lane
2), whereas the ATPase activity of GroELAEX14 was
completely blocked (21). Wild-type GroEL14 was able to form
the hybrid with Tcpn6014, irrespective of reducing or
oxidizing conditions (lanes 4 and 8). The
inability of oxidized GroELAEX14 to form hybrid chaperonin
indicates that ATP binding is not sufficient for hybrid formation and
that the occurrence of ATP hydrolysis on GroEL14 is
essential for hybrid formation.
Isolation and Characterization of Hybrid Chaperonins
The
hybrid between GroEL14 and Thermus chaperonins
was separated from the parent chaperonins with anion-exchange HPLC
(Fig. 4, A and B). The hybrid
chaperonin fraction contained Hybrid (EL-60-10) and Hybrid (EL-60)
(see Fig. 7A, lane 5). In a similar manner, Hybrid (EL-60) was also purified (see Fig. 7A, lane
4). The relative staining intensities of the GroEL band and the
Tcpn60 band in SDS-PAGE (Fig. 4, inset, lanes 1 and 2) were almost the same, again confirming that the
hybrid chaperonins consisted of equal molar amounts of each chaperonin
subunit. The molecular sizes of the isolated hybrid chaperonins were
the same or very close to those of the parent chaperonins, because they
were eluted from a gel-permeation HPLC column at the same retention
time as that of GroEL14 (data not shown). When hybrid
chaperonins formed from GroEL14 and T.holo-cpn were
examined by electron micrograph, two kinds of particles,
GroEL14-like rectangular particles and bullet-shaped particles similar to T.holo-cpn, were observed (data not shown). Hybrid
(EL-60) hydrolyzed ATP at 0.09 unit/mg1 at 37 °C (Fig.
5). Because T.holo-cpn and Tcpn6014
hydrolyzed ATP very slowly at 37 °C (~0.002
unit/mg
1), the ATPase activity of Hybrid (EL-60) would be
mainly attributed to hydrolysis by the GroEL7 ring moiety
in the hybrid. The hybrids produced from GroEL14 and
T.holo-cpn, a mixture of Hybrid (EL-60-10) and Hybrid (EL-60),
exhibited ATPase activity at 0.08 unit/mg
1. As reported
for GroEL14 (e.g., Ref. 26), the addition of
GroES7 or Tcpn107 inhibited the ATPase activity
of GroEL14 (35-40% inhibition). The ATPase activity of
the hybrid chaperonins was also inhibited by GroES7 and
Tcpn107 (55-60% inhibition for Hybrid (EL-60), and ~30% inhibition for the mixture of Hybrid (EL-60-10) and Hybrid (EL-60)).
Chaperone Activity of Isolated Hybrid Chaperonins
We examined
the effect of hybrid chaperonins on the folding of IPMDH from T. thermophilus under a condition in which the yield of spontaneous
folding was only ~10% (Fig. 6). The following
experiments were carried out in the presence of ATP. Under this
condition, GroEL14 alone hardly promoted reactivation (less
than 10% reactivation of IPMDH activity), and GroES7 was
required for effective GroEL-promoted folding. Tcpn107 was
as effective as GroES7 in this GroEL-promoted folding
assay. In contrast to GroEL14, Tcpn6014 alone
was able to promote folding of IPMDH (~35%). Further addition of
Tcpn107 increased the yield of reactivation about twice,
whereas the effect of GroES7 was only marginal. Similar to
GroEL14, Hybrid (EL-60) alone had almost no effect on
folding (less than 10%), and the addition of GroES7 or
Tcpn107 was required for effective folding (80-90%). In
the case of the mixture of Hybrid (EL-60-10) and Hybrid (EL-60), the
folding of IPMDH was promoted moderately (~35%) even in the absence
of GroES7 or Tcpn107, probably due to the endogenous presence of Tcpn107. The inclusion of
GroES7 or Tcpn107 in the solution caused
additional promotion of folding (50-60%). Thus, it is clear that both
Hybrid (EL-60) and Hybrid (EL-60-10) are active in promoting protein
folding.
Parent Chaperonins Were Not Regenerated from Isolated Hybrid Chaperonins
The isolated hybrid chaperonins were very stable. After storage at 4 °C for 3 weeks, about 90% of the hybrid chaperonins were still in the hybrid forms (data not shown). To investigate whether the two heptamer rings of hybrid chaperonins could reexchange each other in the presence of ATP/K+, we incubated the hybrid chaperonins with ATP/K+ and analyzed them by native PAGE. As shown in Fig. 7B, regeneration of the parent chaperonins, GroEL14 and T.holo-cpn (or Tcpn6014), was not observed, irrespective of whether Hybrid (EL-60) or Hybrid (EL-60-10) was used as a starting hybrid chaperonin. Further addition of either GroES7 or Tcpn107 did not change the result (data not shown). This result was unexpected, because if the hybrid chaperonin split in the presence of ATP/K+, as observed for Thermus chaperonin, parent chaperonins should be regenerated more or less as a result of random reassociation of heptamers. Then we examined the effects of heat (70 °C for 10 min) or proteinase K treatment on the stability of the hybrid chaperonins. As shown in Fig. 7, C and D, Thermus chaperonin was resistant to both treatments under the conditions tested, whereas GroEL was destroyed. After the isolated hybrid chaperonins were incubated at 70 °C for 10 min, the hybrid chaperonins disappeared completely, and Thermus chaperonin but not GroEL14 appeared (Fig. 7C, lanes 4 and 5). Treatment with proteinase K also gave the same results (Fig. 7D, lanes 4 and 5). These results indicate that only when the GroEL7 moiety of the hybrid is denatured or proteolyzed, survived heptamer rings of Tcpn60 can reassociate to form Tcpn6014 or T.holo-cpn.
In this report, we demonstrated ATP/K+-dependent formation of the hybrid between GroEL14 and Thermus chaperonin. This is a result of the heptamer exchange reaction between both chaperonins. One can argue the possibility that the hybrids are made up from three stacked heptamers (GroEL14·Tcpn607), that is, that GroEL14 simply binds Tcpn607 as a substrate protein. This possibility was excluded from the analysis of molecular size with gel-permeation HPLC, the observation of molecular shape by electron micrograph, and the estimation of the molar ratio of GroEL:T.cpn60 from phenylthiohydantoin derivatives recovered in Edman degradation and from the staining intensity of the bands in SDS-PAGE (Fig. 4, inset). The random incorporation of each cpn60 monomer into the tetradecamers is also very unlikely. If it really happened, numerous protein bands corresponding to the complexes with various combinations of parent chaperonin monomers should have appeared between the bands of parent chaperonins in native PAGE. However, only a single band appeared between parent chaperonins when the hybrid was formed from GroEL14 and Tcpn6014 (Fig. 2). In addition, under the experiment conditions, bands of monomeric GroEL and monomeric Tcpn60 with meaningful staining intensity were not observed in native PAGE (data not shown). Therefore, there is little possibility, if any, that dissociation into monomers occurs before formation of the hybrid.
Hybrid formation is not restricted to the combination of Thermus chaperonins and GroEL and is also observed between Tcpn6014 and chaperonin from P. denitrificans (10) in the presence of ATP and K+.4 In addition, Burston et al. (27) reported that the hybrid between wild-type GroEL14 and mutant GroEL14, called MR1 (mixed ring), is formed at 42 °C in the presence of ATP and K+. These observations suggest that hybrid formation is not an exceptional event but a rather common reaction among chaperonins from several species. Because the conditions for hybrid formation are nearly physiological, hybrids can be formed even in a living cell. Indeed, when we expressed Tcpn6014 in E. coli cells, a part of the chaperonin was purified as a form of hybrid chaperonin that was separated from Tcpn6014 by anion-exchange HPLC (see "Experimental Procedures").4 Furthermore, the observation that single-ring T. brockii cpn60 dimerizes to a tetradecamer in the presence of both adenine nucleotides and cpn10 (14) also implies that there is a heptamer exchange in the bacterium.
Heptamer Exchange Necessitates the Split of GroEL14Unless GroEL14 and Tcpn6014 split into GroEL7 and Tcpn607, hybrid formation is impossible. The ATP/K+-dependent split of Thermus chaperonin has been established, and Tcpn607 can be readily detected and isolated (17). A stable heptameric form of cpn60 has also been isolated from T. brockii (13, 14) and from mitochondria (15, 16). A possible heptameric form of GroEL has been reported by two groups. Mendoza et al. (28) suggested that a GroEL species, possibly heptamers, is detected in the presence of 3 M urea after the addition of unfolded rhodanese. Mizobata and Kawata (29) observed a GroEL species exhibiting decreased light scattering in the presence of less than 1 M guanidine HCl, and they speculated that the species is a heptamer of GroEL (29). We also observed a decrease in light scattering of GroEL14 in response to the addition of ATP.4 The fact that the hybrid is formed between wild-type GroEL14 and mutant MR1-GroEL14 under the appropriate conditions implies that not only mutant MR1-GroEL14 but also wild-type GroEL14 splits into heptamers (27). Therefore, GroEL14 most likely splits, although the final conclusion on the presence of GroEL7 should be reserved until success in isolating the heptameric form of GroEL has been achieved.
However, the Split May Not Be an Obligatory Step in the Functional Reaction Cycle of ChaperoninOnce the hybrid is formed, it is very stable. Parent chaperonins are not regenerated from hybrid chaperonin even in the presence of ATP/K+ (Fig. 7). If the split into heptamers is an obligatory step in the functional reaction cycle of the chaperonin, parent chaperonin should be formed as a result of a heptamer exchange reaction. Two explanations are possible: (a) split reaction occurs only once before the chaperonin starts the first turnover of the functional cycle; or (b) split reaction occurs in each of the reaction cycles, but reassociation always happens to heterologous combinations (GroEL7-Tcpn607) rather than homologous combinations (GroEL7-GroEL7 or Tcpn607-Tcpn607), thus producing the hybrid again. However, the latter possibility is unlikely, if not impossible, because the parent GroEL14 is not regenerated from the hybrid GroEL7·MR1-GroEL7 in the presence of ATP/K+ (27), and it is not easy to assume that wild-type GroEL7 has a much higher affinity to mutant MR1-GroEL7 than it does to wild-type GroEL7.
A requirement for high concentrations of ATP and K+ is also contradictory to the notion that the split is one of obligatory steps in the chaperonin functional cycle. Steady-state ATPase activity of GroEL14 is inversely dependent on K+; it is saturated at ~5 mM K+ in the presence of 50 µM ATP and at ~300 mM K+ in the presence of 2 µM ATP (26, 30). On the contrary, hybrids were formed only when concentrations of both K+ and ATP were high, and the yield of hybrids was saturated at ~300 mM K+ and ~300 µM ATP (Fig. 1, C and D). If either one of the concentrations was reduced, the yield of hybrids decreased, and no hybrid formation was observed at 5 mM K+/1 mM ATP or at 300 mM K+/2 µM ATP. Therefore, the requirement of K+ for hybrid formation is a different phenomenon than the K+ requirement for steady-state ATPase activity. Although the occurrence of the heptamer exchange reaction has been established, understanding of its functional and physiological significance awaits further study.
We thank Drs. K. Ito and Y. Akiyama for the gift of plasmid pKY206 and Dr. T. Oshima for the gift of IPMDH from T. thermophilus.