From the Department of Biochemistry, Asahikawa Medical College, Asahikawa, 078-8510, Japan
Received for publication, August 29, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Possible roles of the
Lys189-Lys205 outermost loop on the A
domain of sarcoplasmic reticulum Ca2+-ATPase were explored
by mutagenesis. Both nonconservative and conservative substitutions of
Val200 caused very strong inhibition of
Ca2+-ATPase activity, whereas substitutions of other
residues on this loop reduced activity only moderately. All of the
Val200 mutants formed phosphoenzyme intermediate
(EP) from ATP. Isomerization from ADP-sensitive
EP (E1P) to ADP-insensitive EP
(E2P) was not inhibited in the mutants, and a substantially
larger amount of E2P actually accumulated in the mutants
than in wild-type sarcoplasmic reticulum Ca2+-ATPase
at steady state. In contrast, decay of EP formed
from ATP in the presence of Ca2+ was strongly inhibited in
the mutants. Hydrolysis of E2P formed from Pi
in the absence of Ca2+ was also strongly inhibited but was
faster than the decay of EP formed from ATP, indicating
that the main kinetic limitation of the decay comes after loss of ADP
sensitivity but before E2P hydrolysis. On the basis of the
well accepted mechanism of the Ca2+-ATPase, the limitation
is likely associated with the Ca2+-releasing step from
E2P·Ca2. On the other hand, the rate of
activation of dephosphorylated enzyme on high affinity Ca2+
binding was not altered by the substitutions. In light of the crystal
structures, the present results strongly suggest that Val200 confers appropriate interactions of the
Lys189-Lys205 loop with the P domain in the
Ca2+-released form of E2P. Results further
suggest that these interactions, however, do not contribute much to
domain organization in the dephosphorylated enzyme and thus would
be mostly lost on E2P hydrolysis.
Sarcoplasmic reticulum Ca2+-ATPase
(SERCA1a)1 is a 110-kDa
membrane protein and a representative member of P-type ion-transporting ATPases. SERCA1a catalyzes Ca2+ transport coupled with ATP
hydrolysis (Refs. 1 and 2, and for recent reviews, see Refs. 3 and 4).
According to the E1/E2 transport mechanism (Fig.
1) (3-7), the enzyme is activated by the
binding of two Ca2+ ions (E1·Ca2,
steps 1-2) and then autophosphorylated by MgATP to form an
ADP-sensitive phosphoenzyme (E1P, step 3). On formation of
this EP, the bound Ca2+ ions are occluded in the
transport sites. The subsequent isomeric transition to the
ADP-insensitive form (E2P, step 4) will result in a
reduction in affinity and a change in orientation of the Ca2+ binding sites and thus a Ca2+ release into
lumen (step 5). Finally, hydrolysis takes place and returns the enzyme
into an unphosphorylated and Ca2+-unbound form
(E2, step 6). The main kinetic limitation in this cycle is
associated with the mechanism of Ca2+ release before the
hydrolysis of E2P (8, 9). E2P can also be
formed from Pi in the presence of Mg2+ and
absence of Ca2+ by reversal of the hydrolysis of
E2P.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The Ca2+
transport cycle of the
Ca2+-ATPase.
The three-dimensional structure of Ca2+-ATPase with bound Ca2+ (E1·Ca2) and, very recently, the structure without bound Ca2+ and with bound thapsigargin (E2(TG)), were solved by x-ray crystallography at the atomic level (10, 11). The enzyme has three cytoplasmic domains (A, N, and P) that are widely separated in E1·Ca2 and associated in E2(TG). The modeling with a low resolution map of tubular crystals formed with decavanadate (E2V) revealed (10) that three cytoplasmic domains gather to form a most compactly organized single headpiece in E2V (see Fig. 7). Our previous limited and systematic proteolysis experiments showed (12, 13) that E2V is very similar to the Ca2+-released form of E2P in the domain organization and that this EP is the intermediate with the most compactly organized headpiece in the catalytic cycle. The results further indicated that a large motion of the A domain (i.e. rotation by ~90° (10)) and the strong association of the A domain with the P and N domains most likely occur during the isomerization of EP and Ca2+ release and suggested that the stabilization energy provided by intimate contacts between all three cytoplasmic domains in E2P provides energy for moving transmembrane helices and releases the bound Ca2+ ions.
To substantiate such changes in cytoplasmic domain organization and their roles in the Ca2+ transport, it is essential to find the regions and residues involved in the domain-domain interactions and reveal their actual roles in the catalytic steps. The conserved outermost TGES loop (Thr181-Ser184) on the A domain is situated at the interface of the A and P domains in E2V. This loop was previously found to be essential for the isomerization of EP (14) and predicted by iron-catalyzed cleavage with Na+/K+-ATPase to participate in Mg2+ binding with specific residues in the conserved TGDGVND loop (starting from Thr701 with Ca2+-ATPase) on the P domain in E2P and E2 (15). One more outermost loop is located on the outer surface of the A domain in E1·Ca2 but situated at the A-P domain interface in E2V, the Lys189-Lys205 loop that includes the tryptic T2 site (Arg198) (see Fig. 7). We had actually indicated by site-directed chemical modification (16) and mutations (17) as well as by the limited proteolysis (12, 13) that Arg198 comes very close to the phosphorylation site when E2P without bound Ca2+ is formed and that the positive charge of this residue is important for its rapid hydrolysis. These findings suggest that the Lys189-Lys205 loop may have critical roles in domain organization, particularly in the Ca2+-released form of E2P.
In the present study, we therefore further explored possible roles of
the Lys189-Lys205 outermost loop by
mutagenesis and found that Val200 is critical for rapid
processing of E2P, most likely in steps 5 and 6. With the
crystal structures, our results suggest that Val200 confers
appropriate interactions between polar residues on the Lys189-Lys205 loop with those on the P domain
in the Ca2+-released form of E2P and, further,
that these interactions would be mostly lost on the hydrolysis of
E2P.
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EXPERIMENTAL PROCEDURES |
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Mutagenesis and Expression-- Overlap extension PCR (18) was used for the substitution of residues in the Lys189-Lys205 region in rabbit SERCA1a cDNA. The ApaI-KpnI restriction fragments were excised from the PCR products and ligated back into the corresponding region in the full-length SERCA1a cDNA in the pMT2 expression vector (19). The pMT2 DNA was transfected into COS-1 cells by the liposome-mediated transfection method. Microsomes were prepared from the cells as described previously (20). The "control microsomes" were prepared from COS-1 cells transfected with the pMT2 vector containing no SERCA1a cDNA. The amount of expressed SERCA1a was quantified by a sandwich enzyme-linked immunosorbent assay as described previously (21). The expression levels of all the mutants in the microsomes were comparable with those of the wild type.
ATPase Activity--
The rate of ATP hydrolysis was determined
at 25 °C in the presence and absence of 0.5 µM
thapsigargin in a mixture containing 20 µg/ml microsomal protein, 1 µM A23187, 5 mM [-32P]ATP, 7 mM MgCl2, 0.1 M KCl, 50 mM MOPS/Tris (pH 7.0), 0.55 mM CaCl2, and 0.5 mM EGTA. The specific ATPase
activity/mg of expressed SERCA1a protein was calculated from the amount
of expressed SERCA1a and the ATPase activity of expressed SERCA1a,
which was obtained by subtraction of the thapsigargin-sensitive ATPase
activity of the control microsomes from that of the microsomes
expressing SERCA1a. This background level with the control microsomes
was as low as 4% of the activity of microsomes expressing the
wild-type SERCA1a.
Formation and Hydrolysis of EP--
Phosphorylation of SERCA1a
in microsomes with [-32P]ATP or
32Pi and dephosphorylation of
32P-labeled SERCA1a were performed under the conditions
described in the legends to the figures and tables. The reactions were
quenched with ice-cold trichloroacetic acid containing Pi.
Rapid kinetics measurements of phosphorylation and dephosphorylation
were performed with a handmade rapid mixing apparatus (22) and
otherwise as described above. The precipitated proteins were separated
at pH 6.0 by 5% SDS-PAGE according to Weber and Osborn (23). The
radioactivity associated with the separated Ca2+-ATPase was
quantitated by digital autoradiography as described (17). The amount of
EP formed with the expressed SERCA1a was obtained by
subtracting the background radioactivity with the control microsomes.
This background was less than 5% of the radioactivity of EP
formed with the expressed wild-type SERCA1a. The amount of
EP/mg of SERCA1a protein was calculated from the amount of EP thus obtained and the amount of expressed SERCA1a.
Miscellaneous--
Protein concentrations were determined by the
method of Lowry et al. (24) with bovine serum albumin as a
standard. Data were analyzed by nonlinear regression using the program
Origin (Microcal Software, Inc., Northampton, MA). Three-dimensional models were reproduced by the program Swiss-PdbViewer (25).
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RESULTS |
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Effects of Substitutions in Lys189-Lys205
Loop on ATP Hydrolysis--
The specific
Ca2+-ATPase activities of the expressed mutant and
wild-type SERCA1a were determined at 25 °C (Fig.
2). The substitution of
Val200 with Ala resulted in very strong inhibition of the
activity, whereas nonconservative substitutions of Lys189,
His190, Thr191, Pro193,
Val194, Arg198, Ala199,
Gln202, Asp203, Lys204, and
Lys205 caused only slight or moderate reduction, and those
of Asp196, Pro197, and Asn201
caused almost no change or rather a slight increase. Moderate effects
of substitutions of Asp196, Arg198,
Gln202, and Asp203 on the activity were also
reported previously (4, 17, 26), and the results are in essential
agreement. Substitutions of Glu192 and Pro195
caused no loss of function (4). We then focused on Val200,
of which substitutions gave distinguishably strong inhibition, and
substituted with various amino acids including nonpolar and polar
(positively charged, negatively charged, and uncharged) amino acids
(Ala, Ile, Arg, Lys, Asp, Thr, and Gln). The activity was strongly
inhibited in the mutants V200I, V200D, V200T, and V200Q and further
reduced in the mutants V200A, V200R, and V200K (Fig.
3).
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Formation of EP from ATP-- We then performed kinetic analysis on the formation and decay of EP with these Val200 mutants at 0 °C under conditions otherwise similar to those for the ATPase assay. All of the mutants possessed the ability to form EP, and the amount formed was comparable with that of wild type (Table I). As shown in Fig. 4, the time course of E2P formation from ATP was examined in the presence and absence of K+. Under both sets of conditions, the total amount of EP reached its maximum level within 1 s after the addition of ATP and remained unchanged for the time periods of observation (120 s) (data not shown). Thus, the time course actually reflects E2P accumulation from E1P. In the presence of K+, the amount of accumulated E2P at steady state in wild type was very low (Fig. 4A), in agreement with the previous observations on the effects of K+ (27). In contrast, a substantial amount of E2P was accumulated with the mutants, especially V200A and V200I. In the absence of K+, a large amount of E2P accumulated at steady state in wild type as well as in the mutants (Fig. 4B), thus the time courses can be clearly compared. The E2P accumulation apparently proceeded with first-order kinetics, and the apparent rate to reach the steady state (kformation) was estimated (Table II). The rates obtained in the mutants in the absence of K+ were comparable with the rate in wild type, with somewhat lower rates in V200R and V200K. These rates obtained in the mutants are also comparable with their rates in the presence of K+. The results indicate that the E1P to E2P isomerization (i.e. loss of ADP sensitivity) is not significantly inhibited by the Val200 substitutions. Results also suggest that the substitutions (especially in V200A and V200I) affected the equilibrium in step 4 in favor of E2P·Ca2.
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Decay of EP--
Decay of EP formed from ATP (Fig.
5) and hydrolysis of E2P
formed from Pi (Fig. 6) were
then examined at 0 °C under conditions otherwise similar to those
for the ATPase assay (i.e. in the presence of
K+). Decay of EP formed from ATP was determined
first by phosphorylation with [-32P]ATP in the
presence of K+ and Ca2+ for 15 s and then
termination of phosphorylation by the addition of excess EGTA to
prevent further phosphorylation and thus allow the decay of
32P-labeled EP (Fig. 5). The EP decay
was markedly slowed by the Val200 substitutions. The time
courses were apparently fitted very well with a single exponential,
although a substantial amount of E2P (in addition to
E1P) was accumulated in the mutants at the start of the
decay reaction (see Fig. 4A). The results indicate that processing of EP after loss of ADP sensitivity
(i.e. step 5 and/or step 6) was strongly inhibited by the
substitutions. The apparent decay rate (kdecay)
was strongly reduced in V200D, V200Q, V200T, and V200I to ~30-20%
of that of the wild type and was further reduced to ~10% in V200A,
V200K, and V200R (Table II). It should be noted that the extent of
reduction in kdecay agreed well with that of
inhibition of ATPase activity (cf. Table II and Fig. 3). Also, in each mutant the kdecay value is much
lower than the kformation value.
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Hydrolysis of E2P Formed from Pi-- To examine the hydrolysis of E2P without bound Ca2+ (step 6), the enzyme was first phosphorylated by 32Pi without Ca2+ in the absence of K+ and the presence of 35% (v/v) Me2SO, which extremely favors E2P formation (28), and then the phosphorylated samples were diluted with a large volume of a solution containing K+ and non-radioactive Pi (Fig. 6). The conditions were thus made otherwise identical to those used for the E2P formation from ATP in Fig. 4A and the decay of EP formed from ATP in Fig. 5. The hydrolysis of 32P-labeled E2P proceeded with first-order kinetics, and its rate (khydrolysis) was summarized in Table II. The rate was moderately reduced in V200D and largely reduced in V200T and V200Q (8 and 7% that of wild type, respectively). It was further reduced in V200A, V200I, V200K, and V200R (less than 3% that of wild type).
Kinetic Limitation in the Mutants for Decay of EP Formed from ATP-- For the mutants, the above kinetic results show that the decay of EP formed from ATP in the presence of Ca2+ is much slower than the accumulation of E2P from ATP and also that the EP decay is substantially slower than the E2P hydrolysis in the absence of Ca2+ (as is most clearly shown with V200K, V200D, V200T, and V200Q). The results indicate for the mutants that the main kinetic limitation (although not the only one) in the decay of EP formed from ATP comes after loss of ADP sensitivity (step 4) but before E2P hydrolysis (step 6). On the basis of the well accepted mechanism of the Ca2+-ATPase (Fig. 1), it is therefore likely that step 5 associated with Ca2+ release is greatly slowed by the Val200 substitutions and is the main kinetic limitation in the mutants.2
For the mutants, the actual rate of formation of E2P from
E1P on isomerization (kisomerization)
in the presence of K+ can be estimated by assuming the
equilibrium in step 4 without further processing of
EP (because of the subsequent very slow processing as
described above for the mutants). The
kisomerization value thus estimated
for the mutants (s1) by an equation,
kisomerization = kformation × (fraction of E2P at
steady state), was 0.063 (V200A), 0.108 (V200I), 0.011 (V200R), 0.013 (V200K), 0.032 (V200D), 0.036 (V200T), and 0.084 (V200Q). These
values are in fact substantially larger than the
kdecay values (as most clearly shown with V200A,
V200I, V200R, V200T, and V200Q).
Transition from E2 to E1·Ca2-- Ca2+ dependence of phosphorylation with ATP was determined in the presence of K+ at various concentrations of Ca2+ under conditions as described in the legend to Table I and thus similar to those for the ATPase assay. The experiments were performed with the four representative mutants (V200A, V200I, V200R, V200T) and wild-type SERCA1a. The dissociation constant for Ca2+ and Hill coefficient obtained by least square fits for these mutants (0.20-0.29 µM and 1.8-2.0, respectively) were nearly the same as those for wild type (0.27 µM and 1.9, respectively).
To compare the rate of the E2 to
E1·Ca2 transition, the four mutants and wild
type were preincubated in the absence of Ca2+ at pH 6, where equilibrium between E1 and E2 is most
shifted to E2 (30) and then phosphorylated by the
simultaneous addition of saturating concentrations of Ca2+
and ATP under otherwise identical conditions as described above. The
time course of EP formation was well described by the first order kinetics (data not shown). The rates
(s1) obtained with the mutants (0.178 (V200A), 0.122 (V200I), 0.140 (V200R), and 0.204 (V200T)) were almost
the same as or only slightly lower than that of the wild type (0.204).
When ATP was added to the wild-type and mutant enzymes were
preincubated with Ca2+, the EP formation was
much faster and reached its maximal level within 1 s (data not
shown). These results show that the Ca2+-induced
E2 to E1·Ca2 transition, which is
rate-limiting for the EP formation, takes place at almost
the same rate in the mutants and wild type.
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DISCUSSION |
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In the present study, we explored possible roles of the Lys189-Lys205 loop on the A domain by mutagenesis and found that Val200 is critical for rapid processing of E2P, most likely in step 5, and also for rapid hydrolysis of E2P in step 6 in the Ca2+ transport cycle (Fig. 1). During isomerization of EP and Ca2+ release, a large movement of the A domain (i.e. rotation by ~90° (10)) and its strong association with the P and N domains occur to form the most compactly organized cytoplasmic domains in E2P without bound Ca2+ (12, 13). In this E2P, a hydrophobic atmosphere (28, 31-33) is thus realized around the phosphorylation site, and a specific water molecule can now attack the acylphosphate bond. It is likely that the Lys189-Lys205 loop, especially Val200, is critical for the appropriate domain interactions and proper structure in E2P without bound Ca2+.
In E2V that is very similar to E2P without bound
Ca2+ in the cytoplasmic domain organization (12, 13), the
Lys189-Lys205 loop forms an A-P domain
interface (Fig. 7). The side chain of Val200 protrudes toward the P domain and is in van der
Waals contact with the side chain hydrocarbon parts of
Arg198, Gln202, and Asp203, which
interact with polar residues on the P domain by hydrogen bonding or
salt bridge formation (broken green lines in Fig. 7). It is
likely that Val200 coordinates spatial arrangement of these
surrounding residues by the nonpolar interactions so as to produce the
most favorable configuration for appropriate interactions at the
terminal N and O atoms with the polar residues on P domain and thus for
concurrent contribution of these residues to intimate contact between
the A and P domains in E2P without bound
Ca2+.
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In agreement with this view, the substitutions of Val200 by any examined conservative or nonconservative amino acids resulted in strong inhibition of the Ca2+-ATPase activity because of inhibition of the EP decay, whereas substitutions of each of the above surrounding three residues caused only moderately reduced activity (Fig. 2 and Refs. 17 and 26). Actually, the positive charge of Arg198 was previously shown to be important for rapid E2P hydrolysis, and its nonconservative substitutions partially reduce the khydrolysis value (by 62%) (17). Mutations of Glu680 (4) and of Arg678 on the P domain (see Fig. 7) also cause only partial loss of function.3 It is likely that the substitutions at Val200 would seriously affect coordination of the surrounding residues and thus appropriate interactions of the Lys189-Lys205 loop with the P domain.
The observed high Ca2+ affinity and rate of the Ca2+-induced activation in the Val200 mutants being comparable with those in the wild type (see "Results") indicate that the interactions between the Lys189-Lys205 loop and the P domain likely do not contribute much to cytoplasmic domain organization in the unphosphorylated states and thus would be mostly lost on E2P hydrolysis. In fact, in E2(TG) that is very similar to E2 in the cytoplasmic domain organization (12), no hydrogen bonds or salt bridges are found between the Lys189-Lys205 loop and the P domain (Fig. 7). Consistently, the enzyme in E2 state is rapidly attacked and degraded by proteinase K and V8 protease, whereas E2P without bound Ca2+ is completely resistant to all proteinase K, V8 protease, and trypsin at the T2 site (Arg198) (12, 13). E2 is, however, partially resistant to tryptic attack at the T2 site, indicating that the A domain in this state is still partially associated with the P and N domains and has not yet completely rotated back to the widely separated state found in E1·Ca2 (12, 13), as is in fact seen in the E2(TG) structure. Possible interactions between the A and N domains and between the TGES loop and the P domain (possibly through ligation of Mg2+ (15)), may be significant for holding the A domain in such an organized state in E2. On hydrolysis of E2P to E2, the atmosphere around the phosphorylation site again becomes hydrophilic (28, 31-33). It is possible that loss of the interactions between the Lys189-Lys205 loop and the P domain contribute at least in part to such changes in hydrophobicity.
In summary, we conclude that the Lys189-Lys205
loop, especially Val200, is critical for the intimate
contact between the A and P domains and the appropriate cytoplasmic
domain organization in E2P without bound Ca2+.
Although the structure of E2P·Ca2
(ADP-insensitive EP with occluded Ca2+ (8)) is
not yet characterized, the final process of appropriate gathering and
intimate contact of the A domain with the P and N domains will likely
be accomplished in step 5 after the loss of ADP sensitivity, and this
change is likely essential for Ca2+ release into lumen.
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ACKNOWLEDGEMENTS |
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We thank Dr. David H. MacLennan, University of Toronto, for the generous gift of SERCA1a cDNA and Dr. Randal J. Kaufman, Genetics Institute, Cambridge, MA, for the generous gift of expression vector pMT2. We are also grateful to Dr. Chikashi Toyoshima, University of Tokyo, for helpful discussions and reviewing the manuscript.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Biochemistry, Asahikawa Medical College, Midorigaoka-higashi, Asahikawa, 078-8510, Japan. Tel.: 81-166-68-2350; Fax: 81-166-68-2359; E-mail: hisuzuki@asahikawa-med.ac.jp.
Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M208861200
2 The results with the wild type (the very low accumulation of E2P in spite of the slow kdecay) are accounted for by the equilibrium in step 4 favoring E1P·Ca2 (29). To account for this very low E2P accumulation, it is also possible to assume the rate-limiting E1P to E2P isomerization (step 4) followed by rapid Ca2+ release (step 5) and hydrolysis (step 6). In any case, however, the same conclusion as described in text will be made for the mutants that the step after loss of ADP sensitivity but before hydrolysis of E2P is greatly slowed by the Val200 substitutions.
3 K. Yamasaki, T. Daiho, S. Kato, and H. Suzuki, unpublished observation with Arg678 substitutions.
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ABBREVIATIONS |
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The abbreviations used are: SERCA1a, adult fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase; EP, phosphoenzyme; E1P, ADP-sensitive phosphoenzyme; E2P, ADP-insensitive phosphoenzyme, MOPS, 3-(N-morpholino)propanesulfonic acid; TG, thapsigargin; E2V, decavanadate-bound crystal structure.
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