From the Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany and
the ¶ Boston Biomedical Research Institute and Department of
Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts 02114
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ABSTRACT |
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Wild type phosphate carrier (PIC) from Saccharomyces cerevisiae and recombinant PIC proteins with different C-terminal extensions were expressed in Escherichia coli as inclusion bodies. From these, PIC was isolated with the detergent sodium lauroyl sarcosinate in a form, partially monomeric and unfolded. This PIC associates to stable dimers after exchanging the detergent to the polyoxyethylene detergent C12E8 and dialysis. Combining two differently tagged monomers of PIC and following this with affinity chromatography yields defined homo- and heterodimeric forms of PIC, which are all fully active after reconstitution. As a member of the mitochondrial carrier family PIC is supposed to function as a homodimer. We investigated its dimeric nature in the functionally active state after reconstitution. When reconstituting PIC monomers a sigmoidal dependence of transport activity on the amount of inserted protein is observed, whereas insertion of PIC dimers leads to a linear dependence. Heterodimeric PIC constructs consisting of both an active and an inactivated subunit do not catalyze phosphate transport. In contrast, reconstitution of a mixture of active and inactive monomeric subunits led to partially active carrier. These experiments prove (i) that PIC does not function in monomeric form, (ii) that PIC dimers are stable both in the solubilized state and after membrane insertion, and (iii) that transport catalyzed by PIC dimers involves functional cross-talk between the two monomers.
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INTRODUCTION |
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The mitochondrial phosphate carrier (PIC)1 or phosphate transport protein (PTP) catalyzes transport of phosphate into the mitochondrial matrix where the phosphate is utilized for oxidative phosphorylation (1-6). The primary structure of the beef heart PIC was elucidated by protein (7) and DNA/protein sequencing (8) and the PIC gene was cloned and sequenced from Saccharomyces cerevisiae (9). The yeast PIC has been expressed as inclusion bodies in Escherichia coli (10, 11). Methods have been described to solubilize mitochondrial carriers from inclusion bodies including PIC in a functionally active state (11-13).
PIC is a typical member of the structural family of mitochondrial carriers with subunits of six transmembrane segments and a molecular mass of 32 kDa (14, 15). There are several lines of evidence that mitochondrial carriers do not function as monomers but form dimers in the functional state. The first and still one of the most convincing indications up to now was the observation of a binding stoichiometry of one molecule of the tightly binding ligand carboxyatractylate to two monomeric units of the ADP/ATP carrier (16). An even lower binding stoichiometry was observed for the ligands ADP and ATP (17), which in experiments with fluorescent nucleotide analogs led to the suggestion of a tetrameric functional unit of the ADP/ATP carrier (18). By using cross-linking and analytical ultracentrifuge techniques it was shown that the ADP/ATP carrier as well as the mitochondrial uncoupling protein, at least in the solubilized state, forms a homodimer (19, 20). In recent experiments the formation of an intermolecular disulfide bridge between the monomers of the ADP/ATP carrier provided further evidence for the dimeric state of this mitochondrial carrier (21). Studies with PIC in mitochondria demonstrated the requirement of less than one NEM per subunit of PIC (22) and in reconstituted proteoliposomes that a disulfide between Cys-28 of the two monomers reversibly blocks transport (23). Besides these findings, it has been argued also on a theoretical point of view that a dimeric state is favorable for carrier function (24). It is noteworthy that there is a further reason for the acceptance of the dimeric nature of mitochondrial carriers, namely the "consensus minimal unit" of about 12 transmembrane segments which holds true for many carrier proteins (25-27).
The oligomeric state of secondary carriers has been investigated in a number of cases. Evidence has been provided for both the monomeric and the dimeric form of the E. coli lactose permease to be functional (28). However, the dominating evidence suggests that lactose permease is functional as a monomer with 12 transmembrane segments (28). The situation is not better understood for other well studied carriers. Mammalian facilitative sugar carriers, i.e. uniporters (GLUT-family) and Na+-coupled symporters (SGLT-family), were found to function both as monomers and oligomers, and cooperative interactions were suggested as a regulatory mechanism (29-31). There is experimental evidence that also the erythrocyte anion transporter (band 3 protein) may exist as a mixture of dimers and tetramers (32-34), but evidence for a monomeric function of this protein has also been provided (35). Recently, by coexpression and co-reconstitution of functional and nonfunctional monomers of the small secondary carrier EmrE, the oligomeric state of this protein has been demonstrated (36). However, several of the methods used to prove oligomeric associations in these proteins may be questioned (37), and consequently their functional oligomeric structures have not yet been definitely established.
The aim of the present work was to prove that the dimeric state is a prerequisite for function of PIC in the membrane. We used differently tagged PIC monomers to prepare defined heterodimers. The monomers were obtained after heterologous expression in E. coli and solubilization. Analyses of these constructs showed both an inability of monomers to function in phosphate transport and cross-talk between the subunits when integrated into stable dimers.
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EXPERIMENTAL PROCEDURES |
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Materials-- [33P]Phosphate was obtained from Amersham-Buchler (Braunschweig, Germany). Sigma (Deisenhofen, Germany) supplied the following chemicals: mersalylic acid, dithiothreitol, HEPES, PIPES, anti-mouse IgG, alkaline phosphatase conjugate, turkey egg yolk phospholipid, Sigma Fast BCIP/NBT tablets. Dowex 2-X10, sodium lauroyl sarcosinate (SLS), and C12E8 were purchased from Fluka (Deisenhofen, Germany), C8En from Bachem (Bubendorf, Switzerland), Bio-Beads SM-2 from Bio-Rad (Munich, Germany), Sephadex G-75 from Pharmacia (Freiburg, Germany), and N-ethylmaleimide and pyridoxal phosphate were from Merck (Darmstadt, Germany). The micro BCA protein assay was used for protein determination and was purchased from Pierce. Ni-NTA-agarose and Ni-NTA alkaline phosphatase conjugate was purchased from Qiagen (Hilden, Germany). The FLAG system for protein tagging was purchased from Kodak (Rochester, MN). All further chemicals were of analytical grade. All sulfhydryl reagents used were prepared freshly. The reagents were diluted with water or the respective gel filtration buffer. Pyridoxal phosphate in high concentrations was dissolved in 1 M imidazole (pH 6.5).
Generation of Tagged Carrier Proteins--
Cloning of DNA and
subsequent transformation steps were carried out using standard
techniques (38, 39). The 3' part of the mir gene coding for
the phosphate carrier was amplified by polymerase chain reaction using
two oligonucleotide primers annealing upstream (5'-GACTGCTGGTTTGGC-3')
of the KpnI site and downstream of the 3' part thereby
introducing the FLAG tag
(5'-GGTGGTGGTGGTCATGACTACAAGGACGACGATGACAAGTAGGGATCC-3') or the His tag
(5'-GGTGGTGGTGGTCATCATCATCATCATCATTAGGGATCC-3') (tags
underlined), respectively, and a BamHI restriction
site downstream from the stop codon. Polymerase chain reaction (30 s
94 °C, 30 s 50 °C, 60 s 72 °C, 30 cycles) was
carried out using Taq polymerase (Boehringer, Mannheim) and
a Thermo-Cycler 480 (Perkin-Elmer). Plasmid pNYHM131 (13, 40) was used
as template. The polymerase chain reaction products were cut with
BamHI and KpnI and cloned into the plasmid pUC18
for sequencing. Sequencing was carried out using a Pharmacia (Freiburg,
Germany) A.L.F. DNA sequencer and the AutoRead sequencing kit
(Pharmacia, Freiburg, Germany) as recommended by the supplier.
Appropriate fragments were subsequently cloned into plasmid pNYHM131
via BamHI and KpnI restriction and ligation. All
cloning steps were carried out in the E. coli strain DH5.
The expression of the different proteins was carried out in E. coli strain BL21 (DE3) as described below.
Isolation and Purification of the PIC--
Expression strain
BL21 (DE3) carrying plasmids coding for the wild type PIC or a mutant,
respectively, was transformed. A total of 1 liter of 2 × YT
medium (plus 100 mg of carbenicillin) was inoculated with a fresh
overnight colony of transformed BL21 (DE3) and grown to an
OD600 of 0.6 (about 5 h) under vigorous shaking at
37 °C. Expression of PiC was initiated by 1 mM
isopropyl--D-thiogalactopyranoside, and 100 mg of
carbenicillin was added. Growth was continued for 3 h, and the
cells were harvested and stored at
20 °C. All the following steps
were carried out on ice (11). The cell pellet with the expressed PIC
was suspended in TE (10 mM Tris base, 0.1 mM
EDTA, 1 mM DTT, adjusted to pH 7.0 with HCl) and passed
twice through a French pressure cell, followed by centrifugation at 12,100 × g for 10 min. The pellet was homogenized in
10 ml of TE and centrifuged at 1,100 × g for 5 min;
8.8 ml of the supernatant was centrifuged at 12,100 × g for 3 min. The pellet was stored at
20 °C. Isolation
of PIC from the inclusion bodies was carried out as described (12, 13).
The pellet was suspended three times in TE buffer containing 2% Triton
X-114, followed by a centrifugation at 12,100 × g for
2.5 min. The supernatant was discarded. Finally the pellet was
solubilized in 400 µl of TE containing 1.5% SLS. After
addition of 1% C12E8 the SLS-solublized
protein was used for reconstitution directly or for subsequent
dialysis. Functional reconstitution was not possible without addition
of C12E8 or another nonionic detergent (see
"Results" and Table I).
Chemical Modification of Monomeric PIC-- Chemical modification of the monomeric PIC was achieved by incubating the SLS-solubilized PIC (before the addition of C12E8) with a freshly prepared solution of NEM (final concentration 2 mM) for 15 min in the dark at 4 °C. Excess NEM was removed by adding 10 mM DTT, 1% C12E8 was added afterward as described above.
Exchange of Detergent and Dialysis-- For detergent exchange 1% C12E8 was added to the solubilized PIC and dialyzed four times for 12 h against a 1000-fold excess of buffer. Composition of the buffers used was as follows: 400 mM LiCl, 20 mM PIPES, 0.03% NaN3 1 mM DTT (pH 6.5) (first dialysis); 400 mM KCl, 20 mM PIPES (pH 6.5), 0.03% NaN3, 1 mM DTT (second dialysis); 100 mM KCl, 20 mM PIPES, 0.03%, NaN3, 1 mM DTT (pH 6.5) (third dialysis); 300 mM NaCl, 50 mM Na2HPO4 (pH 8.5) (fourth dialysis). After dialysis the protein solutions were centrifuged.
Isolation of the Heterodimer-- The Ni-NTA-agarose column was equilibrated in buffer A (300 mM NaCl, 50 mM Na2HPO4, pH 8.0). After application of the protein the column was washed with buffer B (buffer A containing 10% glycerol (v/v)). Elution was carried out using an imidazole gradient (0-0.5 M imidazole in buffer B). PIC protein was eluted at an imidazole concentration of 0.2 M. The fractions were collected and concentrated using Centricon 30 tubes (Amicon, Eschborn, Germany). After a 5-fold dilution with TBS buffer plus detergent (50 mM Tris-HCl, 150 mM NaCl, 0.1% C12E8 (v/v), pH 7.4) the protein was applied to an anti-FLAG affinity column equilibrated with TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) in a recycling procedure, i.e. the affinity chromatography was repeated five times. After washing the column with TBS the protein was eluted with TBS containing the FLAG-peptide. The eluted fractions were concentrated and used for reconstitution.
Polyacrylamide Gel Electrophoresis and Western Blot Analysis-- SDS-polyacrylamide gel electrophoresis was carried out according to the method of Schägger and von Jagow (41) using gels containing 10% acrylamide and 6 M urea. For analysis under native conditions, urea was replaced by 12% glycerol, and SDS by SLS. For immunological detection of the different constructs the SDS gels were blotted to polyvinylidene difluoride membranes in a semi-dry blotter (Pharmacia, Freiburg, Germany) (38). The FLAG-specific antibody or the Ni-NTA-conjugate was added. In the case of the FLAG antibody the second alkaline phosphatase-conjugated antibody was added subsequently. The color reaction was initiated by adding freshly prepared 5-bromo-4-chloro-3-indolyl phosphate (BCIP/NBT reagent) solution. The reaction was stopped after 10 min by adding 10 mM EDTA.
Reconstitution Procedure-- PIC was reconstituted into pre-formed phospholipid vesicles by using the amberlite method (42). This method was modified (13) with regard to the applied phospholipid/protein and phospholipid/detergent ratio. Maximum exchange rates were obtained with a phospholipid concentration of 16 mg/ml, a phospholipid/protein ratio of 6.25 µg/mg, and a detergent/phospholipid ratio of 0.62 mg/mg. This means that 70 µl of Triton X-114 (10%, v/v), 112 µl of preformed liposomes (10% of egg yolk phospholipids in 50 mM KCl, 20 mM HEPES, KPi 20 mM, pH 6.5), 20 µl of protein solution; HEPES (pH 6.5, final concentration 50 mM) and phosphate (final concentration 30 mM) were added up to 700 µl. A detergent/amberlite ratio of 12 mg/g in combination with 15 column passages was used to remove the detergent (43).
Measurement of Transport Activity and Calculation of
Rates--
The methods were identical to those described for the
analysis of the aspartate/glutamate carrier (44, 45) and PIC (13, 46).
Transport activity was determined using forward exchange experiments
(44). The assay was started by adding labeled phosphate. The time
course of isotope equilibration was fitted to the data points according
to a single exponential function {y = A · (1 e
kt) + B} which delivered the apparent time
constant k [min
1]. The specific activity
(µmol/min · mg of protein) was calculated from k
(min
1), from the final value of the isotope equilibration
(dpm), the specific radioactivity (dpm/nmol), the volume of the
proteoliposome fraction (ml), and the protein concentration (µg/ml)
as published previously (44).
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RESULTS |
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Construction of Different Monomers-- In order to be able to monitor the formation of defined dimeric proteins, the individual monomers and the different types of dimers must be experimentally distinguishable. In the solublized state mitochondrial carriers are assumed to be dimers of identical monomers, PIC monomers had thus to be rendered different artificially. For this purpose, we constructed monomers of the wild type PIC with two different tags, namely the His-tag and the FLAG-tag. These tagged PIC monomers were separately and heterologously expressed in E. coli inclusion bodies, solubilized, and reconstituted. These two constructs, after reconstitution by themselves into proteoliposomes showed specific activities of phosphate transport comparable to the wild type protein (see below).
Reconstitution of Solublized PIC from Different Association States-- We did not succeed in directly reconstituting PIC solubilized from inclusion bodies in SLS as the only detergent, however, after the simple addition of appropriate other detergents, reconstitution of the SLS-solubilized PIC was successful (see "Experimental Procedures and Table II). For proper formation of dimers and for optimum reconstitution, we tried several detergents for replacing SLS as the solublizing agent, some of which are listed in Table I. When considering both protein recovery after the dialysis step, in which protein is lost due to aggregation, as well as the specific transport activity obtained after reconstitution of the dialyzed protein, the polyoxyethylene detergent C12E8 proved to be most favorable. Consequently, this detergent was used in all further experiments. The various constructs and combinations were all active when reconstituted from preparations in which C12E8 was used as detergent (cf. Fig. 3 and Table II).
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Construction and Reconstitution of Heterodimers-- For the experimental strategy used it was necessary to obtain a pure preparation of PIC heterodimers. The first prerequisite, the controlled formation of dimers in solution, was already described above. Another prerequisite is a sufficient stability of the dimers formed. Experiments which proved the stability of the dimeric constructs will be described at the end of "Results." The third prerequisite, finally, is the ability to experimentally select particular heterodimers from the set of different forms of PIC obtained after solublization and dimer formation. When starting with the two differently tagged monomers, after dialysis we obtain a mixture of both types of homodimers ([His-PIC]2 and [FLAG-PIC]2), the desired heterodimer ([His-PIC/FLAG-PIC]), and in addition presumably residual amounts of not correctly folded and assembled monomers ([His-PIC] and [FLAG-PIC]). In order to select the heterodimer from this mixture, we applied two consecutive affinity columns (Fig. 3). In the first step, the starting mixture was applied to a Ni-NTA affinity column. The monomer [FLAG-PIC] and the homodimer [FLAG-PIC]2 was not bound but was directly eluted, as was proven by Western blotting for the two tags (experiment not shown). The bound species which all carry the His tag ([His-PIC], [His-PIC]2, and [His-PIC/FLAG-PIC]) were then eluted by imidazole buffer. After concentration and exchange of buffer this eluate was applied to a FLAG-antibody affinity column, to which the heterodimer [His-PIC/FLAG-PIC] was bound as the only protein species. After elution the protein was reconstituted and analyzed kinetically. As a control, also the other species, i.e. homodimers formed and the eluates from the different columns, were reconstituted and analyzed for phosphate transport (data not shown). In Fig. 4 the kinetics of reconstituted PIC proteins from different steps of purification is shown. Both the specific activity, resembling the functionality of the protein, and the shape of the kinetics, being correlated with the size and integrity of the proteoliposomes (49), was found to be very similar for the different preparations. Table II summarizes the phosphate transport activity of the relevant PIC constructs, whether starting from monomeric or from different dimeric forms.
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The Heterodimeric PIC in the Phospholipid Membrane-- We have used the monomeric and dimeric PIC constructs so far to define the state of aggregation and the reconstitutibility of different forms of the carrier. The major aim of this work, however, to prove whether PIC when inserted into the bilayer membrane is functioning in the dimeric form only, cannot be achieved by this approach, since all kinds of dimers used were similarly active. A discrimination of the actual state of PIC in the membrane requires (i) the construction of a dimer from two different monomers which are characterized by a clearly different state of activity each and (ii) the analysis of the functional properties of this construct after reconstitution into the membrane.
For this purpose, we applied chemical modification by NEM. In contrast to PIC from beef heart, it has been shown for intact PIC from S. cerevisiae that NEM is unable to block its function, since the NEM-sensitive cysteine at position 42, as present in the beef heart PIC, is lacking in the yeast carrier (50). This is of course true for PIC isolated from inclusion bodies, too, when NEM is applied to the reconstituted protein, i.e. when PIC is in the dimeric form and in the native state of conformation (Table III) (11). The activity of S. cerevisiae PIC, however, was completely blocked by NEM when the alkylating reagent was applied to the SLS-solubilized protein, i.e. before reconstitution. Obviously, this is due to the fact that the protein is in the monomeric state and (partially) unfolded under these conditions (see above) which makes at least one of the three available cysteines of PIC from yeast accessible for modification.
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DISCUSSION |
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The definition of the state of aggregation of membrane-embedded carrier proteins was the topic of numerous studies involving techniques of cross-linking, electron microscopy, electrophoresis, chromatography, rotational diffusion, ultracentrifugation, radiation inactivation, as well as reconstitution titration. As a matter of fact, the state of aggregation is not only a question of correct numbers. It is highly relevant for the functional models of solute carriers whether carrier proteins act as monomers, dimers, or higher aggregates. In view of the fact that the three-dimensional structure of not a single solute carrier protein is available so far, solving this question becomes even more interesting. The most advanced structural analysis of a solute carrier correlating the spatial arrangement of particular amino acid residues with carrier function, the lactose permease of E. coli (51), offers an interesting view on the hypothetical substrate translocation pathway. Nevertheless, even in this case the correct arrangement of transmembrane segments as well as the true state of aggregation is not yet completely clear.
The major conclusion reached from reconstituting defined constructs of PIC are that the dimer is functionally active, whereas the monomer is not. Particularly striking was the finding that reconstitution of the monomeric form of PIC resulted in a strongly sigmoidal titration curve, indicating that the first monomers inserted into liposomes are not able to function in phosphate transport. We interpret the sigmoidal shape of the titration curve by the obvious assumption that more than one functionally active monomer must be present in one liposome, in order to be able to form a transport-active complex. It should be noted that the result of the experiment on reconstituting partially inactivated heterodimers does not exclude higher states of aggregation. On the basis of experiments with the mitochondrial ADP/ATP carrier using fluorescent nucleotide analogs, in fact a tetrameric state of the functional unit of this carrier has been suggested (18). However, the observed shape of the titration curves of reconstitution argues against the presence of higher aggregates being essential for transport function. If this would be the case, a sigmoidal shape of the dependence of activity on the amount of added carrier protein would have been expected also for the insertion of dimers into liposomes.
A further conclusion can be drawn from the experiment on reconstitution of heterodimers of both active and inactive subunits. The fact that this construct is inactive not only proves the stability of the dimeric form. It also indicates that monomers do not function by themselves within the complex, i.e. that there is some kind of cross-talk between the two subunits. It may be assumed that only one pathway for phosphate exists within the dimer, and that the inactivation of one subunit is sufficient to render the whole complex inactive. An alternative explanation would be the existence of two independent pathways through the two PIC subunits, the function of each of which depends on the integrity of the corresponding pathway in some kind of mutual interaction. It should be noted that previous kinetic studies both on the mitochondrial aspartate/glutamate and phosphate carrier, respectively, were the basis for defining of the family of mitochondrial carriers with a common kinetic mechanism, namely a simultaneous bisubstrate kinetics (5, 6, 52, 53). This analysis argued for the presence of two substrate pathways in mitochondrial carrier proteins. An experimental basis to decide this question on a molecular level seems to be at hand now by using more sophisticated versions of heterodimers created by methods described in the present publication.
Finally, a further conclusion can be drawn. For a correct interpretation it was necessary to prove that the dimeric constructs were stable in the course of the experiments. Whereas the stability of the dimeric forms in the solublized state, at least in the time range of a few hours, was already proven by the successful application of the strategy for selective isolation, this was not equally simple for the membrane-inserted state. By comparing the activity of defined heterodimers consisting of two active monomers on the one hand, and of both an active and an inactive monomer on the other, we showed that PIC dimers are stable at least within a time range of 22 h. Thus, beside the fact that the dimeric form is essential for function, it has, to our knowledge, been shown for the first time here that there is no dynamic equilibrium between monomers and dimers of mitochondrial carriers in the membrane-inserted state.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. J. Heberle for carrying out the attenuated total reflection-Fourier transform infrared spectroscopy measurements and to H. Sahm for continuous and generous support.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 189) and the Fonds der Chemischen Industrie.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.
§ Present address: Institut für Biochemie, Universität zu Köln, D-50674 Köln, Zülpicherstr. 47, Germany.
To whom correspondence should be addressed: Institut für
Biochemie, Universität zu Köln, D-50674 Köln,
Germany. Tel.: 49-221-470-6461; Fax: 49-221-470-5091; E-mail:
r.kraemer{at}uni-koeln.de.
1 The abbreviations used are: PIC, phosphate carrier; C8En, octyl polyoxyethylene; C12E8, dodecyl octaoxyethylene; DTT, dithiothreitol; NEM, N-ethylmaleimide; SLS, sodium lauroyl sarcosinate; PIPES, 1,4-piperazinediethanesulfonic acid; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium.
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REFERENCES |
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