Variations in the Response of Mouse Isozymes of Adenylosuccinate Synthetase to Inhibitors of Physiological Relevance*

Tudor Borza, Cristina V. Iancu, Evan Pike, Richard B. Honzatko, and Herbert J. FrommDagger

From the Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 5011

Received for publication, October 22, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vertebrates have acidic and basic isozymes of adenylosuccinate synthetase, which participate in the first committed step of de novo AMP biosynthesis and/or the purine nucleotide cycle. These isozymes differ in their kinetic properties and N-leader sequences, and their regulation may vary with tissue type. Recombinant acidic and basic synthetases from mouse, in the presence of active site ligands, behave in analytical ultracentrifugation as dimers. Active site ligands enhance thermal stability of both isozymes. Truncated forms of both isozymes retain the kinetic parameters and the oligomerization status of the full-length proteins. AMP potently inhibits the acidic isozyme competitively with respect to IMP. In contrast, AMP weakly inhibits the basic isozyme noncompetitively with respect to all substrates. IMP inhibition of the acidic isozyme is competitive, and that of the basic isozyme noncompetitive, with respect to GTP. Fructose 1,6-bisphosphate potently inhibits both isozymes competitively with respect to IMP but becomes noncompetitive at saturating substrate concentrations. The above, coupled with structural information, suggests antagonistic interactions between the active sites of the basic isozyme, whereas active sites of the acidic isozyme seem functionally independent. Fructose 1,6-bisphosphate and IMP together may be dynamic regulators of the basic isozyme in muscle, causing potent inhibition of the synthetase under conditions of high AMP deaminase activity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP-forming), EC 6.3.4.4) is present in almost all organisms, the only exceptions being some intracellular prokaryotic parasites (1). Adenylosuccinate synthetases are well conserved through evolution, exhibiting, for instance, ~40% sequence identity between eubacteria and mammals (2, 3). Eukaryotic synthetases differ from their prokaryotic counterparts by the presence of N-terminal leader sequences (~30 residues) and by truncations (~10 residues) at their C termini (2-4). The synthetase catalyzes the first committed step in the de novo biosynthesis of AMP from IMP and, in vertebrates, is also a component of the purine nucleotide cycle (PNC)1 (2, 5-7). Mammals have two different synthetase isozymes (2, 3, 7-11). The basic isozyme (hereafter AdSS1) has a higher Km value for IMP and a lower Km value for L-aspartate than the acidic form (AdSS2). AdSS2 is less susceptible to inhibition by fructose 1,6-bisphosphate (Fru-1,6-P2) than AdSS1 but more strongly inhibited by nucleotides (7, 12). On the basis of these findings alone, investigators assigned AdSS2 to de novo biosynthesis of AMP and AdSS1 to the PNC (2, 6, 12).

The PNC is active in muscle, brain, kidney, liver, and pancreatic islets (5, 13-17) and involves adenylosuccinate synthetase, adenylosuccinate lyase, and AMP deaminase in the following net reaction (Reaction 1):
<UP><SC>l</SC>-aspartate + GTP + H<SUB>2</SUB>O = fumarate + GDP + P<SUB>i</SUB> + NH<SUB>3</SUB></UP>

<UP><SC>Reaction</SC> 1</UP>
The PNC may have multiple roles as follows: 1) shifting the adenylate kinase equilibrium in the direction of ATP formation by converting AMP into IMP; 2) liberating ammonia from amino acids by using L-aspartate as a donor; 3) regulating glycolysis (IMP activates glycogen phosphorylase and both AMP and ammonia activate phosphofructokinase); and 4) providing Krebs cycle intermediates (fumarate) in tissues that lack pyruvate carboxylase (2, 6).

The impact of the PNC on the metabolism of various tissues is unsettled (18-21), as is the assignment of the two isozymes to mutually exclusive metabolic roles (18). Moreover, in muscle, where exercise can increase IMP concentration up to 50-fold, the PNC may work asynchronously; AMP deaminase works only when AdSS1 and/or adenylosuccinate lyase is quiescent (3, 22-25). During recovery (restoration of basal IMP levels) AMP deaminase is inactive (24, 26). Indeed, interactions with myosin activate AMP deaminase, a process regulated by the decrease in the ATP concentration during exercise (24, 26). No study has demonstrated regulation of AdSS1 in the context of the PNC, although slight inhibition of the basic isozyme occurs at high concentrations of IMP (8, 13, 27).

Mouse recombinant AdSS1 has a significantly lower Km for IMP than that reported for the basic isozyme isolated from either rat or rabbit (28). Reported Km values for adenylosuccinate synthetases vary considerably (2, 6, 28) due, in part, to variations in assay protocols and conditions of assay, as well as intrinsic differences in the synthetases themselves (2, 6). The low natural abundance of AdSS2 has been an impediment to its purification and rigorous evaluation (7, 9, 18). Preparations of AdSS2 from malignant cells, such as Novikoff ascites tumor cells (29) and Yoshida sarcoma tumor cells (30), have provided, save in one instance (30), specific activities significantly lower than that of AdSS1 (2, 6). Human and mouse AdSS2 have been cloned (3, 4) and the latter overexpressed in COS (African green monkey kidney) cells, but no kinetic characterization was reported (3). Native states of oligomerization for each isozyme remain ambiguous, as reports of monomeric and dimeric AdSS1 and AdSS2 are in the literature (8, 10, 29-31). In contrast, the synthetase from Escherichia coli is active as a dimer (32, 33) and exists in a monomer-dimer equilibrium (34).

Reported here are first instances of heterologous overproduction and kinetic characterization of mouse AdSS2. The kcat values for recombinant AdSS2 and AdSS1 are almost identical. AdSS2, relative to AdSS1, has a slightly lower Km for IMP and GTP and a significantly higher Km for L-aspartate. High (but physiologically relevant) concentrations of IMP inhibit AdSS1 but not AdSS2. Adenylosuccinate, GDP, and GMP are strong inhibitors of both isozymes, but AMP, which potently inhibits AdSS2, is a weak inhibitor of AdSS1. Furthermore, the kinetic mechanism of AMP inhibition differs for the two isozymes. Fru-1,6-P2 might be a physiologically significant inhibitor of AdSS1 but not of AdSS2. Truncated isozymes (N-terminal leader sequence removed) retain the kinetic properties and state of oligomerization of their full-length counterparts. Mouse isozymes exhibit a monomer-dimer equilibrium, and both GTP and IMP stabilize the dimer. Differences in mouse synthetases reported here do not support mutually exclusive metabolic roles for the two isozymes. Moreover, our findings support the asynchronous operation of the PNC in muscle.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- E. coli strain BL21 (DE3), plasmid pET28b, nickel-nitrilotriacetic acid-agarose, and the thrombin cleavage capture kit were from Novagen, Inc. Restriction enzymes, DNA ligase, and Vent Polymerase were from New England Biolabs. All other reagents were from Sigma unless noted otherwise.

Construction of Full-length and Truncated Synthetases-- The cloning of full-length AdSS1 into the expression plasmid pET28b was described previously (28). cDNA for mouse acidic adenylosuccinate synthetase (AdSS2) was kindly provided by Dr. F. B. Rudolph (Department of Biochemistry and Cell Biology, Rice University, Houston, TX) as a pSPORT1clone (11). A fragment of 1371 bp was amplified using the following primers: forward, 5'-CCCTTGTCATATGTCGATCTCCGAGAGCAGC-3' (NdeI restriction site underlined), and reverse, 5'-CCGCTCGAGTTAGAAGAGCTGAATCATGGACTC-3' (XhoI restriction site underlined). Insertion of the amplified fragment into corresponding sites of the pET28b expression vector resulted in the plasmid pAdSS2a. An NdeI restriction site located into the AdSS2 open reading frame was removed by a silent mutation. Truncated AdSS1 (AdSS1-Tr) and AdSS2 (AdSS2-Tr) were generated using the forward primers 5'-CCCTTGTCATATGACTGGCTCTCGCGTGACCGTG-3' and 5'-CCCTTGTCATATGGGGAACCGGGTGACTGTGGTG-3', respectively (NdeI restriction sites underlined). All constructs were checked by sequencing (Iowa State University DNA sequencing facility).

Sequence Alignments-- DNA and protein sequences were aligned using Multalign (35). Published sequences of mouse AdSS2 (GenBankTM gi, 404056 and 6671520) (11) were compared with other full-length sequences from mouse (GenBankTM gi: 12845574, 20831732, and 12836391) and expressed sequence tags (GenBankTM gi: 3519613, 12089636, 3521474, and 1738606). The published sequence of human AdSS2 (GenBankTM gi: 415848) (4) was compared with other (redundant) sequences (GenBankTM gi: 10438053 and 15214462). The sequence for human AdSS1 came from available full-length clones and expressed sequence tags (GenBankTM gi: 18583312, 16549233, 12889641, 12901898, 16181164, 13408903, 14817011, and 14676160).

Expression and Purification of Recombinant Isozymes-- AdSS1 was produced and purified as described previously (28). The same procedure was used for AdSS1-Tr. AdSS2 was expressed in E. coli BL21 (DE3) at 37 °C in LB media containing 30 µg/ml kanamycin. Cells were collected by centrifugation (4,000 × g for 10 min at 4 °C), resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8, and 1 mM phenylmethanesulfonyl fluoride), and then disrupted by sonication. After centrifugation (24,000 × g for 30 min), the supernatant was loaded onto a nickel-nitrilotriacetic acid-agarose column, previously equilibrated with 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8. The column was washed sequentially with 10 volumes of each of the three buffers, differing from the above only in the concentration of imidazole (20, 30, and then 40 mM). Bound protein eluted with 300 mM imidazole. After dialysis in 50 mM HEPES, 50 mM NaCl, 1 mM dithiothreitol, and 0.5 mM EDTA, pH 7.0, the enzyme was loaded at 0.5 ml/min onto a DEAE-Sepharose column, equilibrated with dialysis buffer. AdSS2 was eluted by a linear gradient (0-200 mM NaCl). AdSS2-Tr was overproduced and purified as above with one modification; cell cultures were maintained at 25 °C after induction. At 37 °C the truncated protein appears in inclusion bodies. Removal of the polyhistidyl tag employed the thrombin cleavage capture kit. Recombinant E. coli adenylosuccinate synthetase was overproduced and purified as described elsewhere (34).

Enzyme Assay-- Protein concentration was determined by the method of Bradford (36), using bovine serum albumin as a standard. Enzyme activity was determined at an absorbance of 280 nm and at 22 °C as described previously (37). A standard assay buffer for AdSS1 contained 40 mM HEPES, pH 6.7, 8 mM magnesium acetate, 150 µM GTP, 250 µM IMP, and 2 mM aspartate. For AdSS2, the assay buffer contained 40 mM HEPES, pH 6.7, 8 mM magnesium acetate, 150 µM GTP, 200 µM IMP, and 8 mM L-aspartate. The reaction was started by the addition of up to 1 µg/ml enzyme. Under these conditions the reaction was linear for 1 min. The Hill coefficient for Mg2+ was determined by varying the concentration of magnesium acetate from 0.2 to 4 mM and from 0.05 to 2 mM for AdSS1 and AdSS2, respectively. Ki values for Fru-1,6-P2, AMP, GDP, and GMP were determined by holding two substrates at saturating levels and varying the concentration of the third substrate over 1-8 Km, at different fixed concentrations of inhibitors ranging over 0.5-2 Ki. In experiments to determine the Ki of IMP inhibition, concentrations of GTP varied from 15 to 200 µM, those of IMP ranged from 40 to 4,000 µM, and concentrations of L-aspartate and Mg2+ were 2 and 8 mM, respectively. Kinetic data were analyzed with the computer program GraFit (38).

Analytical Ultracentrifugation-- Sedimentation equilibrium experiments were performed in a Beckman Optima XL-A analytical ultracentrifuge using an An-60 Ti rotor, rotor speeds of 9,000 and 15,000 rpm, a temperature of 4 °C, and protein concentrations of 0.1-0.5 mg/ml in 20 mM HEPES, pH 7.2, 20 mM NaCl, and 1 mM dithiothreitol. Centrifugation in the presence of ligands (25 µM IMP, 25 µM GTP, 1 µM hadacidin, 2 mM magnesium acetate) employed only one concentration of protein (0.3 mg/ml). Samples were centrifuged for 12 h. Equilibration was verified then by 3 scans recorded at 4-h intervals. Stepwise radial scans were performed at 280 nm, using a step-size of 0.001 cm, with each datum being the average of 30 measurements. Data were analyzed using the "Ideal" model on the Optima XL-A Analysis software (version 2.0). Partial specific volumes of 0.741, 0.743, 0.740, and 0.745 ml/g for AdSS1, AdSS1-Tr, AdSS2, and AdSS2-Tr, respectively, were determined from the amino acid composition and published tables (39). Samples were centrifuged at 40,000 rpm for 15 h to sediment all protein, and then radial scans were recorded to obtain a base-line correction for each cell. Inclusion of the second virial coefficient did not improve fits, indicating that nonideality is not present in the system.

Partial Purification of Mouse Isozymes from Tissue-- Mouse liver and skeletal muscle were disrupted using a Polytron homogenizer (Brinkmann Instruments) in a buffer containing 50 mM HEPES, pH 7.0, 50 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, and 15 µg/ml leupeptin. The homogenate was centrifuged at 25,000 × g for 30 min. The recovered supernatant fluid was heated (60 °C, 1 min) and then centrifuged (25,000 × g, 45 min). The supernatant was subjected to ammonium sulfate fractionation. Proteins that precipitated in the range of 40-60% (w/v) ammonium sulfate were retained. At this point adenylosuccinate synthetase activity was readily detected. Precipitated protein was dialyzed against 25 mM HEPES, 25 mM NaCl, 1 mM dithiothreitol, and 0.5 mM EDTA, pH 7.5, and then loaded onto a DEAE-Sepharose column equilibrated with the same buffer. The column retained the acidic but not the basic isozyme. 1 M NaCl washed the acidic isozyme from the column. Identities of the acidic and basic isozymes were confirmed by Western blots and their kinetic properties.

Antibodies and Western Blots-- Polyclonal antibodies against recombinant AdSS1 were raised in rabbits at the Iowa State University protein facility and were then purified by affinity chromatography, using an Econo-Pac serum IgG purification kit (Bio-Rad). A Sepharose 4B column containing immobilized AdSS2 eliminated cross-reacting antibodies. Protein transfer to a nitrocellulose membrane was performed as recommended by the manufacturer (Bio-Rad) in a buffer containing 25 mM Tris, pH 8.3, 192 mM glycine, 20% methanol, and 0.05% SDS. Detection of antigen-antibody complexation employed the Opti-4CN kit (Bio-Rad).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sequence Comparison and Validation-- Nucleotide sequences of mouse AdSS2, reported here and by Guicherit et al. (11), differ at positions 499 (C in Ref. 11 is now G) and 595 (A becomes G). As a consequence, Arg167 and Thr199 become glycine and alanine, respectively, identical to corresponding residues in human AdSS2 (4). Moreover, Gly167 is invariant among synthetases from 43 organisms, representing 30 major phylogenetic lineages. The published sequence of human AdSS2 (4) also differs with respect to other sequence information; Ala24 should be arginine and an additional residue (proline) comes after position 24. The N-terminal leader sequence in mouse and human AdSS2 then each have 26 amino acid residues (Fig. 1). Sequence identity between AdSS1 and AdSS2 from the same source (mouse or human) is ~75% but exceeds 95% between like isozymes from different sources. N-terminal leader sequences of AdSS1 and AdSS2 from the same source are only 25% identical. In contrast, N-terminal leader sequences between mouse and human AdSS1 are 97% identical, and those between mouse and human AdSS2 are 73% identical.


View larger version (90K):
[in this window]
[in a new window]
 
Fig. 1.   Alignment of amino acid sequences of mouse (M), human (H), and E. coli (purA) synthetases. Conserved amino acids among mouse and human AdSS1 and AdSS2 are boxed. Sequence corrections are in boldface italics. Vertical arrows indicate sites of N-terminal truncation of the mouse isozymes. Active site residues are designated by .

Purity and Yield-- AdSS1-Tr and AdSS2-Tr are shorter by 28 and 26 amino acid residues, respectively, than their full-length counterparts (Fig. 1). The yield of purified AdSS1-Tr was comparable with that of full-length AdSS1 (5-10 mg/liter of cell culture), but substantial quantities of AdSS1 and AdSS1-Tr did appear in inclusion bodies. In contrast, yields of recombinant AdSS2 and AdSS2-Tr were ~ 25 mg/liter of cell culture, ~20% of the total soluble protein. SDS-PAGE of samples revealed a single band of ~50 kDa. Full-length and truncated AdSS2 are stable for several days at 4 °C. AdSS2 is stable with respect to freeze/thaw cycles in buffers supplemented with 30% glycerol. Full-length AdSS1, with or without their N-terminal polyhistidyl tags, have identical kinetic parameters and crystallize under the same conditions (28). Similarly, truncation of the N-terminal polyhistidyl tag from AdSS2 does not change its kinetic properties (data not shown).

Thermal Stability-- In the absence of ligands, the E. coli synthetase is in a monomer-dimer equilibrium (Kd ~10 µM) (34). The presence of active site ligands, such as IMP and GTP, significantly increases thermal stability of E. coli synthetase. Moreover, the thermal stability of the E. coli synthetase increases with protein concentration.2 In the absence of ligands, AdSS1 and AdSS2 are more stable than the E. coli synthetase, but IMP and GTP do not greatly enhance the thermal stability of the mammalian isozymes (Fig. 2). AdSS1-Tr and AdSS2-Tr have the same thermal stability as their full-length counterparts, ruling out an effect due to the N-terminal leader sequence.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   Thermal stability of mouse and E. coli synthetases. Samples of 20 µl of protein solution at a concentration of 0.5 mg/ml protein were incubated for 1 min at different temperatures. Plotted are activities (determined as described under "Experimental Procedures") relative to fully active systems at 40 °C of mouse AdSS1 (open circle  and ), mouse AdSS2 ( and black-square), and the E. coli synthetase (triangle  and black-triangle) in the absence and presence of IMP/GTP (open versus filled symbols, respectively).

Native Molecular Weight of AdSS1 and AdSS2-- Equilibrium sedimentation ultracentrifugation indicates single species of molecular mass 86.0 ± 3.9 and 90.7 ± 2.5 kDa for AdSS1 and AdSS2, respectively. Predicted masses are 101.1 and 100.7 kDa, derived from amino acid sequences of recombinant AdSS1 and AdSS2, respectively. Equilibrium ultracentrifugation of samples in the presence of IMP, GTP, Mg(acetate)2, and hadacidin (N-hydroxy-N-formylglycine, a potent competitive inhibitor with respect to L-aspartate) showed increased molecular masses of 103.6 ± 5.4 and 101.5 ± 4.3 kDa for AdSS1 and AdSS2, respectively. The behavior of AdSS1-Tr and AdSS2-Tr was similar to their full-length counterparts. In the absence of active site ligands, molecular masses were 86.1 ± 5.9 and 84.8 ± 2.7 kDa for AdSS1-Tr and AdSS2-Tr, respectively; and in the presence of substrate/substrate analogues, their molecular masses were 101.1 ± 7.2 and 96.4 ± 0.8 kDa, respectively. Molecular masses of the recombinant mouse isozymes did not change over a protein concentration from 0.1 to 0.5 mg/ml, whereas the molecular mass of the E. coli synthetase varied from 47.8 ± 2.1 to 56.9 ± 2.0 kDa (experiments at 15,000 rpm). Evidently, over the range of protein concentration accessible to analytical centrifugation, AdSS1 and AdSS2 are predominantly dimers, but a small shift from monomer to dimer occurs in the presence of ligands. The Kd value for the mammalian isozymes then, in the absence of ligands, is significantly lower than that of E. coli synthetase (34). The molecular weight (78 kDa) of a yeast synthetase, determined by gel filtration, lies between that of a monomer and dimer (40), whereas plant synthetases are either monomers or dimers, depending on the methodology of mass determination (41).

Km, kcat, pH, and Buffer Effects-- Km values for AdSS1-Tr are comparable with those of AdSS1 (28), but kcat is slightly lower, perhaps due to a small component of misfolded AdSS1-Tr. Relative to AdSS1, AdSS2 has a lower Km for IMP, a similar Km for GTP, and significantly higher Km for L-aspartate (Table I). AdSS2-Tr has a slightly higher Km value for L-aspartate than that of AdSS2. Km values for IMP and L-aspartate of tissue-derived AdSS1 and AdSS2 were similar to those of the recombinant isozymes (data not shown).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Kinetic parameters for full-length and truncated mouse isozymes
KmGTP, KmIMP, and KmAsp are the Michaelis constants for GTP, IMP, and L-aspartate, respectively.

Values of kcat and Km of the E. coli synthetase are sensitive to pH in the range of 7.0-7.7 (42). As the pH in exercising muscle can decrease from 6.9 to 6.4, we determined whether changes in pH influence kinetic parameters of AdSS1 or AdSS2; however, Km values remained constant from pH 6.5 to 7.2. The pH optima for AdSS1 and AdSS2 lie between 6.6 and 6.9. As the pH optima are nearly out of the buffering range of HEPES (pKa 7.5), other more suitable buffers were tested. MES, MOPS, and PIPES buffers support similar turnovers for AdSS2 and the E. coli synthetase. In contrast, AdSS1 in 40 mM MES and PIPES has only 40 and 10%, respectively, of the specific activity of AdSS1 in HEPES.

Metal Requirement-- AdSS2 and the E. coli synthetase reach maximum activities in 2 mM Mg(acetate)2, but AdSS1 requires 8 mM Mg(acetate)2. Concentrations above 10 mM Mg(acetate)2 are inhibitory for all systems. Inhibition is more pronounced when Cl- is a counterion to Mg2+ instead of acetate. Hill coefficients for Mg2+ are 1.1 ± 0.1 for AdSS1 and 1.0 ± 0.2 for AdSS2. The Hill coefficient for Mg2+ is 2 for the E. coli synthetase (43). Crystal structures of AdSS1 (44, 45), AdSS2,3 and the E. coli synthetase (46, 47), however, reveal only one Mg2+ per subunit. Evidently, studies of AdSS1 and AdSS2 will not clarify the role of the "second" Mg2+ inferred by the kinetics of the E. coli synthetase.

Metabolite and Substrate Inhibition-- Mouse synthetases are subject to potent inhibition by adenylosuccinate, GDP, AMP, and GMP (Table II). In contrast to product and GMP inhibition, AMP inhibits AdSS1 and AdSS2 with marked differences. The Ki value for AMP is 12-fold lower for AdSS2 than AdSS1. Moreover, AMP is a competitive inhibitor with respect to IMP for AdSS2 but noncompetitive with respect to IMP for AdSS1.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Inhibition of recombinant mouse AdSS1 and AdSS2

Fru-1,6-P2 is the only intermediate of glycolysis that inhibits mammalian adenylosuccinate synthetases at concentrations below 1 mM (1, 2). Fru-1,6-P2 inhibits AdSS1 more potently than AdSS2 in plots of reciprocal velocity against 1/GTP and 1/L-aspartate (6- and 8-fold, respectively), but inhibition of the two isozymes is nearly the same in plots of reciprocal velocity against 1/IMP. Fru-1,6-P2 is a noncompetitive inhibitor with respect to all substrates.

High concentrations of IMP (>1 mM) at a saturating concentration of GTP (>150 µM) inhibit AdSS1 but not AdSS2; however, IMP inhibits both isozymes at concentrations of GTP below its Km value (Fig. 3). Scheme I represents IMP inhibition of AdSS2 and AdSS1. In the case of AdSS2, Kii Ki, whereas for AdSS1 Kii must assume a value comparable with that of Ki in order to observe IMP inhibition in the presence of saturating concentrations of GTP. In the context of a saturating concentration of L-aspartate and a rapid equilibrium Random Bi Bi kinetic mechanism for IMP and GTP (37, 48), the expression for initial velocity associated with Scheme I is shown in Equation 1,
v=V<SUB><UP>max</UP></SUB>K<SUB>i</SUB>K<SUB>ii</SUB>AB/(K<SUB>ia</SUB>K<SUB>i</SUB>K<SUB>ii</SUB>K<SUB>b</SUB>+K<SUB>a</SUB>K<SUB>i</SUB>K<SUB>ii</SUB>B (Eq. 1)

+K<SUB>i</SUB>K<SUB>ii</SUB>K<SUB>b</SUB>A+K<SUB>i</SUB>K<SUB>ii</SUB>AB+K<SUB>ii</SUB>K<SUB>b</SUB>A<SUP>2</SUP>+K<SUB>i</SUB>A<SUP>2</SUP>B)
where A and B are IMP and GTP concentrations, respectively; Ka and Kb are their corresponding Michaelis constants; Kia is the dissociation constant of IMP from the free enzyme; Ki is the inhibition constant for IMP in the absence of GTP; and Kii is the inhibition constant for IMP in the presence of GTP. When only IMP concentrations vary at saturating concentrations of L-aspartate and GTP, Equation 1 simplifies to Equation 2,
&ngr;=V<SUB><UP>max</UP></SUB>/(1+A/K<SUB>ii</SUB>+K<SUB>a</SUB>/A) (Eq. 2)
The symbols in Equation 2 are as defined in Equation 1. IMP concentrations from 30 to 4000 µM at 250 µM GTP give a Kii of 3.5 ± 0.3 and 4.1 ± 0.6 mM for recombinant AdSS1 and AdSS1 partially purified from skeletal muscle, respectively (Kii values from Equation 2). In comparison, data for recombinant AdSS1, in which concentrations of GTP and IMP vary, fit to Equation 1 with a Ki of 2.3 ± 0.4 mM and a Kii of 3.7 ± 0.6 mM. The agreement between predicted and observed velocities is shown in Fig. 4. High absorbances at 280 nm due to high concentrations of IMP and GTP thwarted the determination of Ki and Kii for IMP inhibition of AdSS2; however, Ki for AdSS2 must exceed 8 mM (data not shown).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Variation in the relief of IMP inhibition of mouse isozymes by GTP. IMP inhibition of AdSS1 at 10 µM GTP () and 150 µM GTP (black-square) exhibits little change, whereas that of AdSS2 differs significantly at 10 µM GTP (triangle ) and 150 µM GTP (black-triangle). Curves are smoothed fits through data points and do not correspond to a kinetic model.


View larger version (10K):
[in this window]
[in a new window]
 
Scheme I.  


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 4.   Comparison of observed and predicted initial velocities for IMP inhibition of mouse AdSS1. GTP and IMP concentrations vary over 10-200 and 40-4000 µM, respectively. The velocities were determined at 40 (open circle ), 70 (), 100 (), 200 (black-square), 800 (triangle ), 1600(black-triangle), 2400(down-triangle), 3200(black-down-triangle ), and 4000 (diamond ) µM IMP. Curves represent theoretical lines obtained from fitted kinetic parameters and Equation 1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

E. coli and mouse synthetases exhibit a monomer-dimer equilibrium, in which GTP and IMP stabilize the dimer. Mouse isozymes in the absence of ligands, however, are more stable than the E. coli synthetase on the basis of thermal stability and analytical ultracentrifugation. The monomer-dimer equilibrium may be a property common to all adenylosuccinate synthetases. The dimer is the active form of the E. coli synthetase, and evidently each of its subunits independently achieves maximum velocity in the presence of saturating substrates (32). Indeed, an arginyl side chain (Arg143 in the E. coli synthetase) critical to the recognition of IMP (34) comes to the active site from a symmetry-related subunit of the dimer and is present in all known sequences of synthetase. Hence, regulatory mechanisms that impair subunit dimerization are conceptually possible but have not been demonstrated for any synthetase in vivo. The increased stability of the mouse dimers relative to E. coli dimer could be exploited, however, in the design of drugs (antibiotics) that target the subunit interface.

N-terminal truncations of the mouse isozymes do not alter dimer stability or kinetics. Therefore, the functional differences exhibited by the two isozymes arise from their core sequences, which are ~75% identical. Nevertheless, the N-leader sequences of AdSS1 and AdSS2 diverge significantly, and yet each is conserved across mammalian species. The latter observation suggests possible differences in regulation, protein-protein interactions, and/or intracellular distribution for the two isozymes. The N-leader sequence for the synthetase from plants, for instance, putatively targets it to the chloroplast (49). The basic isozyme binds to F-actin and reconstructed thin microfilaments (50-51), but no hard evidence as yet implicates the N-leader sequence of AdSS1 in these interactions.

Previous studies (8, 13, 27) have documented IMP inhibition of AdSS1, but have disagreed as to whether GTP antagonizes or reinforces the phenomenon (8, 13). Nonetheless, IMP does bind to the GTP pocket of AdSS1 as evidenced by the crystal structure of an IMP complex (44). Furthermore, all residues of the GTP pocket are identical in AdSS1 and AdSS2. The EA2 complex in Scheme I then may well represent IMP molecules bound to the IMP and GTP pockets within the same subunit of a dimer. High levels of GTP would relieve such inhibition, but AdSS1 remains sensitive to IMP even in the presence of saturating GTP. Hence, AdSS1 has an alternative mechanism that allows IMP to inhibit in the presence of GTP (EBA2 complex of Scheme I). As will be discussed below, the alternative inhibitory site for IMP in AdSS1 may be the symmetry-related IMP pocket of the dimer.

AMP inhibits AdSS2 ~12-fold more strongly than AdSS1, and by a different kinetic mechanism (competitive versus noncompetitive with respect to IMP). Furthermore, in crystalline complexes of the E. coli synthetase (52), AdSS1 (45), and AdSS23 AMP binds only to the IMP pocket. AMP ligation of the IMP pocket accounts for competitive inhibition of AdSS2, but noncompetitive inhibition of AdSS1 suggests one of two alternative mechanisms. (i) AMP promotes the dissociation of an active AdSS1 dimer into inactive monomers. (ii) AMP inhibits one subunit of the AdSS1 dimer by binding to the IMP pocket of the other subunit. The former mechanism is unlikely, given the stabilizing effect of IMP on the dimer (34), but the latter is plausible because of the proximity of IMP pockets in the dimer (Fig. 5). Helix 5 and the IMP loop (the latter binds the 5'-phosphoryl group of AMP) are conformationally dynamic elements in AdSS1 and could interact across the subunit interface. In contrast, helix 5 in AdSS2 is immobilized by hydrogen bond interactions and hence is less likely to transmit conformational changes between active sites.3 IMP and AMP inhibition in AdSS1 then could stem from a common mechanism that involves the interaction of active sites of the dimer. AdSS2 on the other hand seems more like the E. coli synthetase in its properties of IMP and AMP inhibition. Kang et al. (32) have demonstrated functionally independent (non-interacting) subunits in the dimer of the E. coli synthetase.


View larger version (43K):
[in this window]
[in a new window]
 
Fig. 5.   View down the molecular 2-fold axis of AdSS1. Conformational mobility of helix 5 specific to AdSS1, but not observed in structures of AdSS2, may communicate conformational changes in the IMP loop of one active site to that of the other. The IMP pockets and flexible IMP loops (last turn of the C-terminal end of helix 3 and the 10 residues that follow) of the synthetase dimer are adjacent to, but make only weak interactions with the helix 5 pair. Adenylosuccinate (labeled SAMP) occupies the IMP pocket in this particular structure from Ref. 45. This illustration was drawn by MOLSCRIPT (61)

Irrespective of whether subunit interactions in the dimer are the basis for noncompetitive AMP inhibition of AdSS1, an unresolved issue still remains. How does AdSS1 exclude AMP from its IMP pocket and still retain high affinity for IMP? The conformation of the pre-Switch loop of AdSS1 in its ligand-free state differs from that of other synthetases (28), including AdSS2.3 The pre-Switch loop in AdSS1 blocks the 5'-phosphoryl pocket and holds the side chain of Asn256 in an intramolecular hydrogen bond. Hence, IMP and AMP must sacrifice binding energy to overcome the antagonistic conformation of the pre-Switch loop in AdSS1. The 4-5-fold increase in the Km of IMP and the 11-12-fold increase in Ki of AMP are consistent with less favorable interactions of each nucleotide with the IMP pocket of AdSS1 relative to AdSS2. Unlike AMP, however, IMP can in principle recover free energy lost in its binding interaction by forming 6-phosphoryl-IMP; atoms N-7 and N-6 of AMP cannot both hydrogen bond with the side chain of Asn256, whereas atoms N-7 and O-6 of 6-phosphoryl-IMP can and do hydrogen-bond with the side chain of Asn256 (44, 45). As IMP and AMP levels in most tissues are ~60 and 200 µM, respectively (53), fluctuations in the relative concentrations of AMP and IMP would influence the activity of AdSS2, whereas variations in the concentration of IMP alone would influence the activity of AdSS1.

The assignment of AdSS1 and AdSS2 to separate metabolic roles by Nakagawa and co-workers (7, 12) rests largely on different susceptibilities to Fru-1,6-P2 inhibition (7, 12); however, the reported Ki values (0.6 and 1.6 mM for AdSS1 and AdSS2, respectively) are 30-80-fold higher than physiological concentrations of Fru-1,6-P2. Nonetheless, all kinetic parameters reported by Nakagawa and co-workers (7, 10, 12, 30) are much higher than those determined by other groups (2, 6). Stayton et al. (6), for instance, report noncompetitive Fru-1,6-P2 inhibition of AdSS1 from rat (Ki ~130 µM) and rabbit (Ki ~50-100 µM), consistent with the findings here. Although an inhibitory site distinct from the active site accounts for the kinetic mechanism of inhibition, a preliminary crystallographic study reveals Fru-1,6-P2 bound as an analogue of 6-phosphoryl-IMP.3 Fru-1,6-P2 could be a potent competitive inhibitor with respect to IMP at low concentrations of IMP (Ki of 16-20 µM), but when IMP is at saturation, Fru-1,6-P2 must bind elsewhere. The location of this alternative Fru-1,6-P2 site is unknown. The potent mode of Fru-1,6-P2 inhibition is nearly equal for AdSS1 and AdSS2. On the other hand, Fru-1,6-P2 binds to the alternative site of AdSS1 with higher affinity than to that of AdSS2.

As the concentration of Fru-1,6-P2 in most tissues is ~20 µM, but can increase 3-fold in muscle during contraction (54, 55), Fru-1,6-P2 could be a dynamic regulator of AdSS1 in muscle. AdSS1 is the major if not sole form of the synthetase in muscle. The documented 60- and 3-fold increases in the in vivo concentrations of IMP and Fru-1,6-P2 during vigorous exercise should severely limit AdSS1 activity, suggesting an asynchronous PNC. Declining ATP levels in exercising muscle activate AMP deaminase, whereas the concomitant increase in levels of IMP and Fru-1,6-P2 inhibit AdSS1. Cessation of vigorous exercise leads to the restoration of ATP and Fru-1,6-P2 levels, the inactivation of AMP deaminase, and the relief of Fru-1,6-P2 inhibition of AdSS1. As the IMP concentration diminishes, the activity of AdSS1 actually accelerates until IMP is no longer saturating.

Continuous operation of the PNC in muscle is difficult to reconcile with the accumulation of IMP in vivo and the requirement for GTP to drive the AdSS1 reaction. The re-conversion of GDP to GTP is at best unfavorable in the face of diminished concentrations of ATP (24). Hence, the PNC is an unlikely source for the ammonia produced during muscle contraction and probably cannot provide fumarate to the Krebs cycle as originally proposed (5, 13). Indeed, partial and complete deficiency in muscle AMP deaminase does not influence Krebs cycle anaplerosis, phosphocreatine hydrolysis, adenine nucleotide ratios, or exercise performance (21). Moreover, the fate of IMP accumulated during contraction varies with muscle type (56-59); IMP primarily goes to AMP via the PNC, but some is released as inosine and hypoxanthine. These lost nucleotides must be re-synthesized de novo in order to restore adenine nucleotide pools (60). Hence, AdSS1 activity related to de novo purine biosynthesis and the PNC may be inseparable in muscle tissue.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Research Grant NS 10546 and National Science Foundation Grant MCB-9985565.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.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, Biophysics, and Molecular Biology, Molecular Biology Bldg., Iowa State University, Ames, IA 5011. Tel.: 515-294-7103; Fax: 515-294-0453; E-mail: hjfromm@iastate.edu.

Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M210838200

2 T. Borza and H. J. Fromm, unpublished results.

3 C. V. Iancu and R. B. Honzatko, unpublished results.

    ABBREVIATIONS

The abbreviations used are: PNC, purine nucleotide cycle; AdSS1, basic isozyme of mouse adenylosuccinate synthetase; AdSS2, acidic isozyme of mouse adenylosuccinate synthetase; AdSS1-Tr, N-terminal sequence truncated form of the basic isozyme; AdSS2-Tr, N-terminal sequence truncated form of the acidic isozyme; Fru-1, 6-P2, fructose 1,6-bisphosphate; PIPES, 1,4-piperazinediethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Tatusov, R. L., Natale, D. A., Garkavtsev, I. V., Tatusova, T. A., Shankavaram, U. T., Rao, B. S., Kiryutin, B., Galperin, M. Y., Fedorova, N. D., and Koonin, E. V. (2001) Nucleic Acids Res. 29, 22-28[Abstract/Free Full Text]
2. Honzatko, R. B., Stayton, M. M., and Fromm, H. J. (1999) Adv. Enzymol. Relat. Areas Mol. Biol. 73, 57-102[Medline] [Order article via Infotrieve]
3. Guicherit, O. V., Rudolph, F. B., Kellems, R. E., and Cooper, B. F. (1991) J. Biol. Chem. 266, 22582-22587[Abstract/Free Full Text]
4. Powell, S. M., Zalkin, H., and Dixon, J. E. (1992) FEBS Lett. 303, 4-10[CrossRef][Medline] [Order article via Infotrieve]
5. Tornheim, K., and Lowenstein, J. M. (1972) J. Biol. Chem. 247, 162-169[Abstract/Free Full Text]
6. Stayton, M. M., Rudolph, F. B., and Fromm, H. J. (1983) Curr. Top. Cell. Regul. 22, 103-141[Medline] [Order article via Infotrieve]
7. Matsuda, Y., Ogawa, H., Fukutome, S., Shiraki, H., and Nakagawa, H. (1977) Biochem. Biophys. Res. Commun. 78, 766-771[Medline] [Order article via Infotrieve]
8. Muirhead, K. M., and Bishop, S. H. (1974) J. Biol. Chem. 249, 459-464[Abstract/Free Full Text]
9. Van der Weyden, M. B., and Kelly, W. N. (1974) J. Biol. Chem. 249, 7282-7289[Abstract/Free Full Text]
10. Ogawa, H., Shiraki, H., Matsuda, Y., Kakiuchi, K., and Nakagawa, H. (1977) J. Biochem. (Tokyo) 81, 859-869[Medline] [Order article via Infotrieve]
11. Guicherit, O. M., Cooper, B. F., Rudolph, F. B., and Kellems, R. E. (1994) J. Biol. Chem. 269, 4488-4496[Abstract/Free Full Text]
12. Ogawa, H., Shirahi, H., and Nakagawa, H. (1976) Biochem. Biophys. Res. Commun. 68, 524-528[Medline] [Order article via Infotrieve]
13. Goodman, M. V., and Lowenstein, J. M. (1977) J. Biol. Chem. 252, 5054-5060[Abstract]
14. Schultz, V., and Lowenstein, J. M. (1976) J. Biol. Chem. 251, 485-492[Abstract]
15. Bogusky, R. T., Lowenstein, L. M., Goodman, M. V., and Lowenstein, J. M. (1976) J. Clin. Invest. 58, 326-335[Medline] [Order article via Infotrieve]
16. Moss, K. M., and McGivan, J. D. (1975) Biochem. J. 150, 162-169
17. Marynissen, G., Sener, A., and Malaisse, W. J. (1992) Biochem. Med. Metab. Biol. 48, 127-136[Medline] [Order article via Infotrieve]
18. Baugher, B. W., Montonaro, L., Welch, M. M., and Rudolph, F. B. (1980) Biochem. Biophys. Res. Commun. 94, 123-129[Medline] [Order article via Infotrieve]
19. Krebs, H. A., Hems, R., Lund, P., Halliday, D., and Read, W. W. (1978) Biochem. J. 176, 733-777[Medline] [Order article via Infotrieve]
20. Knecht, K., Wiesmüller, K.-H., Gnau, V., Jung, G., Meyermann, R., Todd, K. G., and Hamprecht, B. (2001) J. Neurosci. Res. 66, 941-950[CrossRef][Medline] [Order article via Infotrieve]
21. Tarnopolsky, M. A., Parise, G., Gibala, M. J., Graham, T. E., and Rush, J. W. E. (2001) J. Physiol. (Lond.) 533, 881-889[Abstract/Free Full Text]
22. Meyer, R. A., and Terjung, R. L. (1980) Am. J. Physiol. 239, C32-C38[Abstract/Free Full Text]
23. Mommsen, T. P., and Hochachka, P. W. (1988) Metabolism 37, 552-556[Medline] [Order article via Infotrieve]
24. Hisatome, I., Morisaki, T., Kamma, H., Sugama, T., Morisaki, H., Ohtahara, A., and Holmes, E. W. (1998) Am. J. Physiol. 275, C870-C881[Abstract]
25. Norman, B., Sabina, R. L., and Jansson, E. (2001) J. Appl. Physiol. 91, 258-264[Abstract/Free Full Text]
26. Rundell, K. W., Tullson, P. C., and Terjung, R. L. (1992) Am. J. Physiol. 263, C294-C299[Abstract/Free Full Text]
27. Spector, T., and Miller, R. L. (1976) Biochim. Biophys. Acta 445, 509-517[Medline] [Order article via Infotrieve]
28. Iancu, C. V., Borza, T., Choe, J. Y., Fromm, H. J., and Honzatko, R. B. (2001) J. Biol. Chem. 276, 42146-42152[Abstract/Free Full Text]
29. Clark, S. W., and Rudolph, F. B. (1976) Biochim. Biophys. Acta 437, 87-93[Medline] [Order article via Infotrieve]
30. Matsuda, Y., Shimura, K., Shiraki, H., and Nakagawa, H. (1980) Biochim. Biophys. Acta 616, 340-350[Medline] [Order article via Infotrieve]
31. Fischer, H. E., Muirhead, K. B., and Bishop, S. H. (1978) Methods Enzymol. 51, 207-213[Medline] [Order article via Infotrieve]
32. Kang, C., Kim, S., and Fromm, H. J. (1996) J. Biol. Chem. 271, 29722-29728[Abstract/Free Full Text]
33. Moe, O. A., Malcolm-Baker, J. F., Wang, W., Kang, C., Fromm, H. J., and Colman, R. F. (1996) Biochemistry 35, 9024-9033[CrossRef][Medline] [Order article via Infotrieve]
34. Wang, W., Gorrell, A., Honzatko, R. B., and Fromm, H. J. (1997) J. Biol. Chem. 272, 7078-7084[Abstract/Free Full Text]
35. Carpet, F. (1988) Nucleic Acids Res. 16, 10881-10890[Abstract]
36. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
37. Rudolph, F. B., and Fromm, H. J. (1969) J. Biol. Chem. 244, 3832-3839[Medline] [Order article via Infotrieve]
38. Leatherbarrow, R. J. (2001) GraFit, Version 5 , Erithacus Software Ltd., Horley, UK
39. Laue, T. M., Bhaivari, D. S., Ridgway, T. M., and Pelletier, S. L. (1992) in Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S. E. , Rowe, A. J. , and Horton, J. C., eds) , pp. 90-125, Royal Society of Chemistry, Cambridge, UK
40. Lipps, G., and Krauss, G. (1999) Biochem. J. 341, 537-543[CrossRef][Medline] [Order article via Infotrieve]
41. Prade, L., Cowan-Jacob, S. W., Chemla, P., Potter, S., Ward, E., and Fonne-Pfister, R. J. (2000) J. Mol. Biol. 296, 569-577[CrossRef][Medline] [Order article via Infotrieve]
42. Kang, C., Sun, N., Poland, B. W., Gorell, A., Honzatko, R. B., and Fromm, H. J. (1997) J. Biol. Chem. 272, 11881-11885[Abstract/Free Full Text]
43. Kang, C., and Fromm, H. J. (1995) J. Biol. Chem. 270, 15539-15544[Abstract/Free Full Text]
44. Iancu, C. V., Borza, T., Fromm, H. J., and Honzatko, R. B. (2002) J. Biol. Chem. 277, 26779-26787[Abstract/Free Full Text]
45. Iancu, C. V., Borza, T., Fromm, H. J., and Honzatko, R. B. (2002) J. Biol. Chem. 277, 40536-40543[Abstract/Free Full Text]
46. Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1996) J. Mol. Biol. 264, 1013-1027[CrossRef][Medline] [Order article via Infotrieve]
47. Choe, J. Y., Poland, B. W., Fromm, H. J., and Honzatko, R. B. (1999) Biochemistry 38, 6953-6961[CrossRef][Medline] [Order article via Infotrieve]
48. Cooper, B. F., Fromm, H. J., and Rudolph, F. B. (1986) Biochemistry 25, 7323-7327[Medline] [Order article via Infotrieve]
49. Emanuelsson, O., Nielsen, H., and Von Heijne, G. (1999) Protein Sci. 8, 978-984[Abstract]
50. Ogawa, H., Shiraki, H., Matsuda, Y., and Nakagawa, H. (1978) Eur. J. Biochem. 85, 331-337[Abstract]
51. Manfredi, J. P., Marquetant, R., Magid, A. D., and Holmes, E. W. (1989) Am. J. Physiol. 257, C29-C35[Abstract/Free Full Text]
52. Fonne-Pfister, R., Chemla, P., Ward, E., Girardet, M., Kreuz, K. E., Honzatko, R. B., Fromm, H. J., Schar, H. P., Grutter, M. G., and Cowan-Jacob, S. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9431-9436[Abstract/Free Full Text]
53. Traut, T. W. (1994) Mol. Cell. Biochem. 140, 1-22[Medline] [Order article via Infotrieve]
54. Lee, A. D., and Katz, A. (1989) Biochem. J. 258, 915-918[Medline] [Order article via Infotrieve]
55. Raz, I., Katz, A., and Spencer, M. K. (1991) Am. J. Physiol. 260, E430-E435[Abstract/Free Full Text]
56. Arabadjis, P. G., Tullson, P. C., and Terjung, R. L. (1993) Am. J. Physiol. 264, C1246-C1251[Abstract/Free Full Text]
57. Tullson, P. C., Bangsbo, J., Hellsten, Y., and Richter, E. A. (1995) J. Appl. Physiol. 78, 146-152[Abstract/Free Full Text]
58. Tullson, P. C., Arabadjis, P. G., Rundell, K. W., and Terjung, R. L. (1996) Am. J. Physiol. 270, C1067-C1074[Abstract/Free Full Text]
59. Hellsten, Y., Richter, E. A., Kiens, B., and Bangsbo, J. (1999) J. Physiol. (Lond.) 520, 909-920[Abstract/Free Full Text]
60. Tullson, P. C., and Terjung, R. L. (1991) Am. J. Physiol. 261, C342-C347[Abstract/Free Full Text]
61. Kraulis, J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.



This Article
Abstract
Full Text (PDF)
All Versions of this Article:
278/9/6673    most recent
M210838200v1
Purchase Article
View Shopping Cart
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Copyright Permissions
Google Scholar
Articles by Borza, T.
Articles by Fromm, H. J.
Articles citing this Article
PubMed
PubMed Citation
Articles by Borza, T.
Articles by Fromm, H. J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   Biochemistry and Molecular Biology Education 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.