FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2062450780> ?p ?o ?g. }

• W2062450780 endingPage "6679" @default.
• W2062450780 startingPage "6673" @default.
• W2062450780 abstract "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. 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. purine nucleotide cycle basic isozyme of mouse adenylosuccinate synthetase acidic isozyme of mouse adenylosuccinate synthetase N-terminal sequence truncated form of the basic isozyme N-terminal sequence truncated form of the acidic isozyme 6-P2, fructose 1,6-bisphosphate 1,4-piperazinediethanesulfonic acid 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid 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 (1Tatusov 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. Koonin E.V. Nucleic Acids Res. 2001; 29: 22-28Crossref PubMed Scopus (1539) Google Scholar). Adenylosuccinate synthetases are well conserved through evolution, exhibiting, for instance, ∼40% sequence identity between eubacteria and mammals (2Honzatko R.B. Stayton M.M. Fromm H.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 57-102PubMed Google Scholar, 3Guicherit O.V. Rudolph F.B. Kellems R.E. Cooper B.F. J. Biol. Chem. 1991; 266: 22582-22587Abstract Full Text PDF PubMed Google Scholar). 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 (2Honzatko R.B. Stayton M.M. Fromm H.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 57-102PubMed Google Scholar, 3Guicherit O.V. Rudolph F.B. Kellems R.E. Cooper B.F. J. Biol. Chem. 1991; 266: 22582-22587Abstract Full Text PDF PubMed Google Scholar, 4Powell S.M. Zalkin H. Dixon J.E. FEBS Lett. 1992; 303: 4-10Crossref PubMed Scopus (15) Google Scholar). The synthetase catalyzes the first committed step in thede novo biosynthesis of AMP from IMP and, in vertebrates, is also a component of the purine nucleotide cycle (PNC)1 (2Honzatko R.B. Stayton M.M. Fromm H.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 57-102PubMed Google Scholar, 5Tornheim K. Lowenstein J.M. J. Biol. Chem. 1972; 247: 162-169Abstract Full Text PDF PubMed Google Scholar, 6Stayton M.M. Rudolph F.B. Fromm H.J. Curr. Top. Cell. Regul. 1983; 22: 103-141Crossref PubMed Scopus (82) Google Scholar, 7Matsuda Y. Ogawa H. Fukutome S. Shiraki H. Nakagawa H. Biochem. Biophys. Res. Commun. 1977; 78: 766-771Crossref PubMed Scopus (32) Google Scholar). Mammals have two different synthetase isozymes (2Honzatko R.B. Stayton M.M. Fromm H.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 57-102PubMed Google Scholar, 3Guicherit O.V. Rudolph F.B. Kellems R.E. Cooper B.F. J. Biol. Chem. 1991; 266: 22582-22587Abstract Full Text PDF PubMed Google Scholar, 7Matsuda Y. Ogawa H. Fukutome S. Shiraki H. Nakagawa H. Biochem. Biophys. Res. Commun. 1977; 78: 766-771Crossref PubMed Scopus (32) Google Scholar, 8Muirhead K.M. Bishop S.H. J. Biol. Chem. 1974; 249: 459-464Abstract Full Text PDF PubMed Google Scholar, 9Van der Weyden M.B. Kelly W.N. J. Biol. Chem. 1974; 249: 7282-7289Abstract Full Text PDF PubMed Google Scholar, 10Ogawa H. Shiraki H. Matsuda Y. Kakiuchi K. Nakagawa H. J. Biochem. (Tokyo). 1977; 81: 859-869Crossref PubMed Scopus (30) Google Scholar, 11Guicherit O.M. Cooper B.F. Rudolph F.B. Kellems R.E. J. Biol. Chem. 1994; 269: 4488-4496Abstract Full Text PDF PubMed Google Scholar). The basic isozyme (hereafter AdSS1) has a higher K m value for IMP and a lower K m 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 (7Matsuda Y. Ogawa H. Fukutome S. Shiraki H. Nakagawa H. Biochem. Biophys. Res. Commun. 1977; 78: 766-771Crossref PubMed Scopus (32) Google Scholar, 12Ogawa H. Shirahi H. Nakagawa H. Biochem. Biophys. Res. Commun. 1976; 68: 524-528Crossref PubMed Scopus (20) Google Scholar). On the basis of these findings alone, investigators assigned AdSS2 to de novobiosynthesis of AMP and AdSS1 to the PNC (2Honzatko R.B. Stayton M.M. Fromm H.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 57-102PubMed Google Scholar, 6Stayton M.M. Rudolph F.B. Fromm H.J. Curr. Top. Cell. Regul. 1983; 22: 103-141Crossref PubMed Scopus (82) Google Scholar, 12Ogawa H. Shirahi H. Nakagawa H. Biochem. Biophys. Res. Commun. 1976; 68: 524-528Crossref PubMed Scopus (20) Google Scholar). The PNC is active in muscle, brain, kidney, liver, and pancreatic islets (5Tornheim K. Lowenstein J.M. J. Biol. Chem. 1972; 247: 162-169Abstract Full Text PDF PubMed Google Scholar, 13Goodman M.V. Lowenstein J.M. J. Biol. Chem. 1977; 252: 5054-5060Abstract Full Text PDF PubMed Google Scholar, 14Schultz V. Lowenstein J.M. J. Biol. Chem. 1976; 251: 485-492Abstract Full Text PDF PubMed Google Scholar, 15Bogusky R.T. Lowenstein L.M. Goodman M.V. Lowenstein J.M. J. Clin. Invest. 1976; 58: 326-335Crossref PubMed Scopus (33) Google Scholar, 16Moss K.M. McGivan J.D. Biochem. J. 1975; 150: 162-169Crossref Scopus (28) Google Scholar, 17Marynissen G. Sener A. Malaisse W.J. Biochem. Med. Metab. Biol. 1992; 48: 127-136Crossref PubMed Scopus (4) Google Scholar) and involves adenylosuccinate synthetase, adenylosuccinate lyase, and AMP deaminase in the following net reaction (Reaction 1): L­aspartate+GTP+H2O=fumarate+GDP+Pi+NH3 REACTION1The 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 (2Honzatko R.B. Stayton M.M. Fromm H.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 57-102PubMed Google Scholar, 6Stayton M.M. Rudolph F.B. Fromm H.J. Curr. Top. Cell. Regul. 1983; 22: 103-141Crossref PubMed Scopus (82) Google Scholar). The impact of the PNC on the metabolism of various tissues is unsettled (18Baugher B.W. Montonaro L. Welch M.M. Rudolph F.B. Biochem. Biophys. Res. Commun. 1980; 94: 123-129Crossref PubMed Scopus (10) Google Scholar, 19Krebs H.A. Hems R. Lund P. Halliday D. Read W.W. Biochem. J. 1978; 176: 733-777Crossref PubMed Scopus (46) Google Scholar, 20Knecht K. Wiesmüller K.-H. Gnau V. Jung G. Meyermann R. Todd K.G. Hamprecht B. J. Neurosci. Res. 2001; 66: 941-950Crossref PubMed Scopus (13) Google Scholar, 21Tarnopolsky M.A. Parise G. Gibala M.J. Graham T.E. Rush J.W.E. J. Physiol. (Lond.). 2001; 533: 881-889Crossref Scopus (47) Google Scholar), as is the assignment of the two isozymes to mutually exclusive metabolic roles (18Baugher B.W. Montonaro L. Welch M.M. Rudolph F.B. Biochem. Biophys. Res. Commun. 1980; 94: 123-129Crossref PubMed Scopus (10) Google Scholar). 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 (3Guicherit O.V. Rudolph F.B. Kellems R.E. Cooper B.F. J. Biol. Chem. 1991; 266: 22582-22587Abstract Full Text PDF PubMed Google Scholar, 22Meyer R.A. Terjung R.L. Am. J. Physiol. 1980; 239: C32-C38Crossref PubMed Google Scholar, 23Mommsen T.P. Hochachka P.W. Metabolism. 1988; 37: 552-556Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 24Hisatome I. Morisaki T. Kamma H. Sugama T. Morisaki H. Ohtahara A. Holmes E.W. Am. J. Physiol. 1998; 275: C870-C881Crossref PubMed Google Scholar, 25Norman B. Sabina R.L. Jansson E. J. Appl. Physiol. 2001; 91: 258-264Crossref PubMed Scopus (65) Google Scholar). During recovery (restoration of basal IMP levels) AMP deaminase is inactive (24Hisatome I. Morisaki T. Kamma H. Sugama T. Morisaki H. Ohtahara A. Holmes E.W. Am. J. Physiol. 1998; 275: C870-C881Crossref PubMed Google Scholar, 26Rundell K.W. Tullson P.C. Terjung R.L. Am. J. Physiol. 1992; 263: C294-C299Crossref PubMed Google Scholar). Indeed, interactions with myosin activate AMP deaminase, a process regulated by the decrease in the ATP concentration during exercise (24Hisatome I. Morisaki T. Kamma H. Sugama T. Morisaki H. Ohtahara A. Holmes E.W. Am. J. Physiol. 1998; 275: C870-C881Crossref PubMed Google Scholar,26Rundell K.W. Tullson P.C. Terjung R.L. Am. J. Physiol. 1992; 263: C294-C299Crossref PubMed Google Scholar). 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 (8Muirhead K.M. Bishop S.H. J. Biol. Chem. 1974; 249: 459-464Abstract Full Text PDF PubMed Google Scholar, 13Goodman M.V. Lowenstein J.M. J. Biol. Chem. 1977; 252: 5054-5060Abstract Full Text PDF PubMed Google Scholar, 27Spector T. Miller R.L. Biochim. Biophys. Acta. 1976; 445: 509-517Crossref PubMed Scopus (36) Google Scholar). Mouse recombinant AdSS1 has a significantly lower K mfor IMP than that reported for the basic isozyme isolated from either rat or rabbit (28Iancu C.V. Borza T. Choe J.Y. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2001; 276: 42146-42152Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Reported K m values for adenylosuccinate synthetases vary considerably (2Honzatko R.B. Stayton M.M. Fromm H.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 57-102PubMed Google Scholar, 6Stayton M.M. Rudolph F.B. Fromm H.J. Curr. Top. Cell. Regul. 1983; 22: 103-141Crossref PubMed Scopus (82) Google Scholar, 28Iancu C.V. Borza T. Choe J.Y. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2001; 276: 42146-42152Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) due, in part, to variations in assay protocols and conditions of assay, as well as intrinsic differences in the synthetases themselves (2Honzatko R.B. Stayton M.M. Fromm H.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 57-102PubMed Google Scholar, 6Stayton M.M. Rudolph F.B. Fromm H.J. Curr. Top. Cell. Regul. 1983; 22: 103-141Crossref PubMed Scopus (82) Google Scholar). The low natural abundance of AdSS2 has been an impediment to its purification and rigorous evaluation (7Matsuda Y. Ogawa H. Fukutome S. Shiraki H. Nakagawa H. Biochem. Biophys. Res. Commun. 1977; 78: 766-771Crossref PubMed Scopus (32) Google Scholar, 9Van der Weyden M.B. Kelly W.N. J. Biol. Chem. 1974; 249: 7282-7289Abstract Full Text PDF PubMed Google Scholar, 18Baugher B.W. Montonaro L. Welch M.M. Rudolph F.B. Biochem. Biophys. Res. Commun. 1980; 94: 123-129Crossref PubMed Scopus (10) Google Scholar). Preparations of AdSS2 from malignant cells, such as Novikoff ascites tumor cells (29Clark S.W. Rudolph F.B. Biochim. Biophys. Acta. 1976; 437: 87-93Crossref PubMed Scopus (31) Google Scholar) and Yoshida sarcoma tumor cells (30Matsuda Y. Shimura K. Shiraki H. Nakagawa H. Biochim. Biophys. Acta. 1980; 616: 340-350Crossref PubMed Scopus (16) Google Scholar), have provided, save in one instance (30Matsuda Y. Shimura K. Shiraki H. Nakagawa H. Biochim. Biophys. Acta. 1980; 616: 340-350Crossref PubMed Scopus (16) Google Scholar), specific activities significantly lower than that of AdSS1 (2Honzatko R.B. Stayton M.M. Fromm H.J. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 57-102PubMed Google Scholar, 6Stayton M.M. Rudolph F.B. Fromm H.J. Curr. Top. Cell. Regul. 1983; 22: 103-141Crossref PubMed Scopus (82) Google Scholar). Human and mouse AdSS2 have been cloned (3Guicherit O.V. Rudolph F.B. Kellems R.E. Cooper B.F. J. Biol. Chem. 1991; 266: 22582-22587Abstract Full Text PDF PubMed Google Scholar, 4Powell S.M. Zalkin H. Dixon J.E. FEBS Lett. 1992; 303: 4-10Crossref PubMed Scopus (15) Google Scholar) and the latter overexpressed in COS (African green monkey kidney) cells, but no kinetic characterization was reported (3Guicherit O.V. Rudolph F.B. Kellems R.E. Cooper B.F. J. Biol. Chem. 1991; 266: 22582-22587Abstract Full Text PDF PubMed Google Scholar). Native states of oligomerization for each isozyme remain ambiguous, as reports of monomeric and dimeric AdSS1 and AdSS2 are in the literature (8Muirhead K.M. Bishop S.H. J. Biol. Chem. 1974; 249: 459-464Abstract Full Text PDF PubMed Google Scholar, 10Ogawa H. Shiraki H. Matsuda Y. Kakiuchi K. Nakagawa H. J. Biochem. (Tokyo). 1977; 81: 859-869Crossref PubMed Scopus (30) Google Scholar,29Clark S.W. Rudolph F.B. Biochim. Biophys. Acta. 1976; 437: 87-93Crossref PubMed Scopus (31) Google Scholar, 30Matsuda Y. Shimura K. Shiraki H. Nakagawa H. Biochim. Biophys. Acta. 1980; 616: 340-350Crossref PubMed Scopus (16) Google Scholar, 31Fischer H.E. Muirhead K.B. Bishop S.H. Methods Enzymol. 1978; 51: 207-213Crossref PubMed Scopus (11) Google Scholar). In contrast, the synthetase from Escherichia coliis active as a dimer (32Kang C. Kim S. Fromm H.J. J. Biol. Chem. 1996; 271: 29722-29728Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 33Moe O.A. Malcolm-Baker J.F. Wang W. Kang C. Fromm H.J. Colman R.F. Biochemistry. 1996; 35: 9024-9033Crossref PubMed Scopus (19) Google Scholar) and exists in a monomer-dimer equilibrium (34Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Reported here are first instances of heterologous overproduction and kinetic characterization of mouse AdSS2. Thek cat values for recombinant AdSS2 and AdSS1 are almost identical. AdSS2, relative to AdSS1, has a slightly lowerK m for IMP and GTP and a significantly higherK m 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. 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. The cloning of full-length AdSS1 into the expression plasmid pET28b was described previously (28Iancu C.V. Borza T. Choe J.Y. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2001; 276: 42146-42152Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). 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 (11Guicherit O.M. Cooper B.F. Rudolph F.B. Kellems R.E. J. Biol. Chem. 1994; 269: 4488-4496Abstract Full Text PDF PubMed Google Scholar). 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). DNA and protein sequences were aligned using Multalign (35Carpet F. Nucleic Acids Res. 1988; 16: 10881-10890Crossref PubMed Scopus (4277) Google Scholar). Published sequences of mouse AdSS2 (GenBankTM gi, 404056 and 6671520) (11Guicherit O.M. Cooper B.F. Rudolph F.B. Kellems R.E. J. Biol. Chem. 1994; 269: 4488-4496Abstract Full Text PDF PubMed Google Scholar) 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) (4Powell S.M. Zalkin H. Dixon J.E. FEBS Lett. 1992; 303: 4-10Crossref PubMed Scopus (15) Google Scholar) 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). AdSS1 was produced and purified as described previously (28Iancu C.V. Borza T. Choe J.Y. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2001; 276: 42146-42152Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). 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 mmNaCl, 10 mm imidazole, pH 8, and 1 mmphenylmethanesulfonyl 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 mmNaH2PO4, 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 mmdithiothreitol, 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 (34Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Protein concentration was determined by the method of Bradford (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar), using bovine serum albumin as a standard. Enzyme activity was determined at an absorbance of 280 nm and at 22 °C as described previously (37Rudolph F.B. Fromm H.J. J. Biol. Chem. 1969; 244: 3832-3839Abstract Full Text PDF PubMed Google Scholar). A standard assay buffer for AdSS1 contained 40 mm HEPES, pH 6.7, 8 mmmagnesium 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. K i 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 K m, at different fixed concentrations of inhibitors ranging over 0.5−2 K i. In experiments to determine the K i of IMP inhibition, concentrations of GTP varied from 15 to 200 μm, those of IMP ranged from 40 to 4,000 μm, and concentrations ofl-aspartate and Mg2+ were 2 and 8 mm, respectively. Kinetic data were analyzed with the computer program GraFit (38Leatherbarrow R.J. GraFit, Version 5. Erithacus Software Ltd., Horley, UK2001Google Scholar). 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 mmNaCl, 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 (39Laue T.M. Bhaivari D.S. Ridgway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar). 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. 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. 1m NaCl washed the acidic isozyme from the column. Identities of the acidic and basic isozymes were confirmed by Western blots and their kinetic properties. 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). Nucleotide sequences of mouse AdSS2, reported here and by Guicherit et al. (11Guicherit O.M. Cooper B.F. Rudolph F.B. Kellems R.E. J. Biol. Chem. 1994; 269: 4488-4496Abstract Full Text PDF PubMed Google Scholar), differ at positions 499 (C in Ref. 11Guicherit O.M. Cooper B.F. Rudolph F.B. Kellems R.E. J. Biol. Chem. 1994; 269: 4488-4496Abstract Full Text PDF PubMed Google Scholar 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 (4Powell S.M. Zalkin H. Dixon J.E. FEBS Lett. 1992; 303: 4-10Crossref PubMed Scopus (15) Google Scholar). Moreover, Gly167 is invariant among synthetases from 43 organisms, representing 30 major phylogenetic lineages. The published sequence of human AdSS2 (4Powell S.M. Zalkin H. Dixon J.E. FEBS Lett. 1992; 303: 4-10Crossref PubMed Scopus (15) Google Scholar) 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. 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 (28Iancu C.V. Borza T. Choe J.Y. Fromm H.J. Honzatko R.B. J. Biol. Chem. 2001; 276: 42146-42152Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Similarly, truncation of the N-terminal polyhistidyl tag from AdSS2 does not change its kinetic properties (data not shown). In the absence of ligands, the E. coli synthetase is in a monomer-dimer equilibrium (K d ∼10 μm) (34Wang W. Gorrell A. Honzatko R.B. Fromm H.J. J. Biol. Chem. 1997; 272: 7078-7084Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). 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. 2T. Borza and H. J. Fromm, unpublished results. In the absence of ligands, AdSS1 and AdSS2 are more stable than theE. 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. Equilibrium sedimentation ultracentrifugation indicates single" @default.
• W2062450780 created "2016-06-24" @default.
• W2062450780 creator A5016594483 @default.
• W2062450780 creator A5023871177 @default.
• W2062450780 creator A5024840895 @default.
• W2062450780 creator A5042968935 @default.
• W2062450780 creator A5090163958 @default.
• W2062450780 date "2003-02-01" @default.
• W2062450780 modified "2023-09-27" @default.
• W2062450780 title "Variations in the Response of Mouse Isozymes of Adenylosuccinate Synthetase to Inhibitors of Physiological Relevance" @default.
• W2062450780 cites W119330955 @default.
• W2062450780 cites W12984822 @default.
• W2062450780 cites W1510520747 @default.
• W2062450780 cites W1528360166 @default.
• W2062450780 cites W1535073961 @default.
• W2062450780 cites W1556420131 @default.
• W2062450780 cites W1559638007 @default.
• W2062450780 cites W1560754421 @default.
• W2062450780 cites W1600838929 @default.
• W2062450780 cites W1601759090 @default.
• W2062450780 cites W1839645581 @default.
• W2062450780 cites W1862804945 @default.
• W2062450780 cites W189428430 @default.
• W2062450780 cites W1931867263 @default.
• W2062450780 cites W1956273663 @default.
• W2062450780 cites W1964419886 @default.
• W2062450780 cites W1967216866 @default.
• W2062450780 cites W1969127492 @default.
• W2062450780 cites W1971660446 @default.
• W2062450780 cites W1973947874 @default.
• W2062450780 cites W1979355339 @default.
• W2062450780 cites W1985091943 @default.
• W2062450780 cites W1995158505 @default.
• W2062450780 cites W1997996830 @default.
• W2062450780 cites W2003526654 @default.
• W2062450780 cites W2010459277 @default.
• W2062450780 cites W2011118797 @default.
• W2062450780 cites W2020103336 @default.
• W2062450780 cites W2028231353 @default.
• W2062450780 cites W2034767282 @default.
• W2062450780 cites W2045074917 @default.
• W2062450780 cites W2047873079 @default.
• W2062450780 cites W2055629148 @default.
• W2062450780 cites W2064034368 @default.
• W2062450780 cites W2066192536 @default.
• W2062450780 cites W2073486504 @default.
• W2062450780 cites W2076840863 @default.
• W2062450780 cites W2076954906 @default.
• W2062450780 cites W2088596522 @default.
• W2062450780 cites W2112082641 @default.
• W2062450780 cites W2114051135 @default.
• W2062450780 cites W2117799383 @default.
• W2062450780 cites W2118929896 @default.
• W2062450780 cites W2129095580 @default.
• W2062450780 cites W2139065251 @default.
• W2062450780 cites W2147814596 @default.
• W2062450780 cites W2161912843 @default.
• W2062450780 cites W2169929748 @default.
• W2062450780 cites W2188618846 @default.
• W2062450780 cites W2399503404 @default.
• W2062450780 cites W2412350391 @default.
• W2062450780 cites W2412733985 @default.
• W2062450780 cites W2413185436 @default.
• W2062450780 cites W2415433544 @default.
• W2062450780 cites W2417475375 @default.
• W2062450780 cites W4293247451 @default.
• W2062450780 cites W954380849 @default.
• W2062450780 doi "https://doi.org/10.1074/jbc.m210838200" @default.
• W2062450780 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12482871" @default.
• W2062450780 hasPublicationYear "2003" @default.
• W2062450780 type Work @default.
• W2062450780 sameAs 2062450780 @default.
• W2062450780 citedByCount "15" @default.
• W2062450780 countsByYear W20624507802015 @default.
• W2062450780 countsByYear W20624507802019 @default.
• W2062450780 countsByYear W20624507802020 @default.
• W2062450780 crossrefType "journal-article" @default.
• W2062450780 hasAuthorship W2062450780A5016594483 @default.
• W2062450780 hasAuthorship W2062450780A5023871177 @default.
• W2062450780 hasAuthorship W2062450780A5024840895 @default.
• W2062450780 hasAuthorship W2062450780A5042968935 @default.
• W2062450780 hasAuthorship W2062450780A5090163958 @default.
• W2062450780 hasBestOaLocation W20624507801 @default.
• W2062450780 hasConcept C119795356 @default.
• W2062450780 hasConcept C181199279 @default.
• W2062450780 hasConcept C185592680 @default.
• W2062450780 hasConcept C55493867 @default.
• W2062450780 hasConcept C86803240 @default.
• W2062450780 hasConceptScore W2062450780C119795356 @default.
• W2062450780 hasConceptScore W2062450780C181199279 @default.
• W2062450780 hasConceptScore W2062450780C185592680 @default.
• W2062450780 hasConceptScore W2062450780C55493867 @default.
• W2062450780 hasConceptScore W2062450780C86803240 @default.
• W2062450780 hasIssue "9" @default.
• W2062450780 hasLocation W20624507801 @default.
• W2062450780 hasOpenAccess W2062450780 @default.
• W2062450780 hasPrimaryLocation W20624507801 @default.
• W2062450780 hasRelatedWork W1550996414 @default.

• next