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.
Highlights
Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDPforming), EC 6.3.4.4) is present in almost all organisms, the only exceptions being some intracellular prokaryotic parasites [1]
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
The purine nucleotide cycle (PNC) is active in muscle, brain, kidney, liver, and pancreatic islets [5, 13,14,15,16,17] and involves adenylosuccinate synthetase, adenylosuccinate lyase, and AMP deaminase in the following net reaction (Reaction 1): L-aspartate ϩ GTP ϩ H2O ϭ fumarate ϩ GDP ϩ Pi ϩ NH3
Summary
Materials—E. coli strain BL21 (DE3), plasmid pET28b, nickel-nitrilotriacetic acid-agarose, and the thrombin cleavage capture kit were from Novagen, Inc. After centrifugation (24,000 ϫ 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. 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. Precipitated protein was dialyzed against 25 mM HEPES, 25 mM NaCl, 1 mM dithiothreitol, and 0.5 mM EDTA, pH 7.5, and loaded onto a DEAE-Sepharose column equilibrated with the same buffer. Antibodies and Western Blots—Polyclonal antibodies against recombinant AdSS1 were raised in rabbits at the Iowa State University protein facility and were purified by affinity chromatography, using an Econo-Pac serum IgG purification kit (Bio-Rad). Detection of antigen-antibody complexation employed the Opti-4CN kit (Bio-Rad)
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