Purification of adenylosuccinate lyase from rat skeletal muscle by a novel affinity column. Stabilization of the enzyme, and effects of anions and fluoro analogues of the substrate.
Adenylosuccinate lyase from rat skeletal muscle was purified to apparent homogeneity by a combination of ion-exchange chromatography and affinity chromatography on agarose containing covalently bound adenylophosphonopropionate. The purified enzyme is stable when stored in 20% glycerol at -70 degrees C, and can be thawed and re-frozen with minimal loss of activity. Adenylosuccinate lyase has a specific activity of 11 mumol/min per mg of protein at 25 degrees C. Its subunit Mr is 52,000, by SDS/polyacrylamide-gel electrophoresis, and its apparent native Mr is approx. 200,000, by gel filtration. The purified enzyme has Km values for adenylosuccinate and 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) of 1.5 microM and approximately 1 microM respectively, in Hepes/KOH buffer, pH 7.4. Several monoanions and dianions activate the enzyme at low concentration; several of these inhibit the enzyme at high concentrations. Fluoro analogues of adenylosuccinate and SAICAR were synthesized by using highly purified adenylosuccinate synthase and SAICAR synthase respectively, and erythro-beta-fluoroaspartate in place of aspartate. Both analogues are competitive inhibitors of adenylosuccinate lyase in both of the reactions catalysed by the enzyme, with Ki values well below the Km values for the two substrates.
Casey
P J
PJ
Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02254.
Lowenstein
J M
JM
D000264:Adenylosuccinate Lyase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D000838:Anions / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008190:Lyases / Q000302:isolation & purification*; D008297:Male; D009132:Muscles / Q000201:enzymology*; D009711:Nucleotides / Q000494:pharmacology; D051381:Rats
D000838:Anions; D009711:Nucleotides; D008190:Lyases; D000264:Adenylosuccinate Lyase