Adenylosuccinate lyase from Artemia embryos. Purification and properties

In crude extracts, the molecular form of adenylosuccinate lyase is pH-dependent as studied by gel filtration and sucrose gradient centrifugation. At pH values of 8.7 and 6.5, the enzyme exhibits molecular forms of 200 kDa and larger than 500 kDa, respectively. At pH values of 7.0-7.5, forms of intermediate molecular weight were detected. Interconversion among the different molecular forms of adenylosuccinate lyase was not achieved when the enzyme was subjected to two successive chromatographic steps on Sepharose CL-6B using elution buffers at two different pH values. A unique form of 200 kDa was observed, regardless of the pH of the buffer used, upon either gel filtration or sucrose gradient centrifugation in the presence of 1 M NaCl. The enzyme from Artemia cysts was purified to homogeneity. It had molecular mass of 200 kDa and gave a single band of 56 kDa upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Km values for adenylosuccinate, AMP, and fumarate were 1, 36, and 350 microM, respectively. The enzyme exhibits a Uni Bi-ordered mechanism and is maximally active at a pH value of 8.0-8.5. For maximum activity, the enzyme requires an ionic strength equivalent to 80 mM KCI. The isoelectric point determined by chromatofocusing was 5.04.