Purification and cDNA-derived sequence of adenylosuccinate synthetase from Dictyostelium discoideum.
Adenylosuccinate synthetase (IMP:L-aspartate ligase (GDP), EC 6.3.4.4) plays an important role in purine biosynthesis catalyzing the GTP-dependent conversion of IMP to AMP. The enzyme was purified from the cytosol of Dictyostelium discoideum using GTP-agarose chromatography as the critical step. It has an apparent molecular mass of 44 kDa. Monoclonal antibodies identified several forms of the enzyme with pI values between 8.1 and 9.0. Michaelis-Menten constants (Km) were low for the nucleotide substrates IMP (Km = 30 microM) and GTP (Km = 35 microM) as compared with the value for aspartic acid (Km = 440 microM). These values are in good agreement with constants reported from other organisms. Immunological studies indicated that the protein is predominantly localized in the cytosol and only partially associated with particulate fractions. The enzyme is present throughout the developmental cycle of D. discoideum. Using monoclonal antibodies, the gene was cloned from a lambda gt11 expression library. The complete sequence represents the first reported primary structure of an eucaryotic adenylosuccinate synthetase. Southern blots hybridized with a cDNA probe demonstrate that adenylosuccinate synthetase is encoded by a single gene and contains at least one intron. The deduced amino acid sequence shows 43% identity to adenylosuccinate synthetase from Escherichia coli. Homologous regions include short sequence motifs, such as the glycine-rich loop which is typical for GTP-binding proteins.
Wiesmüller
L
L
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Wittbrodt
J
J
Noegel
A A
AA
Schleicher
M
M
D000265:Adenylosuccinate Synthase / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015139:Blotting, Southern; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D004271:DNA, Fungal / Q000235:genetics; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D006160:Guanosine Triphosphate / Q000378:metabolism; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D004271:DNA, Fungal; D006160:Guanosine Triphosphate; D000265:Adenylosuccinate Synthase