Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the initial step in the conversion of
IMP to
AMP. Two
isoforms of this
enzyme have been observed in vertebrates. A muscle
isozyme is highly abundant in cardiac and skeletal muscle tissue and is thought to play a role in muscle energy metabolism. The non-muscle
isozyme, which is present at low levels in most tissues, likely functions in de novo
AMP biosynthesis. The analysis of the non-muscle
isozyme has been hampered by its low abundance and instability during purification. In this study a genetic selection scheme was used to generate a murine T-
lymphoma cell line which was at least 100-fold enriched for the non-muscle
isozyme, as a result of amplification of the non-muscle
synthetase gene. This cell line made possible the purification of the non-muscle
isozyme, and the subsequent isolation of
isozyme-specific
peptides. Based on
peptide sequence information a degenerate
oligonucleotide probe was designed and used to screen a mouse kidney cDNA library. A 1.5-kilobase
cDNA encoding the non-muscle
isozyme was cloned and found to contain an open reading frame of 1368 base pairs encoding 456
amino acids. Gene transfer experiments showed that the
cDNA encoded a 50-kDa
protein, the size expected for mammalian
synthetases, that correlated with the presence of high levels of
synthetase activity. The deduced amino acid sequence of the mouse non-muscle
synthetase is approximately 75% identical to the previously reported mouse muscle
synthetase. Southern blot analysis of mouse genomic
DNA with the
isozyme-specific
cDNA probes revealed that the
synthetase isozymes are encoded by separate genes. The non-muscle gene is expressed in most tissues but is virtually undetectable in striated muscle tissues. Three different transcripts (1.7, 2.8, and 3.4 kilobases) are detected for the non-muscle
isozyme which show a similar tissue distribution. The availability of a
cDNA for the non-muscle
isozyme of
adenylosuccinate synthetase will facilitate further comparative analyses with the previously cloned muscle
isozyme.