Preparation and characterization of a novel monoclonal antibody specific to human CAD protein.

We have efficiently generated the first monoclonal antibody against the cis-aconitic acid decarboxylase (CAD) protein, which is an enzyme of low stability. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using either purified 6 x His-CAD fusion protein or purified 6 x His-ZNRD1 fusion protein as a control. One monoclonal antibody (MAb) named Z12 (IgG1), which is effective in detecting the recombinant and the cellular protein, was characterized by ELISA and Western immunoblotting. The CAD protein was also detected in gastric cancer and colon cancer tissues by immunohistochemical analysis. Thus, Z12 binds to native CAD protein and should be useful in studies of CAD protein function and expression.

Proteolytic cleavage of the multienzyme polypeptide CAD to release the mammalian aspartate transcarbamoylase. Biochemical comparison with the homologous Escherichia coli catalytic subunit.

We have demonstrated biochemically that the conformation of the proteolytic fragment (mammalian aspartate transcarbamoylase) from the C-terminus of the 240-kDa multienzyme polypeptide carrying the activities carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydroorotase (CAD) is similar to that of the catalytic subunits from Escherichia coli aspartate transcarbamoylase. We have measured the extent of unfolding of the mammalian aspartate transcarbamoylase in guanidinium chloride solutions, and have also demonstrated that the protein cross-reacts with antibodies raised against the E. coli enzyme. CAD is digested by low concentrations of trypsin in the presence of 0.2 mM UTP to release an active aspartate transcarbamoylase domain and a 195-kDa 'nicked CAD' molecule containing active carbamoyl phosphate synthetase. These two products are easily separated by ion-exchange chromatography. Similar proteolytic cleavage and trimming by elastase releases a family of aspartate transcarbamoylase fragments. Direct N-terminal sequencing of the aspartate transcarbamoylase fragments confirms predictions of the most accessible residues in the region linking the aspartate transcarbamoylase and dihydroorotase domains. Only the largest of the four fragments generated by elastase retains phosphorylation site 2. When this largest fragment is phosphorylated, the family of aspartate transcarbamoylase fragments is eluted together from ion-exchange columns in a different fraction from the completely unphosphorylated preparation, demonstrating the affinity of the domains for each other.