Carbonic anhydrase III (CAIII) was shown to be highly oxidatively modified in young rat liver tissue. In addition, the reported phosphotyrosine-phosphatase activity of this isozyme has been shown to be inhibited by reduction with DTT and stimulated by oxidation with glutathione (GSSG). A major focus of this project is to identify the substrate upon which this phosphatase activity acts. Male rat liver contains considerably more CAIII than female liver and, as such, these two tissues provide the basis for a search for potential substrates. Homogenates of male and female rat liver were probed with antibodies versus phosphotyrosine. Many phosphotyrosine-containing proteins were observed on SDS-PAGE without stimulation once sufficient assay sensitivity was obtained, but no significant differences between the sexes was observed. Similarly, no differences were observed by immunoprecipitation with either anti-CAIII or anti-phospho-tyrosine followed by western blotting with the other antibody. Cross-linking experiments on whole male rat liver homogenates failed to yield any proteins closely associated with CAIII as detected by western blotting with a polyclonal anti-CAIII antibody. Rat fibroblast cultures were evaluated for the presence of CAIII by western blotting techniques. In the course of this investigation it was appreciated that rat CAIII and rat CAII (which is expressed at a higher level) are nearly indistinguishable by SDS-PAGE and isoelectric focusing and that CAII cross-reacts with our polyclonal antibody. Subsequently, using sequence information and the published x-ray structures of human CAII and bovine CAIII, several potential epitopes were identified which are surface accessible strands having a high degree of divergence between these two isotypes. Two of these epitopes were selected, synthesized as multiple antigen (dendrimer) peptides (MAP's), and used to raise antibodies in rabbits. Unfortunately, the resulting antibody preparations react poorly with whole CAIII. Recombinant, overexpressed rat liver CAIII was purified from E. coli and found to have negligible phosphatase activity and is unstimulated by treatment with GSSG. This material will be used to generate new polyclonal antibodies in the coming year.
| Selengut, J D; Levine, R L (2000) MDP-1: A novel eukaryotic magnesium-dependent phosphatase. Biochemistry 39:8315-24 |
