Immunochemistry of the subunits of transcarboxylase

Antisera reactive with the subunits of transcarboxylase were produced by injecting rabbits with the 12 SH and 5 SE subunits prepared from trypsin-treated enzyme and with the 12 SH subunit prepared from native transcarboxylase by avidin-Sepharose chromatography. Biotinyl peptides (46 and 65 amino acid residues), released from the enzyme by brief trypsin treatment, formed complexes with avidin and this noncovalent conjugate was used as an antigen to prepare antibodies reactive with the 1.3 SE biotin carboxyl carrier protein. All of these antisera were capable of inhibiting and precipitating intact transcarboxylase. In competitive binding experiments utilizing enzyme inhibition to detect antibody binding, it was found that only the 5 SE subunit was capable of preventing the anti-5 SE sera from inhibiting the enzyme. This technique is useful as a rapid, specific assay for the 5 SE subunit. Both the 5 SE and 12 SH subunits were capable of preventing the anti-12 SH sera from inhibiting the enzyme. To further study this apparent cross-reactivity, the antisera were tested for their capacity to inhibit the partial reactions of transcarboxylase as catalyzed by the isolated subunits. These studies revealed that the anit-12 SH serum inhibits the partial reaction catalyzed by the 12 SH subunit, but preincubation with the 5 SE subunit does not relieve this inhibition, confirming that specific antibody against the 12 SH subunit was formed. These studies also showed that the anti-12 SH serum could inhibit the 5 SE partial reaction, apparently demonstrating cross-reactivity with the 5 SE subunits of the intact enzyme. A panel of immunologic methods was used to characterize the cross-reactivity and quantitate any cross-contamination of the subunit preparations. Passive hemagglutination-hemagglutination inhibition, double immunodiffusion in gels, and competitive precipitation of radioactive antigen all demonstrated cross-reactivity between the isolated subunits and the antisera. Although the subunit preparations apparently are cross-contaminated to a minor extent, this seems insufficient to account for the quantitative aspects of the immunologic cross-reactivity of the subunits with the antisera. Structural homology between the subunits of transcarboxylase is considered to be responsible for the observed cross-reactivity.