Transcarboxylase. An electron microscopic study of the enzyme with antibodies to biotin.
Antibodies prepared against biotin linked to serum albumin have been purified by affinity chromatography using lipoyl-Sepharose (Harmon, F. (1980) Anal. Biochem. 103, 58-63). These antibodies have been used to study the structure of the biotin-containing enzyme, transcarboxylase, by examination of complexes with the enzyme using electron microscopy. The antibodies are observed complexed with the outer subunit of transcarboxylase, which contains the biotin, but they also combine with the central subunit which does not contain biotin. Addition of excess biotin does not prevent combination of the antibodies with the central subunit. Such nonspecific combination does not occur with the outer subunit which has been treated with avidin-Sepharose to remove the biotinyl subunit. Large insoluble intermolecular enzyme complexes are not observed, apparently because the divalent antibodies react with the multiple sites on a single transcarboxylase molecule or because the larger complexes are unstable. Combination of the antibodies with the enzyme weakens the intersubunit linkages, thus, when grids are prepared for electron microscopy, there frequently is much greater dissociation of the enzyme which is complexed than there is with the noncomplexed enzyme. The results have confirmed that the biotinyl subunits remain complexed with the outer subunits when transcarboxylase dissociates.
D000906:Antibodies; D000936:Antigen-Antibody Complex; D001710:Biotin / Q000276:immunology*; D019878:Carboxyl and Carbamoyl Transferases; D046911:Macromolecular Substances; D008764:Methylmalonic Acid; D008854:Microscopy, Electron; D008961:Models, Structural; D008970:Molecular Weight; D011424:Propionibacterium / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation; D014166:Transferases
D000906:Antibodies; D000936:Antigen-Antibody Complex; D046911:Macromolecular Substances; D001710:Biotin; D008764:Methylmalonic Acid; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase