The Microheterogeneity of Rabbit Testosterone-Binding Globulin Is Due to Differential Glycosylation of fts Single Protomer1

Affinizy-purfied rabbit testosterone-binding globulin (rbTeBG) is a homodimer with a molecular weight (Mr) of about 92,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the chemically cross-linked protein. When noncross-l inked rbTeBG is subjected to SDS-PAGE, individual protomers (Mr44,400 ±400 (JJtd Mr 42,000 ± 1300) are resolved. The protomers are present in a ratio of approximately 2 (heavy):) (light). Enzymatic deglycosylation of native rbTeBG or of rbTeBG that had been photoaffinity-labeled with!), 2-3HJ17-hydroxy-4,6-androstadien-3-one was conducted. The products were then identified on immu-noblots using a monoclonal antibody that cross-reacts with rbTeBG, or by fluorography. These analyses indi-cated that rbTeBG contained sialic acid and asparagine (Asn)-linked oligosaccharides and provided evidence for the presence of serine/threonine (0)-linked glycans on the molecule. The preswnptive removal of all oligosaccha-rides by enzymatic or chemical means resulted in the appearance of a single subunit (Mr 37,150±1200). On the basis of this monomeric molecular weight, carbohydrate would contribute 16 % and 11 % to the relative molecular mass of the nondeglycosylated heavy and light subunits, respectively. Therefore, the size heterogeneity of the nondeglycosylated rbTeBG subunits is a result of their differential glycosylation. In addition to size heterogeneity, the rbTeBG subunits are composed of multiple-charge variants. Although enzymatic and chemical methods of glycan removal altered the isoelectric points of the isoforms, none of the treatments yielded a single isoform. Thus, it is possible that moieties other than oligosaccharides are contributing charge to the isoelectric variants of rbTeBG