The microheterogeneity of rabbit testosterone-binding globulin is due to differential glycosylation of its single protomer.
Affinity-purified 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-linked rbTeBG is subjected to SDS-PAGE, individual protomers (Mr approximately equal to 44,400 +/- 400 and Mr approximately equal to 42,000 +/- 1300) are resolved. The protomers are present in a ratio of approximately 2 (heavy):1 (light). Enzymatic deglycosylation of native rbTeBG or of rbTeBG that had been photoaffinity-labeled with [1,2-3H]17 beta-hydroxy-4,6-androstadien-3-one was conducted. The products were then identified on immunoblots using a monoclonal antibody that cross-reacts with rbTeBG, or by fluorography. These analyses indicated that rbTeBG contained sialic acid and asparagine (Asn)-linked oligosaccharides and provided evidence for the presence of serine/threonine (O)-linked glycans on the molecule. The presumptive removal of all oligosaccharides by enzymatic or chemical means resulted in the appearance of a single subunit (Mr approximately equal to 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.
Danzo
B J
BJ
Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2633.
Black
J H
JH
Bell
B W
BW
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D015151:Immunoblotting; D007526:Isoelectric Point; D008297:Male; D008970:Molecular Weight; D011817:Rabbits; D012738:Sex Hormone-Binding Globulin / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D013329:Structure-Activity Relationship
D012738:Sex Hormone-Binding Globulin