Characterization of serum immunoglobulin M of the Antarctic teleost Trematomus bernacchii.
Trematomus bernacchii immunoglobulin M concentration was determined in the serum by ELISA; the mean concentration value was 2.7 mg/ml corresponding to 9.6% of the total serum proteins. Purified IgM was analyzed by SDS-polyacrylamide gel electrophoresis, isoelectrofocusing and 2D electrophoresis. The relative molecular mass of the polymeric form was 830 kDa; that of separated H and L chains was, respectively, 78 and 25 kDa. The isoelectric points of the entire molecule ranged from 4.4 to 6.5, that of isolated H chains was between 4.0 and 6.0. Separated H chains were shown to reaggregate in tetrameric form. The cleavage site of trypsin was at the end of the CH1 domain, as confirmed by the N-terminal amino acid sequence of one of the resultant peptides. Immunoblotting was used to detect carbohydrates in the H and L chains labeled with digoxigenin. Glycosyl residues were detected only in the H chain. The carbohydrate content was evaluated to be 12.8% of the entire chain. Purified Igs were hydrolyzed by N-glycosidase F at different conditions and at least four different hydrolytic sites were revealed by limited deglycosylation. T. bernacchii IgM was also compared to those of five other polar fish species.
Pucci
Biagio
B
Institute of Protein Biochemistry, CNR, Via Marconi 10, 80125 Naples, Italy.
Coscia
Maria Rosaria
MR
Oreste
Umberto
U
D000818:Animals; D000864:Antarctic Regions; D002241:Carbohydrates / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007075:Immunoglobulin M / Q000097:blood* / Q000737:chemistry* / Q000302:isolation & purification; D010473:Perciformes / Q000276:immunology*; D014357:Trypsin / Q000378:metabolism
D002241:Carbohydrates; D007075:Immunoglobulin M; D014357:Trypsin