Characterization of phosphate residues on thyroglobulin.
Follicular 19 S thyroglobulin (molecular weight 660,000) from rat, human, and bovine thyroid tissues contains approximately 10-12 mol of phosphate/mol of protein. These phosphate residues can be radiolabeled when rat thyroid hemilobes, FRTL-5 rat thyroid cells, or bovine thyroid slices are incubated in vitro with [32P]phosphate. Thus labeled, the [32P]phosphate residues comigrate with unlabeled 19 S follicular thyroglobulin on sucrose gradients and gel filtration columns; are specifically immunoprecipitated by an antibody preparation to rat or bovine thyroglobulin as appropriate; and co-migrate with authentic 19 S thyroglobulin when subjected to analytic or preparative gel electrophoresis. Tunicamycin prevents approximately 50% of the phosphate from being incorporated into FRTL-5 cell thyroglobulin. Approximately one-half of the phosphate in FRTL-5 cell or bovine thyroglobulin can also be released by enzymatic deglycosylation and can be located in Pronase-digested peptides which contain mannose, are endo-beta-N-acetylglucosaminidase H but not neuraminidase-sensitive, and release a dually labeled oligosaccharide containing mannose and phosphate after endo-beta-N-acetylglucosaminidase H digestion. The remainder of the phosphate is in alkali-sensitive phosphoserine residues (3-4/mol of protein) and phosphotyrosine residues (approximately 2/mol of protein). This is evidenced by electrophoresis of acid hydrolysates of 32P-labeled thyroglobulin and by reactivity with antibodies directed against phosphotyrosine residues. The phosphoserine and phosphotyrosine residues do not appear to be randomly located through the thyroglobulin molecule since approximately 75-85% of the phosphotyrosine and phosphoserine residues were recovered in a approximately 15-kDa tryptic peptide or a approximately 24-kDa cyanogen bromide peptide, each almost devoid of carbohydrate. 31P nuclear magnetic resonance studies of bovine thyroglobulin confirm the presence and heterogeneity of the phosphate residues on thyroglobulin preparations.
D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008358:Mannose / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D010710:Phosphates / Q000032:analysis*; D010768:Phosphoserine / Q000032:analysis; D019000:Phosphotyrosine; D011402:Pronase / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D013954:Thyroglobulin / Q000032:analysis*; D013961:Thyroid Gland / Q000032:analysis / Q000187:drug effects; D014415:Tunicamycin / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis
D002241:Carbohydrates; D010710:Phosphates; D014415:Tunicamycin; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine; D013954:Thyroglobulin; D008715:Methionine; D011402:Pronase; D008358:Mannose