Abstract
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.
Highlights
660,000) from rat, human, and bovine thyroid tissues structures, each of which is formed by an array of cells contains -10-12 mol of phosphate/mol of protein. surrounding a centralcavity or lumen (1-5)
The phosphate residues are associated with the thyroglobulin molecule even after it is converted to its 330-kDa subunit form after being subjected to sodium dodecyl sulfate (SDS)-gel electrophoresisunder reducing conditions
One-half of the residues are present in endo-H-sensitive glycopeptide(s) whose carbohydrate moiety is of the mannose-rich, B chain type
Summary
AcquavivaS, Silvestro FormisanoS, Domenico LiguoroS, Adriana GalloS, Tassi VittorioS, Pilar Santistebanijll, Michele De Lucaij, Sidney Shifrinij, Herman J. From the SCentro di Endocrimlogin ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di Biologia e Patologin Cellulure e Molecolure “L.Califam,” Naples, Italy, the §Laboratoryof Biochemistry and Metabolism and the Laboratory of Analytical Chemistry, National Institute of Diabetesand Digestive and Kidney Diseases, National Institutes of HeuZth, Bethesda, Maryland 20892
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