Endogenous proteolytic activity and constituent polypeptide chains of sheep and pig 19 S thyroglobulin.
Porcine and ovine 19-S thyroglobulins prepared from frozen glands in several buffers using slice extraction or homogenization, ammonium sulfate precipitation and DEAE-cellulose chromatography or Sepharose 6B gel filtration were contaminated with protease activity of pH optima 4.5 and 8.6, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimum temperatures of autodigestion were 37 degrees C at pH 4.5 and 25 degrees C at pH 8.6. Thyroglobulins prepared from unfrozen glands pH 7.2 in 0.1 M sodium phosphate using slice extraction, ammonium sulfate precipitation and Sepharose 6B gel filtration were devoid of acid proteolytic activity but still underwent autodigestion at pH 8.6. Diisopropylfluorophosphate was a potent inhibitor of the alkaline protease activity of ovine thyroglobulin preparations. In contrast to thyroglobulin obtained from frozen glands the proteins purified from fresh unfrozen glands at pH 7.2 only showed the 19-S and the 12-S species by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Very few bands migrating faster than 12-S were visible. After full reduction and S-alkylation of porcine and ovine thyroglobulins, no qualitative changes were observed in the gel electrophoresis pattern as compared to the unmodified proteins. Species of apparent mol. wt. corresponding to the native 12 S were the major component, strongly suggesting a mol. wt. of about 330 000 for the elementary peptide chains of pig and sheep thyroglobulins.
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007455:Iodine / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D012756:Sheep; D013045:Species Specificity; D013552:Swine; D013696:Temperature; D013954:Thyroglobulin / Q000378:metabolism
D046911:Macromolecular Substances; D013954:Thyroglobulin; D007455:Iodine; D010447:Peptide Hydrolases