Porcine thyroid peroxidase: relationship between the native enzyme and an active, highly purified tryptic fragment.
We previously described the preparation of highly purified porcine thyroid peroxidase by a procedure that involved initial solubilization of the enzyme with trypsin plus detergent. Recently, the complete amino acid sequence of porcine thyroid peroxidase (TPO) was determined by cDNA cloning, and it became of interest to compare the structure of the purified trypsin-solubilized enzyme with that of the native enzyme. For this purpose we employed antibodies to the purified enzyme and to two synthetic peptides representing defined regions of the protein. We also obtained N-terminal amino acid sequence data on TPO fragments separated by gel electrophoresis. Trypsin cleavage sites in the purified enzyme were observed after arg residues 109 and 561, and also at two undetermined sites close to the putative membrane spanning region at the carboxyl end. Major fragments of approximately 60, 32, and 29 kilodaltons were observed when the purified enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. This observation is explained by assuming that the cleavage site after arg residue 561 occurred within a disulfide loop. The Mr of the trypsin-solubilized enzyme is approximately 88,000 compared to approximately 106,000 for the native enzyme. The difference can be accounted for by the loss of approximately 90 residues from the amino terminus and of at least 80 residues from the carboxyl end. Despite the loss of these fragments totaling approximately 18 kilodaltons and cleavage of the peptide bond after arg residue 561, the purified trypsin-solubilized TPO appears to retain full enzyme activity.
Yokoyama
N
N
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Taurog
A
A
D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007453:Iodide Peroxidase / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000378:metabolism*
D010446:Peptide Fragments; D007453:Iodide Peroxidase; D014357:Trypsin