Role of the N-terminus of rat pheochromocytoma tyrosine hydroxylase in the regulation of the enzyme's activity.
Activation of rat pheochromocytoma tyrosine hydroxylase by limited tryptic proteolysis was investigated. The modifications produced upon the enzyme's structure were analyzed with the use of sodium dodecyl sulfate/polyacrylamide gel electrophoresis and tyrosine hydroxylase activity was measured all through the digestion. During the proteolysis the activity of tyrosine hydroxylase was elevated threefold at the same time as a 56-kDa tryptic fragment was formed. When the enzyme was phosphorylated, at its N-terminal region, by a kinase copurified with tyrosine hydroxylase, the major 56-kDa species did not appear to be phosphorylated on the autoradiograph, suggesting that it was derived from the native subunit by cleavage of the N-terminal of the protein. The reactivity of the 2/40/15 anti-(tyrosine hydroxylase) monoclonal antibody with the N-terminal of tyrosine hydroxylase was also investigated, using the Western-blot technique. This antibody reacted with the 62-kDa hydroxylase subunit but not with the 60-kDa tryptic fragment; the amino acid sequences of these two species showed that the 60-kDa fragment lacked the first 16 N-terminal amino acids of the native molecule. These results suggest that the N-terminal region of tyrosine hydroxylase is apparently responsible for an inhibition of the hydroxylase activity and that the first N-terminal amino acids of the hydroxylase are necessary for the recognition of the enzyme by its antibody.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001345:Autoradiography; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010673:Pheochromocytoma / Q000201:enzymology*; D010766:Phosphorylation; D051381:Rats; D014357:Trypsin / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014446:Tyrosine 3-Monooxygenase; D014357:Trypsin