Role of the N‐terminus of rat pheochromocytoma tyrosine hydroxylase in the regulation of the enzyme's activity

Abstract

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.

Amino‐Terminal Analysis of Tryptophan Hydroxylase: Protein Kinase Phosphorylation Occurs at Serine‐58

“…The aromatic amino acid hydroxylases have been hypothesized to be composed of amino-terminal regulatory domains and carboxyl-terminal catalytic domains (Grenett et al, 1987;Abate et al, 1988;Bonnefoy et al, 1988;Abate and Joh, 1991;Daubner et al, 1993;Ribeiro et al, 1993;Vrana et al, 1994;Walker et al, 1994;Yang and Kaufman, 1994;Daubner and Piper, 1995;Ota et al, 1995;D'Sa et al, 1996;Quinsey et al, 1996). Deletion mutants of TPH were designed and constructed in the present experiments to test this hypothesis.…”

“…functional relationship between these aromatic amino acid hydroxylases. In previous studies, the functional domain organization of TH has been characterized (Abate et al, 1988;Bonnefoy et al, 1988;Abate and Job, 1991;Daubner et al, 1993;Ribeiro et al, 1993;Walker et al, 1994;Daubner and Piper, 1995;Ota et al, 1995;Quinsey et al, 1996). Removal of the initial 155-165 amino-terminal residues of TH produced a recombinant enzyme that retained enzymatic activity (Abate et al, 1988;Daubner et al, 1993;Ribeiro et al, 1993;Walker et al, 1994).…”

Purification and characterization of the blue-green rat phaeochromocytoma (PC12) tyrosine hydroxylase with a dopamine-Fe(III) complex. Reversal of the endogenous feedback inhibition by phosphorylation of serine-40

“…in a trypsin-digestion time-course experiment (Bonnefoy et al, 1988). N-Terminal sequence analysis of purified PC12 TH shows a maximum of 100% of the PC12 TH truncated after Arg-36 (J.…”

A Novel Rat Tyrosine Hydroxylase mRNA Species Generated by Alternative Splicing

“…The new mRNA species encodes a putative RTH-2 protein lacking 33 amino acids between residues 75 and 108 of the enzyme. This deletion occurs in the N-terminal region that corresponds to the regulatory domain of the enzyme (Bonnefoy et al, 1988;Daubner et al;1993). This N-terminal region has been described to have an overall inhibitory effect on the enzyme activity including a control on substrate access to the active site and on cofactor binding (Abate and Joh, 1991;Ribeiro et al, 1993;Walker et al, 1994) and to contain four serines located at positions 8, 19, 31, and 40, which could be phosphorylated by different protein kinases and thus regulate TH activity (Campbell et a!., 1986;Haycock, 1993).…”