Limited proteolysis of tyrosine hydroxylase by Ca(2+)-activated neutral protease (calpain).
Limited proteolysis of tyrosine hydroxylase (TH) by calpain, Ca(2+)-activated neutral protease, was studied. Cleavage of TH with calpain in vitro produced molecules having Mrs of approximately 57,000 and 56,000 in SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence, Ser-Pro-Arg-Phe-Val, of the 56-kDa species indicated that calpain cleaved off the N-terminal region (residues 1-30) encoded by the first exon including Ser-8 and Ser-19, the phosphorylation sites by proline-directed protein kinase (PDPK) and by Ca2+/calmodulin-dependent protein kinase II (kinase II), respectively, from the native TH. The removal of the N-terminal region from the native molecule induced a slight but significant activation of TH at pH 7.0. The native TH behaved as the tetramer with an Mr of 240,000. In contrast, calpain-cleaved TH showed the monomeric Mr by gel permeation chromatography and increased Ki for catecholamine which inhibits the native TH in competition to the coenzyme, DL-6-methyl-5,6,7,8-tetrahydropterin (6MPH4). These results imply that calpain cleavage would effectively release TH from the feedback inhibition by removal of the N-terminal region resulting in disruption of the quaternary structure.
Kiuchi
K
K
Radioisotope Center Medical Division, Nagoya University School of Medicine, Japan.
Kiuchi
K
K
Titani
K
K
Fujita
K
K
Suzuki
K
K
Nagatsu
T
T
D000313:Adrenal Medulla / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D014446:Tyrosine 3-Monooxygenase; D002154:Calpain