caa a22 4500 397556918 CHVBK 20180308164805.0 cr unu---uuuuu 161202e199608 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021419 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021419 Selective Cleavage and Modification of the Intersubunit Disulfide Bonds of Bovine Dopamine β-Monooxygenase: Conversion of Tetramer to Active Dimer [Elektronische Daten] [Tetsuo Ishida, Mitauhiro Narita, Mitsuhiro Nozaki, Kihachiro Horiike] Bovine dopamine β-monaooxygenase is a tetramer consisting of two disulfide-linked dimers. To examine the role(s) of the intersubunit disulfide bonds in the protein structure and activity, the enzyme was treated with DTT at pH 7.5 and 25°C under nondenaturing conditions. A 15-min incubation with 0.5 mM DTT selectively cleaved half of the intersubunit disulflde bonds. The cleavage did not affect the activity or tetrameric structure of the enzyme. Upon chemical modification of the reduced cysteine residues with 0.1 M iodoacetamide (IAA) for 60 min, half of the tetramer was converted to a dimeric species. The resulting dimeric and tetrameric species exhibited similar kinetic properties, and the Vmax values were decreased by 30% compared to that of the native enzyme. Upon treatment with IAA alone, no dimer species was detected but the enzyme lost 30% of the original activity. Cys514 and Cys516 were selectively modifled by the treatment with DTT and IAA. From these results, we concluded that: (i) chemical modification of the intermolecular disulfide bonds strongly destabilizes the intersubunit interaction; (ii) breakage of the intersubunit interaction does not affect the activity. The reduction mechanism of the intersubunit disulfide bonds and the roles of the intersubunit interactions are discussed. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence chemical modification nationallicence disulfide bonds nationallicence dithiothreitol nationallicence dopamine β-monooxygenase nationallicence intersubunit interaction nationallicence Ishida Tetsuo Department of Biochemistry Shiga Unversity of Medical Science Seta, Ohtsu Shiga 520-21 aut Narita Mitauhiro Department of Urology Shiga Unversity of Medical Science Seta, Ohtsu Shiga 520-21 aut Nozaki Mitsuhiro Department of Biochemistry Shiga Unversity of Medical Science Seta, Ohtsu Shiga 520-21 aut Horiike Kihachiro Department of Biochemistry Shiga Unversity of Medical Science Seta, Ohtsu Shiga 520-21 aut The Journal of Biochemistry Oxford University Press 120/2(1996-08), 346-352 0021-924X 120:2<346 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021419 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021419 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Ishida Tetsuo Department of Biochemistry Shiga Unversity of Medical Science Seta, Ohtsu Shiga 520-21 aut NATIONALLICENCE 700 1- Narita Mitauhiro Department of Urology Shiga Unversity of Medical Science Seta, Ohtsu Shiga 520-21 aut NATIONALLICENCE 700 1- Nozaki Mitsuhiro Department of Biochemistry Shiga Unversity of Medical Science Seta, Ohtsu Shiga 520-21 aut NATIONALLICENCE 700 1- Horiike Kihachiro Department of Biochemistry Shiga Unversity of Medical Science Seta, Ohtsu Shiga 520-21 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/2(1996-08), 346-352 0021-924X 120:2<346 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford