Open AccessMonoamine Oxidase B Isolated from Bovine Liver Exists as Large Oligomeric Complexes in Vitro
Author(s) -
Shiloff Bryan A.,
Behrens Patricia Q.,
Kwan SauWah,
Lee Jong H.,
Abell Creed W.
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.0041r.x
Subject(s) - size exclusion chromatography , octyl glucoside , chemistry , protein quaternary structure , enzyme , chromatography , enzyme assay , dimer , gel permeation chromatography , molecular mass , ultracentrifuge , biochemistry , monoamine oxidase , protein subunit , organic chemistry , gene , polymer
The quaternary structure and subunit composition of bovine liver monoamine oxidase B (MAO B) was investigated using size‐exclusion chromatography, sucrose gradient centrifugation and electron microscopy. Purified enzyme was subjected to Superdex gel filtration column chromatography in the presence of the non‐ionic detergents, n ‐octyl β‐ d ‐glucopyranoside (octyl glucoside) and Triton X‐100R‐PC (Triton). The specific activity and elution profiles indicate that the enzyme exists as a dimer and preferentially functions as larger oligomeric complexes. Distribution of the oligomeric forms of MAO B was found to be dependent upon protein concentration. Dilution of the enzyme, however, had little or no effect upon the specific activity profiles. In Triton and octyl glucoside, plots of specific activity versus molecular mass displayed a sigmoidal shape. The chromatographic data suggest that detergent‐solubilized bovine liver MAO B exists as cooperative oligomeric enzyme complexes. Similarly, sucrose density gradient centrifugation of purified MAO B exhibited a direct correlation between enzyme activity and molecular mass of the MAO complexes. MAO B activity was found to be widely distributed throughout the sucrose gradient and the highest enzyme activity was contained in the high‐density sucrose layers. MAO B specific activity is dependent upon the size of the protein complexes and, therefore, oligomerization of the enzyme may play a role in the regulation of MAO B. Transmission electron microscopy of purified MAO B was performed using protein prepared by octyl glucoside extraction. Purified enzyme was applied to Formvar‐coated copper grids and negatively stained with methylamine tungstate. MAO‐B‐specific monoclonal antibody (MAO B‐1C2) conjugated to colloidal gold was used as a probe. Contrast enhancement of the electron microscopy data showed that detergent‐depleted enzyme tends to aggregate in a linear arrangement of oligomeric complexes. Our data suggest that the MAO B oligomeric complexes are hexamers which contain threefold rotational symmetry. The individual complexes have globular morphology and the hexamers appear to be composed of a trimer of MAO B homodimers.