Roles of bovine serum albumin and copper in the assay and stability of ammonia monooxygenase activity in vitro

Abstract

We investigated the effects of bovine serum albumin (BSA) on both the assay and the stability of ammoniaoxidizing activity in cell extracts of Nitrosomonas europaea. Ammonia-dependent O 2 uptake activity of freshly prepared extracts did not require BSA. However, a dependence on BSA developed in extracts within a short time. The role of BSA in the assay of ammonia-oxidizing activity apparently is to absorb endogenous free fatty acids which are present in the extracts, because (i) only proteins which bind fatty acids, e.g., BSA or ␤-lactoglobulin, supported ammonia-oxidizing activity; (ii) exogenous palmitoleic acid completely inhibited ammonia-dependent O 2 uptake activity; (iii) the inhibition caused by palmitoleic acid was reversed only by proteins which bind fatty acids; and (iv) the concentration of endogenous free palmitoleic acid increased during aging of cell extracts. Additionally, the presence of BSA (10 mg/ml) or CuCl 2 (500 M) stabilized ammoniadependent O 2 uptake activity for 2 to 3 days at 4؇C. The stabilizing effect of BSA or CuCl 2 was apparently due to an inhibition of lipolysis, because both additives inhibited the increase in concentrations of free palmitoleic acid in aging extracts. Other additives which are known to modify lipase activity were also found to stabilize ammonia-oxidizing activity. These additives included HgCl 2 , lecithin, and phenylmethylsulfonyl fluoride.

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

“…For example, ammonia oxidation is often strongly but reversibly inhibited by metal-binding agents, and some of the most potent of these are copper-selective compounds such as allylthiourea (9). The selectivity of these compounds for copper as well as the fact that AMO activity can be stimulated and stabilized by copper ions in cell extracts (6,7) suggests that AMO is a copper-dependent enzyme. Many organic compounds also reversibly inhibit ammonia oxidation through their action as alternative substrates for AMO.…”

“…The initial oxidation of ammonia is catalyzed by membrane-bound AMO while hydroxylamine (NH 2 OH), the immediate product of ammonia oxidation, is further oxidized to nitrite (NO 2 Ϫ ) by the periplasmic enzyme hydroxylamine dehydrogenase (HAO) (5). Although N. europaea is the most extensively studied AOB, studies of AMO in this bacterium and AOB in general have historically been hampered by the labile nature of this enzyme (6)(7)(8). However, even though AMO has not yet been obtained in a highly purified active state, considerable insights into the activities and structure of this important enzyme have been obtained from whole-cell studies of N. europaea using different classes of inhibitors (5,9).…”

Influence of ultrasound irradiation on the adsorption of bovine serum albumin on copper

“…BSA has also been suggested as a corrosion inhibitor and its effects on oxide passivation phenomena have been probed [8,9]. Other investigations have been specifically focused on the interactions of BSA with copper, which is helpful for the understanding of biological phenomena related to covalent copper adduct formation [10][11][12].…”

“…Interactions between these two components are regarded as important in biological systems [10]. For this purpose, BSA was allowed to adsorb on the surface of naturally oxidised pure copper immersed in pH 7 buffer electrolyte.…”

Inhibition of Membrane-Bound Methane Monooxygenase and Ammonia Monooxygenase by Diphenyliodonium: Implications for Electron Transfer

“…Although there are other enzymes that are able to oxidize small alkanes (32,46,47), they are distinct in structure and properties from AMO and pMMO. These two enzymes thus represent a novel mechanism for alkane oxidation, one that has yet to be elucidated due to the difficulty in isolating pure samples of either enzyme with high activity (4,17,28,31,49,51).AMO and pMMO initiate oxidative metabolism of NH 3 and CH 4 in nitrifying and methanotrophic bacteria, respectively (3,19). Both types of bacteria obtain energy solely from the subsequent oxidation of the products of the monooxygenase reaction.…”