Methylamine oxidase from Arthrobacter P1 as a prototype of eukaryotic plasma amine oxidase and diamine oxidase.

Methylamine oxidase (MAOx) from Gram-positive soil bacterium Arthrobacter P1 catalyzes the oxidation of CH3NH2 to H2C = O and NH4+ via reduction of O2 to H2O2. Past work indicates that MAOx is similar to mammalian plasma amine oxidase (PAO) and diamine oxidase (DAO), plant DAO, and yeast peroxisomal amine oxidase (YAO). All have Mr congruent to 170,000 and are composed of 2 identical subunits, each of which contains 1 atom of Cu(II) and one molecule of quinonoid cofactor. Herein, we report further evidence as to the striking similarity of these enzymes, and describe properties of MAOx which offer insights into understanding the eukaryotic oxidases. It is our belief that the structure of the quinone cofactor, and the Cu(II) site in MAOx are identical to these sites in PAO and DAO.

Studies on the active site of pig plasma amine oxidase.

Amine oxidase from pig plasma (PPAO) has two bound Cu2+ ions and at least one pyrroloquinoline quinone (PQQ) moiety as cofactors. It is shown that recovery of activity by copper-depleted PPAO is linear with respect to added Cu2+ ions. Recovery of e.s.r. and optical spectral characteristics of active-site copper parallel the recovery of catalytic activity. These results are consistent with both Cu2+ ions contributing to catalysis. Further e.s.r. studies indicate that the two copper sites in PPAO, unlike those in amine oxidases from other sources, are chemically distinct. These comparative studies establish that non-identity of the Cu2+ ions in PPAO is not a requirement for amine oxidase activity. It is shown through the use of a new assay procedure that there are two molecules of PQQ bound per molecule of protein in PPAO; only the more reactive of these PQQ moieties is required for activity.

Evidence for methoxatin (pyrroloquinolinequinone) as the cofactor in bovine plasma amine oxidase from resonance Raman spectroscopy.

Resonance Raman spectra of the 2,4-dinitrophenylhydrazine derivatives of bovine plasma amine oxidase [amine:oxygen oxidoreductase (deaminating) (copper-containing), EC 1.4.3.6] have been measured. Detailed comparisons to the spectra of the corresponding derivatives of methoxatin (pyrroloquinolinequinone), pyridoxal, and other aldehydes and diones provide further evidence that covalently bound methoxatin or a closely similar derivative is the organic cofactor in copper-containing amine oxidases.

Attempts surgically to induce chorionic fusion between embryos.

Intercornual fistulas were made in the uteri of 19 ewes in an attempt to induce chorionic fusion between fetuses. Ewes were 28 to 40 days pregnant with at least one fetus per horn. Sections were removed from the dorsomedial wall of each uterine horn, and left and right horns were sutured together around the openings to produce the fistulas. In some cases a doughnut-shaped sheet of silastic was inserted between the horns, and in most cases the chorioallantoic membranes of the respective fetuses were sutured to one another prior to suturing the uterine walls. In the 16 animals in which intercornual fistulas were constructed in the area of the intercornual ligament, no chorionic anastomosis was induced. In one of the three animals in which fistulas were produced distal to that ligament, the fistula remained patent 25 days post-surgery, and membranes of two female fetuses were joined, through the opening, by a narrow strand of chorionic tissue. We concluded that this technique was not a promising one for induction of choriovascular anastomosis.

Inactivation of beef plasma amine oxidase by sulfide.

Sulfide irreversibly inactivates beef plasma amine oxidase in a time-dependent reaction. Mercaptoacetic acid and 2-mercaptoethanol do not inactivate the enzyme. The sulfide complex displayed an intense absorption band at 360 nm (epsilon = 6000 M-1 cm-1, per mol of copper) that is assigned as sigma S leads to Cu(II) ligand to metal charge-transfer transition. However, this band slowly decreased in intensity; the final spectrum resembles the spectrum of the dithionite-reduced enzyme. Bleaching at approximately 450-500 nm specifically indicates that the organic cofactor is reduced. EPR parameters for the sulfide complex differ significantly from those observed for the native amine oxidase. Superhyperfine structure, attributable to coordinated nitrogens, is clearly evident. Time-dependent reduction of Cu(II) that parallels the kinetics and absorbance changes was also observed by EPR. The amine oxidaseazide complex was inactivated by sulfide at a considerably slower rate than the resting enzyme. Since azide is known to coordinate to Cu(II) in beef plasma amine oxidase, the data strongly suggest that enzyme-bound copper is the site of action for inhibition by sulfide.