Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity

Abstract

1. Isoelectric focusing studies on human placental diamine oxidase showed the pl value of the active enzyme to be 6.5. This infonnation was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH-gradient elution and this, together with affinity chromatography on concanavalin A-Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration ofdiamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultrantrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx.

Human kidney diamine oxidase: heterologous expression, purification, and characterization

“…EPR parameters derived from simulated spectra (g^=2.043, g || =2.265, A || =162 G) are consistent with published values for CAOs from various sources [1]. Crabbe et al [34] reported a gv alue of 2.05 from Q-band EPR spectra of purified human placental DAO; however, the presence of manganese prevented the determination of other copper parameters. Manganese was not removed by passing the protein over a Chelex 100 column, leading those investigators to conclude the human placental DAO was a Cu(II)-Mn(II) metalloprotein, with an apparent stoichiometry of 2.0 mol copper and 2.4 mol manganese per mol enzyme dimer.…”

“…The $10 kDa discrepancy between the predicted and observed molecular weights suggests the expressed protein is substantially glycosylated (vide infra). Literature values for the apparent molecular weight of the natural human DAO have been reported as 70, 90, and 105 kDa by SDS/PAGE [33,34,35]. As evident in Fig.…”

“…Gel filtration on a calibrated AcA 34 Ultrogel column gave an apparent molecular weight of 113 kDa, unrealistically low for the homodimer (data not shown). Interestingly, Crabbe et al [34] reported native molecular weights for the purified human placental DAO as 70 kDa by Sephadex G-200 gel filtration and 69.5 kDa by polyacrylamide gel electrophoresis. However, sedimentation equilibrium ultracentrifugation data in the same work indicated a native molecular weight of 235 kDa.…”

Some Aspects of the Enzymic Inactivation of Sympathomimetic Amines

“…While some are known to contain this metal as an essential constituent [Barker et al, 1979], attempts to find it in rat BAT have failed , Doubts stem from the fact that some copper chelating agents are also carbonyl reagents. In addition, cyanide, which is also a chelator of copper [Crabbe et al, 1976] only inhibits some SSAO enzymes [Lyles and Callingham, 1975;Lewinsohn, 1984], Attempts to determine the substrate selectivities of the SSAOs have not produced a clear picture, except to show that many of these enzymes prefer the physiologically in significant amine, benzylamine. In the case of human plasma and smooth muscle, ben zylamine is the major and so far the only substrate.…”

New Methods of Simulation of Mn(II) EPR Spectra: Single Crystals, Polycrystalline and Amorphous (Biological) Materials

“…Mn(II) has been detected unequivocally in living organisms. Examples are Mn(II)-metalloproteins, such as pyruvate carboxylase (Sutton et al, 1966), plant lectins (Galbraith & Goldstein, 1970), dioxygenase enzyme isolated from Bacillus brevis (Que et al, 1981), arginase isolated from liver tissue (Hirsch-Kolb et al, 1971), Mn(II) protein glutamine synthetase isolated from sheep brain (Wedler et al, 1982), placental diamine oxidase (Crabbe et al, 1976), clam shells and sea shells (Blanchard & Chasteen, 1976;White et al, 1982). (See, among others, the review article by McEuen (1982), which summarizes information on Mn-metalloproteins and several Mn-activated enzymes.)…”