Comparison of the osteogenic potential of OsteoSelect demineralized bone matrix putty to NovaBone calcium-phosphosilicate synthetic putty in a cranial defect model.

The purpose of this study was to compare the osteogenic potential of a synthetic and a demineralized bone matrix (DBM) putty using a cranial defect model in New Zealand white rabbits. Paired, bilateral critical-size defects (10 mm) were prepared in the frontal bones of 12 rabbits and filled with either OsteoSelect DBM Putty or NovaBone calcium-phosphosilicate putty. At days 43 and 91, 6 rabbits were killed and examined via semiquantitative histology and quantitative histomorphometry. Defects filled with the DBM putty were histologically associated with less inflammation and fibrous tissue in the defect and more new bone than the synthetic counterpart at both time points. Histomorphometric analysis revealed that the defects filled with DBM putty were associated with significantly more bone formation at day 43 (70.7% vs 40.7%, P = 0.043) and at day 91 (70.4% vs 39.9%, P = 0.0044). The amount of residual implant was similar for both test groups at each time point.

A comparative study of the binding and inhibition of four copper-containing amine oxidases by azide: implications for the role of copper during the oxidative half-reaction.

Copper amine oxidases (CuAOs) catalyze the oxidative deamination of primary amines operating through a ping-pong bi-bi mechanism. In this work, azide (an exogenous monodentate ligand) was used to probe the role of copper during the oxidative half-reaction of CuAO catalysis. The effects of azide on both the reductive and oxidative half-reactions of pea seedling amine oxidase (PSAO), the recombinant human kidney diamine oxidase (rhDAO), Arthrobacter globiformis amine oxidase (AGAO), and Pichia pastoris amine oxidase (PPLO) have been examined. For the reductive half-reaction, defined as the oxidation of amine substrate to an aldehyde, azide was discovered to exhibit either noncompetitive or competitive inhibition with respect to the amine, depending on the enzyme source. With regard to the oxidative half-reaction, defined as the reoxidation of the enzyme via reduction of O(2) to H(2)O(2), azide has been determined to exhibit competitive inhibition with respect to O(2) in PSAO with a calculated K(i) value that is in excellent agreement with the experimentally determined K(d) value for the Cu(II)-N(3)(-) complex. Azide was found to exhibit mixed-type/partially competitive inhibition with respect to substrate O(2) in rhDAO, with an apparent K(i) that is similar to the K(d) value for the Cu(II)-N(3)(-) complex. The competitive inhibition for PSAO and the partially competitive inhibition for rhDAO are consistent with O(2) interacting directly with copper during enzymatic reoxidation. For the enzymes AGAO and PPLO, pure noncompetitive and mixed-type/partially competitive inhibition is observed. K(i) values for reductive and oxidative half-reactions are equivalent and are lower than measured K(d) values for the Cu(II)-N(3)(-) complexes in oxidized and substrate-reduced forms of these enzymes. Given these observations, it appears that substantial inhibition of the reductive half-reaction occurs at the concentrations of azide used for the oxidative half-reaction experiments, thereby complicating kinetic interpretation. At this time, the data do not permit us to distinguish between two possibilities: (1) inhibition by azide with respect to O(2) is intrinsically competitive in CuAOs, but this effect cannot always be deconvolved experimentally from the effects of azide on the reductive half-reaction; or (2) CuAOs differ in some steps of their reoxidation mechanisms.

Reversible inhibition of copper amine oxidase activity by channel-blocking ruthenium(II) and rhenium(I) molecular wires.

Molecular wires comprising a Ru(II)- or Re(I)-complex head group, an aromatic tail group, and an alkane linker reversibly inhibit the activity of the copper amine oxidase from Arthrobacter globiformis (AGAO), with K(i) values between 6 muM and 37 nM. In the crystal structure of a Ru(II)-wire:AGAO conjugate, the wire occupies the AGAO active-site substrate access channel, the trihydroxyphenylalanine quinone cofactor is ordered in the "off-Cu" position with its reactive carbonyl oriented toward the inhibitor, and the "gate" residue, Tyr-296, is in the "open" position. Head groups, tail-group substituents, and linker lengths all influence wire-binding interactions with the enzyme.

Using xenon as a probe for dioxygen-binding sites in copper amine oxidases.

Potential dioxygen-binding sites in three Cu amine oxidases have been investigated by recording X-ray diffraction data at 1.7-2.2A resolution for crystals under a high pressure of xenon gas. Electron-density difference maps and crystallographic refinement provide unequivocal evidence for a number of Xe-binding sites in each enzyme. Only one of these sites is present in all three Cu amine oxidases studied. Structural changes elsewhere in the protein molecules are insignificant. The results illustrate the use of xenon as a probe for cavities, in which a protein may accommodate a dioxygen molecule. The finding of a potential dioxygen-binding cavity close to the active site of Cu amine oxidases may be relevant to the function of the enzymes, since the formation of a transient protein-dioxygen complex is a likely step in the catalytic mechanism. No evidence was found for xenon binding in a region of the molecule that was previously identified in two other Cu amine oxidases as a potential transient dioxygen-binding site.

Cyanide as a copper and quinone-directed inhibitor of amine oxidases from pea seedlings ( Pisum sativum) and Arthrobacter globiformis: evidence for both copper coordination and cyanohydrin derivatization of the quinone cofactor.

The interactions of cyanide with two copper-containing amine oxidases (CuAOs) from pea seedlings (PSAO) and the soil bacterium Arthrobacter globiformis (AGAO) have been investigated by spectroscopic and kinetic techniques. Previously, we rationalized the effects of azide and cyanide for several CuAOs in terms of copper coordination by these exogenous ligands and their effects on the internal redox equilibrium TPQ(amr)-Cu(II) right harpoon over left harpoon TPQ(sq)-Cu(I). The mechanism of cyanide inhibition was proposed to occur through complexation to Cu(I), thereby directly competing with O(2) for reoxidation of TPQ. Although cyanide readily and reversibly reacts with quinones, no direct spectroscopic evidence for cyanohydrin derivatization of TPQ has been previously documented for CuAOs. This work describes the first direct spectroscopic evidence, using both model and enzyme systems, for cyanohydrin derivatization of TPQ. K(d) values for Cu(II)-CN(-) and Cu(I)-CN(-), as well as the K(i) for cyanide inhibition versus substrate amine, are reported for PSAO and AGAO. In spite of cyanohydrin derivatization of the TPQ cofactor in these enzymes, the uncompetitive inhibition of amine oxidation is determined to arise almost exclusively through CN(-) complexation of Cu(I).

Gold electrodes wired for coupling with the deeply buried active site of Arthrobacter globiformis amine oxidase.

Diethylaniline-terminated oligo(phenyl-ethynyl)-thiol (DEA-OPE-SH) wires on Au-bead electrodes facilitate electron tunneling to and from the deeply buried topaquinone (TPQ) cofactor in Arthrobacter globiformis amine oxidase (AGAO). Reversible cyclic voltammograms were observed when AGAO was adsorbed onto this DEA-OPE-SAu surface: the 2e-/2H+ reduction potential is -140 mV versus SCE.

Inhibition of six copper-containing amine oxidases by the antidepressant drug tranylcypromine.

Potential inhibitory effects of the clinically utilized monoamine oxidase inhibitor tranylcypromine (TCP) on mammalian, plant, bacterial, and fungal copper-containing amine oxidases have been examined. The following enzymes have been investigated: human kidney diamine oxidase (HKAO), bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), and Pichia pastoris lysyl oxidase (PPLO). Only BPAO, EPAO, and AGAO were found to lose significant levels of activity when incubated with varying amounts of TCP. Inhibition of BPAO was completely reversible, with dialysis restoring full activity. TCP inhibition of AGAO was also found to be ultimately reversible; however, dialysis did not remove all bound compounds. Chemical displacement with either substrate or a substrate analogue successfully removed all bound TCP, indicating that this compound has a high affinity for the active site of AGAO. The notable lack of TCP inhibition on HKAO argues against the inhibition of diamine oxidase as a potential source for some of the deleterious side effects occurring in patients treated with this antidepressant. The marked differences observed in behavior among these enzymes speaks to the importance of intrinsic structural differences between the active sites of copper amine oxidases (CAO) which affect reactivity with a given inhibitor.