Amine oxidase-like activity of polyphenols. Mechanism and properties.

Polyphenols in several oxidation systems gained amine oxidase-like activity, probably due to the formation of the corresponding quinones. In the presence of Cu(II), o- and p-phenolic compounds exhibited amine oxidase-like activity, whereas only the o-phenolic compounds showed the activity in the presence of 1,1-diphenyl-2-picrylhydrazyl radical. The activity was determined by measuring the conversion of benzylamine to benzaldehyde by HPLC. Moreover, gallic acid, chlorogenic acid, and caffeic acid, which are plant polyphenols, converted the lysine residue of bovine serum albumin to alpha-amino-adipic semialdehyde residue, indicating lysyl oxidase-like activity. We also characterized the activity of pyrocatechol, hydroquinone, and pyrogallol in the presence of Cu(II). The oxidative deamination was accelerated at a higher pH, and required O2 and transition metal ions. Furthermore, EDTA markedly inhibited the reaction but not beta-aminopropionitrile, which is a specific inhibitor of lysyl oxidase. Catalase significantly inhibited the oxidation, implying the participation of hydroxyl radical in the reaction, but superoxide dismutase stimulated the oxidation, probably due to its radical formation activity. We discussed the mechanism of the oxidative deamination by polyphenols and the possible significance of the activity for biological systems.     

A peptide model of the copper-binding region of lysyl oxidase

The copper-binding site of lysyl oxidase remains extremely poorly characterized and although models have been suggested for copper(II) coordination by three histidine ligands, as has been found for other copper-containing amine oxidases, there has been no experimental confirmation of these suggestions. In this work, two synthetic peptides with 24 and 34-amino acid residues, respectively, were chosen from the highly conserved histidine-rich sequence previously suggested as the copper-binding region of lysyl oxidase. These peptides each bind one equivalent of Cu(II), at the same site in the two peptides. Spectroscopic (NMR, electron paramagnetic resonance (EPR), CD, visible absorption and fluorescence) techniques were employed to investigate the nature of the resulting complexes. The results indicate that at neutral pH three histidine ring nitrogen atoms and one carboxylate oxygen atom coordinate as the in-plane ligands of the copper, which is in an approximately tetragonally-distorted octahedral geometry. Modeling of the copper-peptides using the consistent force field (CFF91) produces a minimum energy configuration with three histidines and one water molecule as the copper ligands. CD, EPR and fluorescence results are reported for lysyl oxidase and compared with results for the peptides.     

Oxidation of peptidyl lysine by copper complexes of pyrroloquinoline quinone and other quinones. A model for oxidative pathochemistry.

Various o- and p-quinones were assessed as oxidants of peptidyl lysine in elastin and collagen substrates in the presence and absence of divalent copper as paradigms of protein-lysine 6-oxidase (lysyl oxidase) which contains both quinone and copper cofactors. Pyrroloquinoline quinone was among the most active in the absence and the most active of the o- and p-quinones tested in the presence of copper. The optimal rate of elastin oxidation occurred at a 2:1 PQQ/Cu(II) ratio while Cu(II) itself oxidized elastin relatively slightly. Elastin oxidation by 2:1 PQQ/Cu(II) required aerobic conditions consistent with oxygen-dependent turnover of this catalytic pair. Dimethylsulfoxide and catalase individually or in combination inhibited elastin oxidation by PQQ/Cu(II) by approx. 50%, suggesting that oxygen free radical species participate in the reaction. Amino-acid analysis of elastin and collagen substrates oxidized by 2:1 PQQ/Cu and then reduced with borohydride revealed that alpha-aminoadipic-delta-semialdehyde and lesser amounts of covalent cross-linkages were generated by this oxidant. In contrast, lysine oxidase produced aldehydes and significantly greater quantities of cross-linkage products, consistent with the known specificity of the enzyme. These data, thus, indicate the potential for free quinones, such as PQQ, particularly when stimulated by appropriate metal ions, to act as adventitious oxidants of lysine side-chains in proteins.     

Cu(I)-dependent biogenesis of the galactose oxidase redox cofactor

Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h). EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.     

Lysyl oxidase coupled with catalase in egg shell membrane.

The activity of lysyl oxidase was found in egg shell membrane (ESM) of hens. The activity was determined by measuring the enzymatic conversion of n-butylamine and Nalpha-acetyl-L-lysine to n-butyraldehyde and Nalpha-acetyl-L-allysine, respectively. ESM lysyl oxidase was significantly inhibited by beta-aminopropionitrile, chelating agents, and deoxygenation, consistent with the known properties of lysyl oxidase. Nevertheless, ESM lysyl oxidase was insoluble in urea solution, suggesting that it complexes with ESM. These findings support previous reports indicating the presence of lysine-derived cross-links in ESM and the necessity of lysyl oxidase located in the isthmus of the hen oviduct for the biosynthesis of ESM. Lysyl oxidase secreted around the egg white from the isthmus may initiate the cross-linking reaction of ESM protein, and remain as the constituent of ESM. Moreover, the H(2)O(2) released by lysyl oxidase in ESM was completely decomposed by coexisting catalase activity. ESM lysyl oxidase activity was greatly elevated in the presence of H(2)O(2), probably due to the O(2) produced by catalase. These findings indicate that lysyl oxidase is coupled with catalase in ESM. This coupling enzyme system was considered to be involved in the biosynthesis of ESM and to protect the embryo against H(2)O(2).     

Reaction pathway of bovine aortic lysyl oxidase

The catalysis of amine oxidation by lysyl oxidase has been probed to assess for the likely order of substrate binding and product release and to discriminate between mechanistic alternatives previously proposed for other copper-dependent amine oxidases using molecular oxygen as a substrate. Lineweaver-Burk plots revealed a pattern of parallel lines when the oxidation of n-butylamine was followed at different fixed concentrations of oxygen consistent with a "ping-pong" kinetic mechanism in which the aldehyde is produced and released before the binding of oxygen, the second substrate. Initial burst experiments revealed the ability of lysyl oxidase to form and release n-butyraldehyde in amounts stoichiometric with functional active site content in the absence of oxygen, consistent with the ping-pong kinetics obtained. Reciprocal plots of n-butylamine oxidation in the presence of fixed concentrations of the reaction products were consistent with a Uni Uni Uni Bi ping-pong kinetic mechanism with the aldehyde being the first, H2O2 the second, and ammonia the last departing product. Moreover, spectral studies of the oxidation of p-hydroxybenzylamine by lysyl oxidase indicated that the enzyme does not process the amine substrate to a noncovalently bound p-hydroxybenzaldimine intermediate subsequently to be hydrolyzed to p-hydroxybenzaldehyde. The kinetic mechanism of lysyl oxidase thus appears to be similar to those described for diamine oxidase and pig plasma monoamine oxidase.     

The Formation of lysine tyrosylquinone (LTQ) is a self-processing reaction. Expression and characterization of a Drosophila lysyl oxidase

Recent work in our laboratory has established methods for the expression and purification of a recombinant form of Drosophila lysyl oxdidase (rDMLOXL-1) [Molnar, J., Ujfaludi, Z., Fong, S. F. T., Bollinger, J. A., Waro, G., Fogelgren, B., Dooley, D. M., Mink, M., and Csiszar, K. (2005) J. Biol. Chem. 280, 22977-22985]. Previous investigations on the expression and purification of recombinant forms of lysyl oxidase [Kagan, H. M., Reddy, V. B., Panchenko, M. V., Nagan, N., Boak, A. M., Gacheru, S. N., and Thomas, K. (1995) J. Cell. Biochem. 59, 329-338] and lysyl oxidase-like proteins [Jung, S. T., Kim, M. S., Seo, J. Y., Kim, H. C., and Kim, Y. (2003) Protein Expression Purif. 31, 240-246] [Molnar, J., Fong, K. S. K., He, Q. P., Hayashi, K., Kim, Y., Fong, S. F. T., Fogelgren, B., Szauter, K. M., Mink, M., and Csiszar, K. (2003) Biochim. Biophys. Acta 1647, 220-224] have been reported in the literature. However, this is the first time that an expression system has been developed yielding sufficient amounts of a recombinant lysyl oxidase for detailed characterization. rDmLOXL-1 is secreted into the medium from S2 cells, and the protein is readily purified by Cibacon blue affinity chromatography yielding 10 mg of protein per liter of medium. The protein, as initially purified, is inactive and has no detectable copper or cofactor present. Following aerobic dialysis against copper, the protein is active and displays an electronic absorption spectrum with lambda(max) at 504 nm, consistent with the presence of an organic cofactor. Addition of phenylhydrazine to the copper-loaded protein produced a high-affinity adduct with lambda(max) at 454 nm. Comparison of the resonance Raman spectra of this adduct and a phenylhydrazine-labeled model compound of lysine tyrosylquinone (LTQ) establishes that the cofactor in the active, copper-containing enzyme is LTQ. Collectively, the data demonstrate that LTQ biogenesis most likely occurs by self-processing chemistry, requiring only the precursor protein, copper, and oxygen. Electron paramagnetic resonance and circular dichroism spectroscopy were used to characterize the Cu(II) site in rDmLOXL-1. The data are consistent with a tetragonal Cu(II) site with nitrogen and oxygen ligands. Recombinant DmLOXL-1 displayed significant activity toward tropoelastin and a wide variety of amines including polyamines and diamines. beta-aminoproprionitrile (betaAPN), a well-known irreversible inhibitor of mammalian lysyl oxidases, is also a potent inhibitor of rDmLOXL-1. Results from this investigation have important implications for the lysyl oxidase family.     

Evidence for a functional role for histidine in lysyl oxidase catalysis

The pH-dependent kinetics of lysyl oxidase catalysis was examined for evidence of an ionizable enzyme residue which might function as a general base catalyzing proton abstraction previously shown to be a component of the mechanism of substrate processing by this enzyme. Plots of log Vmax/Km for the oxidation of n-hexylamine versus pH yielded pKa values of 7.0 +/- 0.1 and 10.4 +/- 0.1. The higher pKa varied with different substrates, reflecting ionization of the substrate amino group. A van't Hoff plot of the temperature dependence of the lower pKa yielded a value of 6.1 kcal mol-1 for the enthalpy of ionization. This value as well as the pKa of 7.0 are consistent with those of histidine residues previously implicated as general base catalysts in enzymes. Incubation of lysyl oxidase with low concentrations of diethyl pyrocarbonate, a histidine-selective reagent, at 22 degrees C and pH 7.0 irreversibly inhibited enzyme activity by a pseudo first-order kinetic process. The inactivation of lysyl oxidase correlated with spectral and pH-dependent kinetic evidence for the chemical modification of 1 histidine residue/mol of enzyme, the pKa of which was 6.9 +/- 0.1, within experimental error of that seen in the plot of log Vmax/Km versus pH. Enzyme activity was restored by incubation of the modified enzyme with hydroxylamine, consistent with the ability of this nucleophile to displace the carbethoxy group from N-carbethoxyhistidine. The presence of the n-hexylamine substrate largely protected against enzyme inactivation by diethyl pyrocarbonate. These results thus indicate a functional role for histidine in lysyl oxidase catalysis consistent with that of a general base in proton abstraction.     

Structural and catalytic properties of copper in lysyl oxidase

The spectral and catalytic properties of the copper cofactor in highly purified bovine aortic lysyl oxidase have been examined. As isolated, various preparations of purified lysyl oxidase are associated with 5-9 loosely bound copper atoms per molecule of enzyme which are removed by dialysis against EDTA. The enzyme also contains 0.99 +/- 0.10 g atom of tightly bound copper per 32-kDa monomer which is not removed by this treatment. The copper-free apoenzyme, prepared by dialysis of lysyl oxidase against alpha,alpha'-dipyridyl in 6 M urea, catalyzed neither the oxidative turnover of amine substrates nor the anaerobic production of aldehyde at levels stoichiometric with enzyme active site content, thus contrasting with the ping pong metalloenzyme. Moreover, the spectrum of the apoenzyme was not measurably perturbed upon anaerobic incubation with n-butylamine, while difference absorption bands were generated at 250 and 308 nm in the spectrum of the metalloenzyme incubated under the same conditions. A difference absorption band also developed at 300-310 nm upon anaerobic incubation of pyrroloquinoline quinone, the putative carbonyl cofactor of lysyl oxidase, with n-butylamine. Full restoration of catalytic activity occurred upon the reconstitution of the apoenzyme with 1 g atom of copper/32-kDa monomer, whereas identical treatment of the apoenzyme with divalent salts of zinc, cobalt, iron, mercury, magnesium, or cadmium failed to restore catalytic activity. The EPR spectrum of copper in lysyl oxidase is typical of the tetragonally distorted, octahedrally coordinated Cu(II) sites observed in other amine oxidases and indicates coordination by at least three nitrogen ligands. The single copper atom in the lysyl oxidase monomer is thus essential at least for the catalytic and possibly for the structural integrity of this protein.     

Expression of lysyl oxidase from cDNA constructs in mammalian cells: The propeptide region is not essential to the folding and secretion of the functional enzyme

Rat aortic lysyl oxidase cDNA was expressed under a metallothionein promoter in Chinese hamster ovary cells using a dihydrofolate reductase selection marker. One methotrexate-resistant cell line, LOD-06, generated by transfecting with full-length cDNA, yielded lysyl oxidase proteins consistent with the 50 kDa proenzyme and a 29 kDa mature catalyst. A second cell line, LOD32–2, was generated by transfection with a truncated cDNA lacking sequences which code for the bulk of the propeptide region. Both cell lines secreted apparently identical, 29 kDa forms of mature lysyl oxidase each of which catalyzed the deamination of human recombinant tropoelastin and alkylamines, consistent with the known specificity of lysyl oxidase. The secreted enzyme forms were inhibited by chemical inhibitors of lysyl oxidase activity, including β-aminopropionitrile, phenylhydrazine, ethylenediamine, α,α′-dipyridyl, and diethyl-dithiocarbamate. Sensitivity to these agents is consistent with the presence of copper and carbonyl cofactors in the expressed enzymes, characteristic of lysyl oxidase purified from connective tissues. These results indicate the lack of essentiality of the deleted proprotein sequence for the proper folding, generation of catalytic function, and secretion of lysyl oxidase. © 1995 Wiley-Liss, Inc.     

Reaction of enzyme-bound 5-deazaflavin with peroxides. Pyrimidine ring contraction via an epoxide intermediate

Reaction of peroxides with 5-deazaflavin bound to glucose oxidase, lactate oxidase, or D-amino acid oxidase results in the formation of 5-deazaflavin 4a, 5-epoxide. The reaction of D-amino acid oxidase with m-chloroperoxybenzoate is an exception since the reagent reacts rapidly with the protein moiety to form m-chlorobenzoate which then binds noncovalently near the unmodified coenzyme. Epoxide bound to glucose oxidase is converted to deazaFAD X X in a reaction similar to that observed previously with oxynitrilase and glycolate oxidase. With lactate oxidase the epoxide is quite stable in the absence of light. With D-amino acid oxidase, denaturation of the protein is accompanied by the release of the epoxide into solution where it decomposes in a manner similar to that observed with model epoxide compounds at neutral pH. Reaction of deazaFAD X X with phosphodiesterase and alkaline phosphatase yields deazariboflavin X X. The same compound has been formed in model studies by exposing 5-deazariboflavin 4a,5-epoxide to alkaline conditions. Structural studies indicate that this reaction involves contraction of the pyrimidine ring to yield 4-ribityl-6,7-dimethyloxazolo[ 4,5-b ]quinolin-2(4H)-one. Model reaction studies are consistent with a mechanism initiated by alkaline hydrolysis of the pyrimidine ring at position 4 followed by two additional steps which proceed at neutral pH. A similar mechanism for the enzyme reactions appears likely since analogous intermediates are detected in the glycolate oxidase and the model reactions. The results suggest that position 4 of the coenzyme in oxynitrilase, glycolate oxidase, and glucose oxidase must be accessible to solvent and that the protein moiety must facilitate the initial hydrolysis of the pyrimidine ring since the enzyme reactions occur at neutral pH. Failure to observe formation of deazaFMN X X with lactate oxidase is attributed, at least in part, to the inaccessibility of the pyrimidine ring to solvent.     

Active site rearrangement of the 2-hydrazinopyridine adduct in Escherichia coli amine oxidase to an azo copper(II) chelate form: a key role for tyrosine 369 in controlling the mobility of the TPQ-2HP adduct

Adduct I (lambda(max) at approximately 430 nm) formed in the reaction of 2-hydrazinopyridine (2HP) and the TPQ cofactor of wild-type Escherichia coli copper amine oxidase (WT-ECAO) is stable at neutral pH, 25 degrees C, but slowly converts to another spectroscopically distinct species with a lambda(max) at approximately 530 nm (adduct II) at pH 9.1. The conversion was accelerated either by incubation of the reaction mixture at 60 degrees C or by increasing the pH (>13). The active site base mutant forms of ECAO (D383N and D383E) showed spectral changes similar to WT when incubated at 60 degrees C. By contrast, in the Y369F mutant adduct I was not stable at pH 7, 25 degrees C, and gradually converted to adduct II, and this rate of conversion was faster at pH 9. To identify the nature of adduct II, we have studied the effects of pH and divalent cations on the UV-vis and resonance Raman spectroscopic properties of the model compound of adduct I (2). Strikingly, it was found that addition of Cu2+ to 2 at pH 7 gave a product (3) that exhibited almost identical spectroscopic signatures to adduct II. The X-ray crystal structure of 3 shows that it is the copper-coordinated form of 2, where the +2 charge of copper is neutralized by a double deprotonation of 2. These results led to the proposal that adduct II in the enzyme is TPQ-2HP that has migrated onto the active site Cu2+. The X-ray crystal structure of Y369F adduct II confirmed this assignment. Resonance Raman and EPR spectroscopy showed that adduct II in WT-ECAO is identical to that seen in Y369F. This study clearly demonstrates that the hydrogen-bonding interaction between O4 of TPQ and the conserved Tyr (Y369) is important in controlling the position and orientation of TPQ in the catalytic cycle, including optimal orientation for reactivity with substrate amines.     

Ultrastructural immunolocalization of lysyl oxidase in vascular connective tissue

The localization of lysyl oxidase was examined in calf and rat aortic connective tissue at the ultrastructural level using polyclonal chicken anti-lysyl oxidase and gold conjugated rabbit anti-chicken immunoglobulin G to identify immunoreactive sites. Electron microscopy of calf aortic specimens revealed discrete gold deposits at the interface between extracellular bundles of amorphous elastin and the microfibrils circumferentially surrounding these bundles. The antibody did not react with microfibrils which were distant from the interface with elastin. There was negligible deposition of gold within the bundles of amorphous elastin and those few deposits seen at these sites appeared to be associated with strands of microfibrils. Lysyl oxidase was similarly localized in newborn rat aorta at the interface between microfibrils and nascent elastin fibers. Gold deposits were not seen in association with extracellular collagen fibers even after collagen-associated proteoglycans had been degraded by chondroitinase ABC. However, the antibody did recognize collagen-bound lysyl oxidase in collagen fibers prepared from purified collagen to which the enzyme had been added in vitro. No reaction product was seen if the anti-lysyl oxidase was preadsorbed with purified lysyl oxidase illustrating the specificity of the antibody probe. The present results are consistent with a model of elastogenesis predicting the radial growth of the elastin fiber by the deposition and crosslinking of tropoelastin units at the fiber-microfibril interface.     

Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole), PVIm, gels were prepared by γ-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption–desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.     

Coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in cytochrome c oxidase. Studies with the carbon monoxide inhibited enzyme

A study is presented on the coupling of electron transfer with proton transfer at heme a and Cu(A) (redox Bohr effects) in carbon monoxide inhibited cytochrome c oxidase isolated from bovine heart mitochondria. Detailed analysis of the coupling number for H(+) release per heme a, Cu(A) oxidized (H(+)/heme a, Cu(A) ratio) was based on direct measurement of the balance between the oxidizing equivalents added as ferricyanide to the CO-inhibited fully reduced COX, the equivalents of heme a, Cu(A), and added cytochrome c oxidized and the H(+) released upon oxidation and all taken up back by the oxidase upon rereduction of the metal centers. One of two reductants was used, either succinate plus a trace of mitochondrial membranes (providing a source of succinate-c reductase) or hexaammineruthenium(II) as the chloride salt. The experimental H(+)/heme a, Cu(A) ratios varied between 0.65 and 0.90 in the pH range 6.0-8.5. The pH dependence of the H(+)/heme a, Cu(A) ratios could be best-fitted by a function involving two redox-linked acid-base groups with pK(o)-pK(r) of 5.4-6.9 and 7.3-9.0, respectively. Redox titrations in the same samples of the CO-inhibited oxidase showed that Cu(A) and heme a exhibited superimposed E'(m) values, which decreased, for both metals, by around 20 mV/pH unit increase in the range 6.0-8.5. A model in which oxido-reduction of heme a and Cu(A) are both linked to the pK shifts of the two acid-base groups, characterized by the analysis of the pH dependence of the H(+)/heme a, Cu(A) ratios, provided a satisfactory fit for the pH dependence of the E'(m) of heme a and Cu(A). The results presented are consistent with a primary involvement of the redox Bohr effects shared by heme a and Cu(A) in the proton-pumping activity of cytochrome c oxidase.     

Alterations in copper and collagen metabolism in the Menkes syndrome and a new subtype of the Ehlers-Danlos syndrome

Cultured fibroblasts of 13 patients with the Menkes syndrome and two with a new subtype (type IX) of the Ehlers-Danlos syndrome (E-D IX patients) showed many very similar abnormalities in their copper and collagen metabolism. Both cell types had markedly increased copper concentrations and 64Cu incorporation, and this cation accumulated in metallothionein or a metallothionein-like protein, as previously established for Menkes cells. Histochemical staining indicated that copper was distributed diffusely throughout the cytoplasm in both cell types, this location being consistent with the accumulation in metallothionein. Both fibroblast types also had markedly low lysyl oxidase activity and distinctly increased extractability of newly synthesized collagen, whereas no abnormalities were present in cell viability, duplication rate, prolyl 4-hydroxylase activity, or collagen synthesis rate. A high negative correlation (P less than 0.001) was found in the pooled group of Menkes and E-D IX cells between cellular copper concentration (r = 0.804) or 64Cu incorporation (r = 0.863) and the logarithm of lysyl oxidase activity. There was also a high positive correlation (P less than 0.001) between cellular copper concentration and incorporation (r = 0.869). One of the two E-D IX patients was also shown to have similar changes in lysyl oxidase activity and collagen extractability in the skin biopsy specimen, suggesting that the abnormalities observed in cultured cells are similar to those present in vivo. The only distinct abnormality found in the cells of the parents of the E-D IX patients was an increased 64Cu incorporation in those of the mother, this finding being consistent with X-linked inheritance of the disorder.     

Purification and properties of four species of lysyl oxidase from bovine aorta

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49-60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000-33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of alpha-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu(2+), and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.     

Copper(II) complexes of a new N-picolylated bis benzimidazolyl diamide ligand: Synthesis, crystal structure and catechol oxidase studies

Copper(II) complexes of a new bis benzimidazole diamide ligand N-picolyl-N,N′-bis(2-methylbenzimidazolyl)hexanediamide [Pic-GBHA = L₂] have been synthesized and characterized. One of the compound [Cu(L₂)(NO₃)₂] has been structurally characterized. The copper atom is bound to two benzimidazolyl nitrogen atoms, two amide carbonyl oxygen atoms and a bidentate nitrate ion, resulting in a distorted octahedral geometry. EPR spectra obtained at low temperature indicate a tetragonal geometry in the solution state. Complexes display a quasi-reversible redox wave due to the Cu(II)/Cu(I) reduction process having fairly cathodic E_1/2. These Cu(II) complexes were utilized to carry out oxidation of ditertbutylcatechol (DTBC) in methanol using molecular oxygen as the oxidant in. Low temperature EPR study of the oxidation reaction implicates the formation of an active copper species with fairly low A_‖ value. The presence of picolyl groups on the ligand also serve as a proton sponge giving 2-3 times higher rates of reaction in comparison to the non-picolylated ligand, implying a role of free basic groups in the pH control of enzymatic oxidation of catechols by catechol oxidase and tyrosinase.     

Reactions of bovine serum amine oxidase with NN-diethyldithiocarbamate. Selective removal of one copper ion.

NN-Diethyldithiocarbamate (DDC) was able to bind, at 1.0 mM concentration, only about 50% the Cu(II) ions of bovine plasma amine oxidase. Under reducing conditions, this Cu(II) was removed with inactivation of the enzyme. Up to 90% activity could be recovered by treatment with excess Cu(II). The organic cofactor, sensitive to carbonyl reagents, was reduced in the half-Cu-depleted protein and no longer bound phenylhydrazine. The fully reacted protein, in the presence of 10 mM-DDC, lost 50% Cu(II) upon storage at -20 degrees C, but in this case the residual Cu(II) was in the DDC-bound form and the cofactor was in the oxidized state, as it could still bind phenylhydrazine. In the presence of DDC, the rate of reaction with phenylhydrazine was always low, even at 50% DDC saturation, and all derivatives showed identical modifications of the optical and e.p.r. spectra with respect to the phenylhydrazone of the native protein. It is concluded that the two Cu(II) ions are not equivalent, that removal of a single Cu(II) is sufficient to inhibit the re-oxidation of the organic cofactor, and that both Cu(II) ions are in some way involved in the reaction with phenylhydrazine. After reaction with DDC, the optical and e.p.r. spectra of 63Cu(II)-amine oxidase and of 63Cu(II)-carbonic anhydrase [Morpurgo, Desideri, Rigo, Viglino & Rotilio (1983) Biochim. Biophys. Acta 746, 168-175] are very similar and show distorted equatorial co-ordination to Cu(II) of two sulphur atoms and two magnetically equivalent nitrogen atoms.     

Immunological studies of bovine aorta lysyl oxidase: evidence for two forms of the enzyme.

Evidence is presented that beef aorta contains two forms of lysyl oxidase which we have designated as lysyl oxidase A and B. The two forms of the enzyme can be separated by DEAE-cellulose chromatography. Immunogical tests show that lysyl oxidase A and B have distinct antigenic determinants. Immunoelectrophoresis at pH 8.6 showed that the aorta lysyl oxidase A and B differed in net charge. Antisera to pure lysyl oxidase A formed a precipitin line with lysyl oxidase A but did not react with lysyl oxidase B in the Ouchterlony double immunodiffusion test. These findings show that it will now be necessary to separate the two forms of enzymes for certain types of biochemical studies of lysyl oxidase.