A microassay for lysyl oxidase activity.

Pinnell and Martin's [(1968) Proc. Nat. Acad. Sci. USA61, 708–716] standard assay for lysyl oxidase is modified to determine activity in small samples. Data collected by both methods are comparable, and the microassay has the advantages of being economical and rapid.     

An ultrafiltration assay for lysyl oxidase

A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware.     

Simplified assay for lysyl oxidase activity.

A simplified method for the assay of lysyl oxidase activity was developed. The method is based on the measurement of tritiated water released by enzyme action from labeled protein-bound lysine and hydroxylysine. Trichloroacetic acid (TCA) supernates of the incubation mixtures are passed through small Dowex 50 (H⁺) columns and the effluents are counted. For rapid screening purposes an indication of the presence of enzyme activity in enzyme preparations can be obtained by measuring the radioactivity present in aliquots of the TCA supernates as such and by measuring the radioactivity after drying at 60°C, taking the difference between the two as a measurement of enzyme activity.     

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.     

Immunological characterization of bovine lysyl oxidase

Antibodies to homogeneously purified bovine aortic lysyl oxidase were prepared in chickens. The chicken anti-lysyl oxidase antiserum effectively inhibited bovine aortic lysyl oxidase activity. Non-immune antiserum from chickens, goats and humans was found to enhance bovine aortic lysyl oxidase activity, while non-immune rabbit serum inhibited enzyme activity. A competitive ELISA was developed to monitor immunoreactive lysyl oxidase during purification. Chromatography of bovine lysyl oxidase on Sephacryl S-200, the final step in purification, revealed two peaks of immunoreactive lysyl oxidase. The large molecular weight peak appears to contain inactive multimeric forms of the enzyme.     

Development of a peroxidase-coupled fluorometric assay for lysyl oxidase

Lysyl oxidase catalyzes the oxidation of peptidyl lysine in elastin and collagen and also acts upon nonpeptidyl amines, although the enzyme becomes slowly inactivated while processing nonpeptidyl substrates. In spite of this complexity, it has been possible to devise a continuously monitored peroxidase-coupled fluorometric assay for the oxidation of simple amines by lysyl oxidase. In the present study, optimal assay conditions have been explored and found to include assay temperatures of 50 to 60°C, the presence of urea in the assay, and the use of diaminopentane as substrate. Although the assay is subject to interference by contaminating macromolecules in enzyme fractions, a linear assay response to enzyme concentration is obtained with highly purified lysyl oxidase with a limiting sensitivity of 0.3 μg of enzyme per assay.     

The Pro-regions of lysyl oxidase and lysyl oxidase-like 1 are required for deposition onto elastic fibers

These studies were undertaken to determine how lysyl oxidase (LOX) and lysyl oxidase like-1 (LOXL) enzymes are targeted to their substrates in the extracellular matrix. Full-length LOX/LOXL and constructs containing just the pro-regions of each enzyme localized to elastic fibers when expressed in cultured cells. However, the LOXL catalytic domain without the pro-region was secreted into the medium but did not associate with matrix. Ligand blot and mammalian two-hybrid assays confirmed an interaction between tropoelastin and the pro-regions of both LOX and LOXL. Immunofluorescence studies localized both enzymes to elastin at the earliest stages of elastic fiber assembly. Our results showed that the pro-regions of LOX and LOXL play a significant role in directing the deposition of both enzymes onto elastic fibers by mediating interactions with tropoelastin. These findings confirmed that an important element of substrate recognition lies in the pro-domain region of the molecule and that the pro-form of the enzyme is what initially interacts with the matrix substrate. These results have raised the interesting possibility that sequence differences between the pro-domain of LOX and LOXL account for some of the functional differences observed for the two enzymes.     

Repeat polypeptide models of elastin as substrates for lysyl oxidase

Synthetic repeat polypeptides analogous in sequence to the valine-rich regions of elastin have been tested as substrates for purified bovine aorta lysyl oxidase. These polypeptides, HCO(phi-Pro-Gly-Gly)n-Val-OMe, HCO(Val-Pro-Gly-phi-Gly)n-Val-OMe, and HCO-Val-(Ala-Pro-Gly-phi-Gly-Val)n-OMe, where phi = Val or Lys at approximately a 4:1 ratio and where n greater than or equal to 40, are models of the tetra-, penta-, or hexapeptide repeat sequences found in elastin. alpha-Aminoadipic delta-semialdehyde is generated in each of these upon incubation with lysyl oxidase at 37 degrees C, whereas the aldol and anhydrolysinonorleucine bifunctional cross-linkages were formed only in the incubation of enzyme with polypentapeptide. Incubation of the polypentapeptide at 55 degrees C, which enhances coacervation of the peptide, increases aldehyde formation and generates a much higher ratio of cross-linkages to aldehyde than occurred at 37 degrees C. These results demonstrate that lysyl oxidase can oxidize lysine in synthetic polypeptides and suggest important conformational aspects of lysyl oxidase substrates which may control substrate potential as well as the ability of peptidyl aldehyde, once formed by the enzyme, to condense to cross-linkage products.     

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.     

Induction of human monocyte motility by lysyl oxidase

Lysyl oxidase highly purified from calf aorta was found to be a potent chemotactic agent for unstimulated human peripheral blood mononuclear cells, determined in in vitro assays in Boyden chambers. A typical chemotactic bell-shaped curve was observed, with a maximal migratory response of 237% of control occurring at 10⁻¹⁰ M lysyl oxidase. The chemotactic response was prevented by prior heat inactivation of the enzyme, by treatment of the enzyme with β-aminopropionitrile or ethylenediamine, which are active site-directed inhibitors of lysyl oxidase, and by a competing, lysine-containing peptide substrate of lysyl oxidase. The chemoattractant reponse to lysyl oxidases was characterized by both chemokinetic and chemotactic components. These results raise the possibility that extracellular lysyl oxidase may have important roles to play in biology in addition to its established function in the crosslinking of elastin and collagen.     

Functional importance of lysyl oxidase family propeptide regions

The lysyl oxidase family of proteins is primarily known for its critical role in catalyzing extracellular oxidative deamination of hydroxylysine and lysine residues in collagens, and lysine residues in elastin required for connective tissue structure and function. Lysyl oxidases have additional important biological functions in health and disease. While the enzyme domains are highly conserved, the propeptide regions are less uniform, and have biological activity, some of which are independent of their respective enzymes. This review summarizes what has been published regarding the functions of the propeptide regions of this family of proteins in the context of extracellular matrix biosynthesis, fibrosis and cancer biology. Although much has been learned, there is a need for greater attention to structure/function relationships and mechanisms to more fully understand these multifunctional proteins.     

The crystal structure of Pichia pastoris lysyl oxidase

Pichia pastoris lysyl oxidase (PPLO) is unique among the structurally characterized copper amine oxidases in being able to oxidize the side chain of lysine residues in polypeptides. Remarkably, the yeast PPLO is nearly as effective in oxidizing a mammalian tropoelastin substrate as is a true mammalian lysyl oxidase isolated from bovine aorta. Thus, PPLO is functionally related to the copper-containing lysyl oxidases despite the lack of any significant sequence similarity with these enzymes. The structure of PPLO has been determined at 1.65 A resolution. PPLO is a homodimer in which each subunit contains a Type II copper atom and a topaquinone cofactor (TPQ) formed by the posttranslational modification of a tyrosine residue. While PPLO has tertiary and quaternary topologies similar to those found in other quinone-containing copper amine oxidases, its active site is substantially more exposed and accessible. The structural elements that are responsible for the accessibility of the active site are identified and discussed.     

Characterization of a model compound for the lysine tyrosylquinone cofactor of lysyl oxidase

We characterized a model compound for the lysine tyrosylquinone (LTQ) cofactor of lysyl oxidase which is one of the mammalian copper-dependent amine oxidases. The model compound, 4-butylamino-5-methyl-o-quinone, was prepared from n-butylamine and 4-methylcatechol by the oxidation with sodium iodate and characterized by spectroscopic analyses. The absorption maximum at 494 nm is consistent with that of lysyl oxidase. The model compound was capable of deaminating benzylamine to benzaldehyde at 37 degrees C in buffered aqueous acetonitrile. The aldehyde production was markedly elevated in the presence of the Cu(II)-EDTA complex but inhibited by free Cu(II). The catalytic cycle was observed at pH 10 in the presence of Cu(II), and the pH activity profile showed a broad optimum at about pH 9.0. In the presence of beta-aminopropionitrile and upon deoxygenation with N2 aldelyde, production was decreased. The important features of the reaction were consistent with the enzymatic reaction.     

A single-step large-scale purification of pyruvate oxidase

Pyruvate oxidase is an Escherichia coli peripheral membrane flavoprotein which catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2. Pyruvate oxidase, like several other peripheral membrane enzymes, can be activated either by binding to lipid amphiphiles or by limited protease digestion. This paper reports a rapid and convenient method for effecting the large-scale purification of pyruvate oxidase from crude enzyme preparations using a Triton X-114 phase separation technique. It appears likely that this purification procedure can be used successfully with the family of enzymes which respond to both lipid and protease activation.     

A micromethod for the purification and quantification of organic acids of the tricarboxylic acid cycle in plant tissues

A rapid and convenient microscale method for the purification and quantification of several of the organic acids of the tricar?ylic acid cycle is described. It has been applied to a variety of plant tissues, and its use in studying the organic acid content of the soybean plant is described in detail.     

Megakaryocyte polyploidy is inhibited by lysyl oxidase propeptide

Megakaryocytes (MKs), the platelet precursors, undergo an endomitotic cell cycle that leads to polyploidy. Lysyl oxidase propeptide (LOX-PP) is generated from lysyl oxidase (LOX) pro-enzyme after proteolytical cleavage. We recently reported that LOX, a known matrix cross-linking enzyme, contributes to MK lineage expansion. In addition, LOX expression levels are ploidy-dependent, with polyploidy MKs having minimal levels. This led us to test the effects of LOX-PP on the number and ploidy of primary MKs. LOX-PP significantly decreases mouse bone marrow MK ploidy coupled with a reduction in MK size. MK number is unchanged upon LOX-PP treatment. Analysis of LOX-PP- or vehicle-treated MKs by western blotting revealed a reduction in ERK1/2 phosphorylation and in the levels of its downstream targets, cyclin D3 and cyclin E, which are known to play a central role in MK endomitosis. Pull-down assays and immunochemistry staining indicated that LOX-PP interacts with α-tubulin and the mictotubules, which can contribute to decreased MK ploidy. Thus, our findings defined a role for LOX-PP in reducing MK ploidy. This suggests that high-level expression of LOX in aberrantly proliferating MKs could play a part in inhibiting their polyploidization via LOX-PP.