Induction and characterization of a novel amine oxidase from the yeast Kluyveromyces marxianus.

An amine oxidase from the yeast Kluyveromyces marxianus was induced, purified and completely characterized; it was shown to belong to the class of copper-containing amine oxidases (E.C. 1.4.3.6). The enzyme was induced by putrescine and, very strongly, by copper(II); structural-functional characterization of the enzyme was performed, including determination of molecular weight, glycosylation, copper and TPQ content, isoelectric point, K(M) and k(CAT) (with benzylamine as substrate), pH, temperature and ionic strength effect on catalysis, substrate and inhibitor specificity. A 700 bp clone was isolated containing the cDNA that encodes for the C-terminus of the enzyme; the amino acid sequence deduced (the first available for a benzylamine oxidase from yeast) was compared to that of other copper amine oxidases from microorganisms and higher organisms. From the results obtained, the putrescine/benzylamine oxidase from Kluyveromyces marxianus was found to have a good homology with other enzymes of this class from microorganisms, and particularly with AO I from Aspergillus niger. Nonetheless, some features resulted closer to those of animal amine oxidases and histaminases. Some potential biotechnological applications are proposed. The cDNA Accession No. is AJ320485.

Semicarbazide-sensitive amine oxidase in pig heart.

A semicarbazide-sensitive amine oxidase (SSAO) (E.C.1.4.3.6) has been purified from pig heart. Western blot analysis showed that the enzyme reacts with a polyclonal antibody raised against homogeneous crystalline pig plasma benzylamine oxidase (BAO). A subunit molecular mass of 97 KDa obtained by SDS electrophoresis is identical to the plasma enzyme. The purification procedure consisted of sequential DEAE cellulose, octyl-Sepharose, Con A-Sepharose and hydroxyapatite columns. Two peaks of activity were obtained on octyl-Sepharose which were found to be kinetically and immunologically indistinguishable. The specific activity of the purified enzyme was 0.045 mumol/min/mg of protein at 37 degrees C and the Km for benzylamine was estimated to be 63 microM. The enzyme was inhibited by carbonyl reagents such as semicarbazide but was insensitive to the effect of pargyline.

Nature of the organic cofactor of pig plasma benzylamine oxidase.

The identification of the organic cofactor of pig plasma benzylamine oxidase is described. Acid hydrolysis of the enzyme in argon in the presence of phenylhydrazine (6 h at 115 degrees C in 0.027 M sulphuric acid) allowed the isolation of an adduct which was purified by HPLC and identified as phenylhydrazone of pyridoxal by spectrophotometry, spectrofluorimetry and mass spectrometry.

A method for the isolation and identification of pyridoxal phosphate in proteins.

A method for the isolation and identification of covalently bound pyridoxal phosphate (PLP) contained in some enzymatic proteins is presented. The method involves acid hydrolysis of the protein in the presence of phenylhydrazine, separation of the adduct by elution from Sep-Pak C18 cartridges, isolation by HPLC, and either direct analysis by mass spectrometry with direct electron impact or conversion into trimethylsilyl derivatives followed by gas chromatography-mass spectrometry. Under the prescribed conditions of hydrolysis, PLP forms its phenylhydrazone and is released from the protein and hydrolyzed to the phenylhydrazone of pyridoxal, which shows a typical fragmentation in direct electron impact and in gas chromatography-mass spectrometry after silylation. The yield in phenylhydrazone of pyridoxal is on the order of 50% (+/- 5% SE, n = 15) when PLP is added to 10 mg of protein in amounts ranging from 20 to 40 nmol. Analysis of pig plasma benzylamine oxidase by this procedure confirms the presence of covalently bound pyridoxal phosphate in this enzyme.

Purification of benzylamine oxidase from cultured porcine aortic smooth muscle cells.

Soluble benzylamine oxidase (BzAO) from cell homogenates and the conditioned culture medium of porcine aortic smooth muscle cells was purified by anionic HPLC methods and characterized with regard to enzyme kinetics and inhibition by semicarbazide, phenelzine, cuprizone, diethyldithiocarbamate (DDC), and p-chloromercuriphenylsuphonate (PCMPS). BzAO from both the cell homogenates and the conditioned culture medium had an Mr of 130,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using [methylene-14C]benzylamine hydrochloride as substrate, BzAO from cell homogenates and the conditioned culture medium had Km values of 5.1 and 6.1 microM, respectfully, and Vmax values of 89 and 53 nmol.mg protein-1.h-1. Both enzymes were sensitive to inhibition by semicarbazide and phenelzine, but insensitive to inhibition by the copper chelating agent DDC. BzAO isolated from the conditioned culture medium was more sensitive to inhibition by lower concentrations of cuprizone and PCMPS than the enzyme isolated from cell homogenates. Antisera raised against BzAO from cell homogenates reacted with BzAO from the conditioned culture medium and from porcine plasma.

Benzylamine oxidase from brain microvessels.

Copper-containing benzylamine oxidase with a specific activity of 200 units was isolated from bovine brain microvessels. It was shown that the content of the enzyme in microvessels was significantly higher as compared with large blood vessels such as heart aorta. Some physico-chemical properties of the enzyme were determined. The enzyme was inhibited by high concentrations of the substrate as well as thiol reagents and beta-aminopropionitrile fumarate. On the basis of EPR and optical spectra of the enzyme its copper was considered to be 'non-blue' type.

The presence of an inhibitor of benzylamine oxidase in human blood plasma.

The presence of an inhibitor of benzylamine oxidase in human blood plasma was observed. It may be removed by use of either a DEAE-cellulose column or a Sephadex G-200 column.

Benzylamine oxidase in normal and atherosclerotic human aortae.

Assays of serum benzylamine oxidase (BzAO) have led some workers to postulate a relationship between elevated BzAO activity and diseases characterized by proliferating connective tissue. The present study was designed to determine whether BzAO activity of a cellular tissue is also affected. BzAO was assayed in homogenates of normal and atherosclerotic human aortae. Characterization done in normal aortae showed that BzAO is not a classical monoamine, diamine, polyamine, or lysyl oxidase, nor is it a ceruloplasmin. The enzyme is heat stable at 60 degrees C and is associated primarily with the microsomal fraction on density centrifugation. Compared with phenylethylamines and indoleamines, benzylamine is the best substrate. BzAO is sensitive to inhibition by hydrazines and chymotrypsin but not trypsin, and is insensitive to Triton X-100 and sulfhydryl-group blockade. BzAO activity of atherosclerotic plaque (expressed per gram wet weight or per milligram protein) was decreased markedly compared to that in adjacent, nonplaque regions and in normal aortae. However, on a per milligram DNA basis, the BzAO activity of plaque did not differ from that of nonplaque tissue. We conclude that there is a decreased cell population density in plaque, a contention supported by kinetic analysis. Plaque BzAO showed a decreased Vmax with no change in the Km of benzylamine compared with nonplaque tissue. Thus, if a relationship exists between BzAO activity and proliferating connective tissue, it is not apparent at the level of the cellular enzyme in atherosclerotic aortae of man.

Microbial oxidation of amines. Distribution, purification and properties of two primary-amine oxidases from the yeast Candida boidinii grown on amines as sole nitrogen source.

1. The yeast Candida boidinii was grown on glucose as carbon source with a range of amines and amino acids as nitrogen sources. Cells grown on amines contained elevated activities of catalase. If the amines contained N-methyl groups, formaldehyde dehydrogenase, formate dehydrogenase and S-formylglutathione hydrolase were also elevated in activity compared with cells grown on (NH(4))(2)SO(4). 2. Cells grown on all the amines tested, but not those grown on urea or amino acids, contained an oxidase attacking primary amines, which is referred to as methylamine oxidase. In addition, cells grown on some amines contained a second amine oxidase, which is referred to as benzylamine oxidase. 3. Both amine oxidases were purified to near homogeneity. 4. Benzylamine oxidase was considerably more stable at 45 and 50 degrees C than was methylamine oxidase. 5. Both enzymes had a pH optimum in the region of 7.0, and had a considerable number of substrates in common. There were, however, significant differences in the substrate specificity of the two enzymes. The ratio V/K(app.) (m) increased with increasing n-alkyl carbon chain length for benzylamine oxidase, but decreased for methylamine oxidase. 6. Both enzymes showed similar sensitivity to carbonyl-group reagents, copper-chelating agents and other typical ;diamine oxidase inhibitors'. 7. The stoicheiometry for the reaction catalysed by each enzyme was established. 8. The kinetics of methylamine oxidase were examined by varying the methylamine and oxygen concentrations in turn. A non-Ping Pong kinetic pattern with intersecting double-reciprocal plots was obtained, giving K(m) values of 10mum for O(2) and 198mum for methylamine. The significance of this unusual kinetic behaviour is discussed. Similar experiments were not possible with the benzylamine oxidase, because it seemed to have an even lower K(m) for O(2). 9. Both enzymes had similar subunit M(r) values of about 80000, but the benzylamine oxidase behaved as if it were usually a dimer, M(r) 136000, which under certain conditions aggregated to a tetramer, M(r) 288000. Methylamine oxidase was mainly in the form of an octamer, M(r) 510000, which gave rise quite readily to dimers of M(r) 150000, and on gel filtration behaved as if the M(r) was 286000.

Lysyloxidase and benzylamine oxidase in pig aorta.

Three enzymes were isolated from pig aorta: a benzylamine oxidase differing from the plasma type, and two different types of lysyloxidase. Some properties of the two types of lysyloxidase are described. The three enzymes were inhibited by cupric copper chelating agents and by carbonyl reagents. They did not cross-react with the antibodies to pure pig plasma benzylamine oxidase.

[Soluble benzylamine oxidase from aorta]. / Rastvorimaia benzilaminoksidaza iz tkani aorty.

A new method for purification of soluble benzylamine oxidase from bovine aorta with specific activity of more than 100 units is described. The optical and magnetic properties of the enzyme have been studied. The enzyme contains type II copper, the environment of which is sensitive to pH, substrate, inhibitors and chelators. An addition of substrate and inhibitors results in a formation of free radicals on the enzyme. The properties of the enzyme are compared to those of copper-containing amine oxidases from other sources.