Diamine oxidase: molecular weight and subunit analysis.

Electrophoretically pure hog kidney diamine oxidase has been isolated by an improved procedure and subjected to molecular weight and subunit analyses. Sedimentation/diffusion and sedimentation equilibrium ultracentrifugation clearly show that the native enzyme has a molecular weight of 172,000. Acrylamide gel electrophoresis indicates that the enzyme consists of two apparently identical subunits of 91,000 daltons each. The native enzyme contains two firmly bound Cu(II) ions. The isolation procedure described provides diamine oxidase in 50–60% yield of activity and of the highest specific activity yet reported (1.2 units/mg).     

Optimized Preparation and Determination of Pea Seedling Diamine Oxidase

The level of diamine oxidase in pea seedling stems has been determined as a function of time after germination in both etiolated and non-etiolated plants. The maximum amount of enzyme per plant is obtained between 11 and 13 days. The amount of activity per gram of tissue appears to be proportional to the rate of growth. We describe an efficient method of isolation of pea seedling stem diamine oxidase from 12-day-old etiolated seedlings, a procedure that brings the enzyme to purity after a 97-fold purification. A new assay procedure for pea seedling diamine oxidase is detailed and compared to previously used methods. The kinetic parameters for three common substrates have also been determined. SDS-acrylamide gel electrophoresis, gel filtration chromatography and copper analyses have been used to determine that pea seedling diamine oxidase exists as a dimer of two apparently identical subunits, the dimer molecular weight being about 190,000. The isoelectric point of this enzyme was determined to be 6.5.     

Optimized preparation and determination of pea seedling diamine oxidase

The level of diamine oxidase in pea seedling stems has been determined as a function of time after germination in both etiolated and non-etiolated plants. The maximum amount of enzyme per plant is obtained between 11 and 13 days. The amount of activity per gram of tissue appears to be proportional to the rate of growth. We describe an efficient method of isolation of pea seedling stem diamine oxidase from 12-day-old etiolated seedlings, a procedure that brings the enzyme to purity after a 97-fold purification. A new assay procedure for pea seedling diamine oxidase is detailed and compared to previously used methods. The kinetic parameters for three common substrates have also been determined. SDS-acrylamide gel electrophoresis, gel filtration chromatography and copper analyses have been used to determine that pea seedling diamine oxidase exists as a dimer of two apparently identical subunits, the dimer molecular weight being about 190,000. The isoelectric point of this enzyme was determined to be 6.5.     

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

Human kidney diamine oxidase has been overexpressed as a secreted enzyme under the control of a metallothionein promoter in Drosophila S2 cell culture. This represents the first heterologous overexpression and purification of a catalytically active, recombinant mammalian copper-containing amine oxidase. A rapid and highly efficient purification protocol using chromatography on heparin affinity, hydroxyapatite, and gel filtration media allows for the recovery of large quantities of the recombinant enzyme, which is judged to be greater than 98% homogenous by SDS/PAGE. The availability of large quantities of highly purified enzyme makes it now possible to investigate the spectroscopic, mechanistic, functional, and structural properties of this human enzyme at the molecular level. Visible absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman spectroscopic results are presented. The recombinant enzyme contains the cofactors 2,4,5-trihydroxyphenylalaninequinone and copper at stoichiometries of up to 1.1 and 1.5 mol per mol homodimer, respectively. In addition, tightly bound and stoichiometric calcium ions were identified and proposed to occupy a second metal-binding site. The apparent molecular weight of the recombinant protein, determined by analytical ultracentrifugation, suggests 20-26% glycosylation by weight. Detailed kinetic studies indicate the preferred substrates (k(cat)/K(M)) of human diamine oxidase are, in order, histamine, 1-methylhistamine, and putrescine, with K(M) values of 2.8, 3.4, and 20 microM, respectively. These results, demonstrating the substrate preference for histamine and 1-methylhistamine, were unanticipated given the available literature. The pH dependence of k(cat) for putrescine oxidation gives two apparent p K(a) values at 6.0 and 8.2. Tissue-specific expression of the human diamine oxidase gene was investigated using an mRNA array. The relevance of this work to earlier work and the suggested physiological roles of the human enzyme are discussed.     

Purification of human placenta diamine oxidase.

Diamine oxidase (histaminase) is produced at very high levels by the decidual cells of the placenta. The presence of diamine oxidase has been demonstrated in human neutrophils. Purification of human placenta diamine oxidase was performed by four subfractionation steps and led to the isolation of one polypeptide whose molecular weight was 84,000, as assessed by SDS PAGE. Using polyclonal antibodies raised against the purified enzyme, we have demonstrated that the neutrophil diamine oxidase is immunochemically identical to the placental diamine oxidase. Development of immunological methods will be useful for detection and quantitation of diamine oxidase in neutrophils during the inflammation process.     

Pig kidney diamine oxidase. A new method of purification.

Diamine oxidase has been purified from pig kidney by a new method to rapidly obtain larger amounts of pure enzyme with a good yield. The enzyme obtained gives only one band in SDS gel electrophoresis. The specific activity and the absorption spectra were identical to those of already preparations homogeneous reported by different methods of purification.     

Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration

The effect of a single administration of ethanol (2 g/kg body weight) on hepatic and renal diamine oxidase activity was studied in fasted rats. Diamine oxidase activity significantly increased in liver and kidney 6 h after ethanol intubation. Pyrazole (an inhibitor of alcohol dehydrogenase), cycloheximide or actinomycin D (inhibitors of macromolecular syntheses), as well as prior adrenalectomy, prevented the ethanol-induced stimulation of diamine oxidase in the liver, but not in the kidney. The results demonstrated that the enhancement of diamine oxidase activity in the liver was due to an enzyme induction mediated by alcohol metabolism as well as by adrenals. In contrast, the stimulation of diamine oxidase activity in the kidney did not depend on synthesis of new enzyme molecules and was not mediated by ethanol metabolism or adrenal hormones.     

The amine oxidases of human placenta and pregnancy plasma

1. The purification of monoamine oxidase and diamine oxidase from normal human term placental tissue is described. 2. The properties of these enzymes are reported and compared with the properties of unpurified human pregnancy plasma. 3. This comparison shows that the amine oxidase of pregnancy plasma has properties corresponding to purified placental diamine oxidase, suggesting a placental origin for the plasma enzyme system. 4. Detailed kinetic study of the purified placental diamine oxidase suggests that it has a Ping Pong sequence, a mechanism of action and rate-limiting step similar to the diamine oxidase of pig kidney. 5. It is suggested that the enzyme system is important in protecting the foeto-placental unit from excesses of biogenic amines.     

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.     

Cloning, sequence analysis, and characterization of the 'lysyl oxidase' from Pichia pastoris

Lysyl oxidase from Pichia pastoris has been successfully overexpressed. EPR and resonance Raman experiments have shown that copper and TPQ are present, respectively. Lysyl oxidase from P. pastoris has a similar substrate specificity to the mammalian enzyme (both have been shown to oxidize peptidyl lysine residues) and is 30% identical to the human kidney diamine oxidase (the highest of any non-mammalian source). This enzyme also has a relatively broad substrate specificity compared to other amine oxidases. Molecular modeling data suggest that the substrate channel in lysyl oxidase from P. pastoris permits greater active site access than observed in structurally-characterized amine oxidases. This larger channel may account for the diversity of substrates that are turned over by this enzyme.     

On the nature of chromophore in pig kidney diamine oxidase.

The nature of the 500-nm chromophore in pig kidney diamine oxidase was investigated by absorption spectroscopy and fluorescence in the presence of various chelating or carbonyl-specific reagents. From the spectroscopic measurements the following conclusions can be drawn. First, the 500-nm absorption band is not due to copper, the reduction of which is not related to the disappearance of this band. Second, phenylhydrazine and cycloserine give rise, upon reaction with the enzyme, to absorptions very similar to those of a pyridoxal enzyme, aspartate aminotransferase. Third, these enzyme derivatives are unexpectedly non-fluorescent. Copper removal, obtained after prolonged incubation of cycloserine-treated enzyme in the presence of reducing and chelating agents, leads to a fluorescence similar to that of cycloserine-aspartate transminase. It is proposed that copper is coordinated to the postulated pyridoxal phosphate of diamine oxidase through the pyridine nitrogen.     

Inhibition of plant and mammalian diamine oxidases by hydrazine and guanidine compounds

1. Pig kidney and pea seedling diamine oxidases have similar sensitivity to methylhydrazine and phenylhydrazine as inhibitors. 2. Inhibition of pig kidney and pea seedling enzymes by hydrazine and guanidine compounds is time dependent. To reveal full inhibitory potency, methylhydrazine and aminoguanidine need longer preincubation with plant diamine oxidase as compared with mammalian diamine oxidase. 3. Impromidine, a known H2 histamine receptor agonist with guanidine and imidazole structures, and aminoguanidine have higher inhibitory activity towards pig kidney enzyme in comparison with the pea seedling one. 4. Impromidine inhibits pig kidney diamine oxidase in a noncompetitive manner. The Ki value is 6.6 muM. 5. The 24 hr dialysis of rat intestinal diamine oxidase preincubated with phenylhydrazine or impromidine only partially recovered the enzymic activities. 6. Impromidine inhibits mouse intestinal diamine oxidase in vivo.     

Spectroscopic and kinetics studies of the inhibition of pig kidney diamine oxidase by anions

The role of copper in pig kidney diamine oxidase has been probed by examining the effects of potential Cu(II) ligands on the spectroscopic and catalytic properties of the enzyme. In the presence of azide and thiocyanate, new absorption bands are evident at 410 nm (epsilon = 6300 M-1 cm-1) and 365 nm (epsilon = 3000 M-1 cm-1), respectively. These bands are assigned as ligand-to-metal charge-transfer transitions, N3-/SCN- leads to Cu(II). One anion/Cu(II) is coordinated in an equitorial position. Anion binding can be completely reversed by dialysis. The equilibrium constants for diamine oxidase-anion complex formation are 134 M-1 (N3-) and 55 M-1 (SCN-). Azide and thiocyanate are linear uncompetitive inhibitors with respect to the amine substrate when O2 is present at saturating concentrations. Taken together, the data are consistent with a functional role for Cu(II) in diamine oxidase catalysis.     

Induction of diamine oxidase activity in rat kidney during compensatory hypertrophy

Diamine oxidase (EC 1.4.3.6) activity, measured as [¹⁴C]Δ¹-pyrroline formation from [¹⁴C] putrescine, was studied in homogenates of rat kidney during compensatory hypertrophy after unilateral nephrectomy. Acetaldehyde and to a lesser degree phenobarbital, at concentrations which did not modify the activity of a preparation of hog kidney diamine oxidase, increased Δ¹-pyrroline formation in kidney homogenate, which suggests that aldehyde-metabolizing enzymes present in this tissue may interfere with yield of Δ¹-pyrroline and that the use of acetaldehyde may give better information on kidney diamine oxidase activity. Other inhibitors of aldehyde-metabolizing enzymes such as chloral hydrate, disulfiram, and pyrazole cannot be used for diamine oxidase determination since they stimulated or depressed this enzyme activity. In rat kidney undergoing compensatory hypertrophy the levels of putrescine, spermidine, and spermine increased rapidly and were followed by an increase in diamine oxidase activity that presented a first peak on day 2 and a second peak on day 6. The administration of cycloheximide or actinomycin D to nephrectomized rats prevented the increase in diamine oxidase activity. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 14 h in normal and hypertrophic kidney. These results suggest that the increase in diamine oxidase activity in renal hypertrophy was due to the synthesis of new enzyme rather than to a slowing of its degradation.     

Diamine oxidase from horse kidney: ionic strength dependence of stability and activity

Diamine oxidase was prepared from horse kidney by a procedure involving heat denaturation at 50 degrees C, ammonium sulfate fractionation, chromatography on hydroxyapatite and on G-200 Sephadex columns. This procedure gave about 1000 fold purification over the crude kidney cortex homogenate. The enzyme preparations thus obtained are stable only at high ionic strength. The effect on enzyme activity of salt concentration and various stabilizing agents have been investigated. The horse kidney diamine oxidase is irreversibly inhibited by carbonyl reagents and shows substrate specificity quite similar to other animal diamine oxidases.     

Aminoacetone oxidase from goat liver. Formation of methylglyoxal from aminoacetone

An enzyme which oxidizes aminoacetone to methylglyoxal has been purified from the particulate fraction of goat liver. Polyamines, such as spermidine and spermine, are also good substrates for this enzyme. The pH optimum for aminoacetone oxidation was found to be 8.2. The apparent Km values of the enzyme for aminoacetone and spermidine were 0.009 and 0.095 mM, respectively. The subunit molecular weight of the enzyme was 93,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of the native enzyme was 186,000 by gel filtration. The enzyme is highly sensitive to carbonyl group reagents. The enzyme is not inhibited by monoamine and diamine oxidase inhibitors.     

Rat kidney L-alpha-hydroxy acid oxidase: isolation of enzyme with one flavine coenzyme per two subunits.

L-alpha-Hydroxy acid oxidase (listed as EC 1.4.3.2, L-amino acid: O2 oxidoreductase) has been purified 100-fold from rat kidney to apparent homogeneity by gel electrophoresis. A subunit molecular weight of 47,500 was found by sodium dodecyl sulfate gel electrophoresis, but in contrast to previous reports, the enzyme has been found to have a molecular weight of ca. 200,000 by Sephadex gel filtration and by dodecyl sulfate gel electrophoresis of the enzyme cross-linked with dimethyl suberimidate. A somewhat higher value was found by sedimentation equilibrium, but a tetrameric structure for the active enzyme is definitely established. The enzyme was found to contain the FMN coenzyme at a concentration of one FMN/102,000 daltons or one flavine/two subunits, a highly unusual finding. This ratio was determined from spectroscopic analysis of the FMN in lyophilized samples of the enzyme and by titration of the coenzyme with the flavine specific enzyme inactivator 2-hydroxy-3-butynoate. The enzyme has the same specific activity as a crystalline sample of the enzyme reported to have twice as much flavine/milligram.     

Purification and characterization of mouse kidney beta-glucuronidase.

Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.     

Determination of diamine oxidase in lentil seedlings by enzymic activity and immunoreactivity

A competitive radioimmunoassay for the quantitation of diamine oxidase (EC 1.4.3.6) from Lens culinaris is reported. Specific antibodies raised in rabbits immunized with a homogeneous preparation of the enzyme were incubated with purified ¹²⁵I-enzyme and with either unlabeled diamine oxidase or plant material. Antigen-antibody complexes were isolated from the mixture by incubation with Staphylococcus protein A. The sensitivity of the test was about 5 nanograms in terms of enzyme protein. This assay was applied to the determination of the enzyme in extracts from lentil shoots grown either in the dark or in the light. Diamine oxidase activity and enzyme protein (as determined by radioimmunoassay) were measured during 7 days after germination. Both enzymic activity and enzyme protein declined slowly in the dark and rapidly in the light. These results indicate that fluctuation of the enzymic activity in this organ, both in the light and in the dark, are mediated via changes in the amount of the enzyme protein and not via the action of an inhibitor.     

The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.