Oxidation of some seleno-diamines by pig kidney diamine oxidase

The enzymatic deamination of Se-lanthionamine (Se-L), Se-homolanthionamine (Se-HL) and Se-homocystamine (Se-HC) catalyzed by pig kidney diamine oxidase (PKAO) has been studied to collect information about the possible metabolic reactions of selenocompounds in comparison with the sulfur ones. The results indicate that: Se-L, Se-HL and Se-HC are substrates for PKAO. The first product of Se-L, Se-HL and Se-HC oxidative deamination, in analogy with other thio and seleno diamines, may be the cyclized aminoaldehyde. The final product of Se-L, Se-HL and Se-HC degradation are ammonia, elemental selenium and C2 or C3 compounds.     

Inhibition of pig kidney diamine oxidase by substrate analogues

1. The oxidation of p-dimethylaminomethylbenzylamine was followed spectrophotometrically by measuring the change in E(250) caused by the p-dimethylaminomethylbenzaldehyde produced. 2. This reaction was inhibited by substrate analogues such as isothiouronium, guanidino, dimethylsulphonium and trimethylammonium compounds. 3. The inhibition by both mono- and bis-onium compounds has been studied and a comprehensive theory is developed to explain both the type and degree of inhibition produced.     

Analytical-band centrifugation of the active form of pig kidney diamine oxidase

In this communication it is shown that pig kidney diamine oxidase undergoes an association-dissociation reaction which is under the influence of the concentration of oxygen, one of the substrates. The sedimentation constant of the active unit was measured using the analytical-band centrifugation of the active enzyme-substrate complex.     

Synthesis and oxidation of aminoalkyl-onium compounds by pig kidney diamine oxidase

1. The preparation of a series of compounds derived from diamines by replacing one amino group by a dimethylsulphonium, isothiuronium, trimethylammonium, NN'-dimethylimidazolium or N-methylpyridinium species is described. 2. The behaviour of these compounds as substrates of pig kidney diamine oxidase is reported. All but the trimethylammonium compounds proved to be substrates. 3. Many of these compounds showed potent inhibition at high substrate concentration and this was studied. 4. On the basis of these and other observations a scheme for enzyme-substrate interaction is suggested.     

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.     

A reinvestigation of the substrate specificity of pig kidney diamine oxidase

1. The substrate specificity of pig kidney diamine oxidase was reinvestigated with a purer enzyme preparation than has previously been used for this purpose. 2. All substrates were extensively purified before use, and methods of preparation or sources are given, together with R(F) values. 3. The substrate specificity determined differed somewhat from that reported by previous workers and, in addition, the behaviour of several compounds not previously used as substrates is described. 4. A model for enzyme-substrate interaction embodying these observations is formulated. It is suggested that a negatively charged substrate-binding group is situated at 6.0-9.0 A from the oxidizing site. The binding and oxidizing sites are separated by a hydrophobic or methylene-binding site.     

Immunochemical reactivity of native and modified preparations of pig kidney diamine oxidase

This paper describes the antigenicity of pig kidney diamine oxidase [EC 1.4.3.6] and the possible role of constituent amino acids in the epitope structure of the enzyme. The loss of 62% of the biological activity after DAO-anti-DAO antibodies interaction was attributed to the steric hindrance caused by binding of antibody to the enzyme molecule. A gradual loss in antigenicity during ultraviolet (UV) irradiation was observed without any significant conformational change, demonstrating the destruction of antigenic determinants. However, ethoxyformylation of nine histidyl residues with complete inactivation caused no change in immunoreactivity. The results indicate that the antigenic sites and catalytic sites are located at different positions along the polypeptide chain. Moreover, the results of lysine residue modification were suggestive of possible involvement of lysine in the antigenic determinants of DAO.     

The effect of potassium-deficiency on diamine oxidase activity in Pea

Pea plants grown in nutrient solution in which K⁺ ions were equimolarly replaced with Na⁺, NH₄ ⁺ or Rb⁺ did not show morphological symptoms of potassium-deficiency. The activity of diamine oxidase in these plants was higher than in controls. Similarly higher diamine oxidase activity was found in plants grown in a complete nutrient solution supplemented with putrescine.     

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.     

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.     

Role of disulfides in biological activity and conformational stability of pig kidney diamine oxidase: evidence for two disulfide states

Six disulfides are found to be present in pig kidney diamine oxidase and all of these are available to reducing agents under nondenaturating conditions. Disulfide reduction with dithiothreitol followed by carbamidomethylation indicated two states of disulfides, each containing three groups, distinguishable by pH dependence. The first group of three disulfides has a functional role in catalytic activity. The another class of three disulfides showed accessibility only at higher pH values and appears to be important in maintaining the three dimensional structure of the molecule. The disulfides for these two activities appear to be independent of each other. Almost similar behaviour was noticed with copper depleted apo-enzyme.     

Oxidation of p-dimethylaminomethylbenzylamine by pig kidney diamine oxidase. A new method for spectrophotometric assay

1. The oxidation of some para-substituted benzylamines by diamine oxidase produces the corresponding aldehydes. This was studied to develop a spectrophotometric method for following the enzyme reaction, as the aldehydes produced absorb strongly at 250nm where the substrates are almost optically transparent. 2. p-Dimethylaminomethylbenzylamine was the most useful substrate and full details of its preparation are given. The synthesis of its related oxidation product, p-dimethylaminomethylbenzaldehyde, is also described. 3. The effects of variations in pH, ionic strength, temperature and oxygenation on the reaction are described and the usefulness of the method is illustrated by several applications and assessed by comparison with the standard spectrophotometric assay.