The depression of the synthesis of pea diamine oxidase due to light and the verification of its participation in growth processes using competitive inhibitors

The time courses of the synthesis of diamine oxidase in pea plants grown for 14 days either in the light or in the dark are similar with the highest increase in activity occurring in the cotyledons and in the shoots during the first 6 to 8 days. Plants grown in the dark showed a 2- to 3-fold higher enzyme activity than plants grown in the light. Pea diamine oxidase could bein vivo efficiently inhibited by substrate analogues 1,4-diamino-2-butanone and 1,5-diamino-3-pentanone. The first compound inhibited proportionally to its concentration the growth of etiolated pea plants, but its instability makes an unequivocal interpretation of the results difficult. On the other hand, 1,5-diamino-3-pentanone a stable and more efficient diamine oxidase inhibitor depressed the growth of pea seedlings only at concentrations as high as 5 mM and 10 mM, at which the growth of cress seedlings not containing diamine oxidase was also strongly depressed. The results obtained indicate that tryptamine oxidation catalyzed by diamine oxidase is not involved in the main metabolic pathway leading from tryptophan to indoleacetate in pea plants.     

The effects of copper nutrition and developmental state on the biosynthesis of diamine oxidase in clover leaves

The biosynthesis of diamine oxidase (DAO; EC 1.4.3.6) in leaf blades of subterranean clover (Trifolium subterraneum L. cv Seaton Park) was followed by labeling whole plants with ¹⁴CO₂. A pulse-chase experiment where DAO was immunoprecipitated with anti-DAO antibodies showed that only leaf primordia and the youngest emerged leaves were able to synthesize the enzyme. The amount of DAO in young leaves of clover grown with a range of Cu treatments was determined by its enzymic activity and by single radial immunodiffusion against anti-DAO antibodies; both parameters were highly correlated with the Cu concentration of the leaf. Further, anti-DAO antibodies reacted against apo-DAO prepared in vitro indicating that apo-DAO was absent from Cu-deficient leaves. These results suggest that the biosynthesis of DAO in young clover leaves is controlled by the Cu concentrations of the leaves. Poly(A) mRNA purified from leaf primordia and young emerging leaves of plants with either a high Cu or low Cu supply was translated in wheat germ and rabbit reticulocyte cell-free systems. No differences between the two Cu treatments could be seen in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the translation products after fluorography. However, anti-DAO antibodies did not detect any DAO synthesized in vitro from either treatment.     

Light modulation of maize leaf phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH₄)₂SO₄ fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition.     

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.     

Biosynthesis of phytoene from isopentenyl pyrophosphate by a Neurospora enzyme system.

An enzyme system catalyzing the conversion of isopentenyl pyrophosphate to phytoene has been isolated from Neurospora crassa mycelia. This enzyme system shows an absolute requirement for Mg^?, but no other cofactors. Cultures of N. crassa exhibit a low level of phytoene synthesizing activity when grown in the dark. A 2-min in vivo blue light irradiation results in a ninefold increase in activity after 24 h. This increase is dependent on the duration of the light treatment and is inhibited by cycloheximide. A similar blue light-induced elevation of phytoene synthesizing activity was demonstrated in an albino-1 mutant. This enzyme activity was not found in either dark-grown or irradiated cultures of an albino-2 or an albino-3 mutant.     

Phytochrome-mediated regulation of a monooxygenase hydroxylating cinnamic acid in etiolated pea seedlings

Cinnamic acid is hydroxylated by the mixed-function oxidase trans-cinnamic acid 4-hydroxylase (CA4H). The hydroxylation reaction involves the transfer of electrons from reduced pyridine nucleotides via the enzyme NADPH cytochrome P-450 reductase to the terminal oxidase cytochrome P-450. This multi-enzyme complex is localized in the microsomal fraction. Isopycnic and velocity gradient centrifugation suggest that in the apical bud of etiolated pea seedlings this complex is restricted to the endoplasmic reticulum membranes. CA4H activity which develops in dark germinating pea seedlings was found to be stimulated by light, an effect mediated by phytochrome. CA4H and NADPH cytochrome c reductase activities, cytochromes P-450 and b 5 contents were measured in seedlings submitted to either short pulses of red and far-red light, or to continuous far-red or blue irradiation. The results are discussed in terms of a specific effect of phytochrome on the different parts of the multi-enzyme complex.     

Development of three copper metalloenzymes in clover leaves

Subterranean clover (Trifolium subterraneum L. cv Seaton Park) was grown in solution cultures containing adequate nitrogen both with and without Cu. After Cu deficiency had developed, Cu²⁺ was added to some deficient plants and Cu content, protein content, and activities of three Cu metalloenzymes (diamine oxidase [EC1.4.3.6], ascorbate oxidase [EC1.10.3.3] and o-diphenol oxidase [EC1.10.3.1]) were assayed in young and recently matured leaf blades over 11 days during the development of the next three leaves.

Copper deficiency had little effect on protein concentrations, but markedly depressed enzyme activities and Cu concentration in all leaf blades assayed. Within 4 d of adding Cu²⁺ to Cu-deficient plants, Cu concentrations of all the leaf blades increased to adequate values. Enzyme activities only increased to control levels in leaves which had not yet emerged at the time that Cu²⁺ was added.

The results suggest that active holoenzymes of diamine oxidase, ascorbate oxidase, and o-diphenol oxidase can only be synthesized in leaf blades during very early stages of their development.

     

Immunoaffinity purification and characterization of diamine oxidase from Cicer

Antiserum specific for diamine oxidase (DAO;EC 1.4.3.6) from Lens culinaris cross-reacted with DAO from several other members of the Leguminosae when tested by agar double diffusion. Antibodies purified by affinity chromatography were used to make an immunoadsorbent for the one-step purification of DAO from various species of the Leguminosae. This technique has made it possible to purify in one step the already characterized DAO from pea and lentil, and the unknown diamine oxidase from Cicer arietinum. This enzyme was partially characterized; it showed a pH optimum of 7.5 with putrescine as substrate and followed typical Michaelis-Menten kinetics with a K_m of 2.4 × 10^?4 M. Copper ligands and carbonyl group-directed reagents inhibited the enzyme.     

Alternatiol-O-methyltransferase fromAlternaria alternata: Partial purification and relation to polyketide synthesis

Alternaria alternata produces the polyketide mycotoxins alternariol (AOH) and alternariol monomethylether (AME) during the stationary growth phase when cultured in darkness. AME is formed by methylation of AOH by an alternariol-O-methyltransferase (AOH-MT). This methyltransferase was purified to near homogeneity from dark grown cultures ofA. alternata resulting in a 240-fold purification. The major protein in the enriched fraction of AOH-MT had a mass of 43,000 Da and was shown to bind the cofactorS-adenosyl-[³H]methionine by photoaffinity labeling, suggesting that this polypeptide contained the active site. WhenA. alternata was cultured in white light, the accumulation of AOH and AME was reduced to less than 4% of the production in darkness which is in agreement with earlier results. This reduction in polyketide content was accompanied by a reduced AOH-MT activity in extracts from light grown cultures. However, the activity of AOH-MT in mycelia grown in light was only reduced to 30% of the activity in dark grown cultures. Thus, it seems that the main target for light suppression of polyketide accumulation inA. alternata is either the activity or formation of the enzyme synthesizing AOH or the precursor availability for AOH synthesis.     

Protein degradation in lemna with particular reference to ribulose bisphosphate carboxylase: I. The effect of light and dark

Ribulose bisphosphate carboxylase from Lemna minor resembles the structure reported for the enzyme from other plants. When grown in the light, the enzyme appears to undergo little or no degradation, as measured by a double-isotope method. This situation is similar to that reported for wheat and barley, but is unlike that reported for maize, where the enzyme degrades at the same rate as total protein. Prolonged periods of darkness usually induce leaf senescence, characterized by the rapid degradation of chlorophyll and protein, with ribulose bisphosphate carboxylase undergoing preferential degradation. In L. minor there is selective protein degradation in the dark, but chlorophyll and ribulose bisphosphate carboxylase are stable when fronds are kept in the darkness for up to 8 days. It appears that Lemna is not programmed to senesce, or at least that darkness does not induce senescence in Lemna. Although there is no evidence for in vivo degradation or modification of ribulose bisphosphate carboxylase during prolonged periods of darkness, extracts from fronds which have been kept in the dark for periods in excess of 24 hours convert ribulose bisphosphate carboxylase to a more acidic form. The properties of the dark-induced system which acts on ribulose bisphosphate carboxylase, suggest that it may be a mixed function oxidase. The proposition that the selectivity of protein degradation is genetically determined, so that the rate at which a protein is degraded is determined by its charge or size, was tested for fronds grown in the light or maintained in the dark. There was no significant correlation between protein degradation and either charge or size, in light or dark.     

Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V–VII were rather resistant to degradation. From the relative inaccessibility of subunits V–VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.     

Spermidine oxidase in human pregnancy serum. Probable identity with diamine oxidase.

Diamine oxidase was previously measured in human pregnancy serum with putrescine or histamine as substrate. We have now documented the presence of spermidine oxidase activity in pregnancy serum by means of a specific radioactive assay with [14C]spermidine as substrate and Dowex 50 cation-exchange chromatography to separate products from substrate. The apparent Km of a partially purified preparation of this enzyme for spermidine was 10.9 microM and the Ki for aminoguanidine was 0.8 microM. The pH optimum (pH 9.0) and temperature optimum (55 degrees C) were identical with those for diamine oxidase. Spermidine oxidase activity and diamine oxidase activity eluted in a concerted fashion when pregnancy serum was subjected to cadaverine-Sepharose chromatography, gel filtration and ion-exchange chromatography. Spermidine oxidase became detectable in serum during pregnancy in the human approx. 8 weeks after the last menstrual period and increased with gestational age in concert with the increase in diamine oxidase activity, reaching a plateau at 20 weeks of gestation. Foetal-cord serum displayed virtually no activity of either enzyme. A 400-fold-purified preparation of diamine oxidase retained the same diamine oxidase/spermidine oxidase ratio as exhibited by crude pregnancy serum. These data suggest that in pregnancy serum, unlike foetal bovine serum, spermidine oxidase and diamine oxidase activity may be a single enzyme protein.     

Light promotes an increase of cytokinin oxidase/dehydrogenase activity during senescence of barley leaf segments

Following a study of the relationship between cytokinin oxidase/dehydrogenase (CKX) and senescence in darkened barley leaf segments, we have now investigated the influence of light on the in vitro activity of CKX. Seedlings of Hordeum vulgare L. were grown for 8 d under a light/dark regime of 18 h white light and 6 h darkness. Then apical parts of 7 cm length were cut from the first foliage leaves and their bases were placed in water. In segments kept in the dark, the CKX activity measured by cleavage of N₆-(Δ₂-isopentenyl)adenine rose from 0.1 pkat (g FW)⁻¹ to 0.8 pkat (g initial FW)⁻¹ within the first 4 d of incubation. In contrast, in segments kept under the light/dark regime it reached a value of 8.6 pkat (g initial FW)⁻¹ over the same time period. The chlorophyll a content declined slightly slower during light/dark cycling than in darkness. In contrast to segments and isolated laminae, corresponding attached laminae exhibited less CKX activity after 2 d under light/dark conditions than after 2 d in the dark. The activity in attached laminae of first foliage leaves of plants growing in light/dark cycling increased strongly only when the plants were older than 4 weeks. In line with this, the CKX activity in attached laminae of flag leaves of barley growing in fields increased in a late developmental state. The senescence of darkened isolated laminae of Zea mays L. and Phragmites australis (Cav.) Trin. ex Steudel was associated with an enhancement of CKX activity too. Because in most cases a positive correlation between CKX activity and senescence was found, it is likely that the enzyme promotes senescence by destroying cytokinins, which help to keep Poaceae leaves green. Light may promote not only cytokinin degradation but also the formation of bioactive cytokinins in leaf segments.     

Purification and properties of a polyamine oxidase from Zea mays

Polyamine oxidase, purified 260-fold from maize shoots, was light yellow in colour. Maximum light-absorption was at 450 nm and was decreased by the addition of either sodium dithionite or spermidine, but not by putrescine. Under aerobic conditions, the enzyme could use p-benzoquinone as an electron acceptor. Cu²⁺ inhibited the enzyme activity, while SO₃⁻ was stimulatory. Several metal-binding agents and thiol reagents were without effect.     

Release of intestinal diamine oxidase by histamine in rats

Diamine oxidase activity was measured in the intestinal mucosa, lymph, and in the serum of rats, to determine whether histamine, a substrate of diamine oxidase, liberates this enzyme from its mucosal storage site(s). Histamine induced a sharp rise in intestinal lymph flow, lymph protein, and lymph diamine oxidase, lasting less than 1 h after the histamine injection. The rise in lymph diamine oxidase activity was dose dependent over a narrow concentration range (0.05-0.2 mmol/kg, i.v. and 0.15-0.6 mmol/kg i.d.). It did not correlate with the dose dependent increase in lymph flow or lymph protein. A single maximal intraduodenal dose of histamine caused a 41.6-fold increase in the lymph diamine oxidase activity and a 2.4-fold increase in the serum enzyme level temporarily. A second injection of histamine, 2 h after the first, resulted in a comparatively smaller increase in the lymph enzyme. The extent of the reduction was dependent on the magnitude of the first injection. The results suggest that histamine causes a limited liberation of diamine oxidase from the intestinal mucosa. The function of this enzyme release may be a protective response by the mucosa to reduce toxic levels of free histamine, either liberated by the mucosal tissue or absorbed from the intestinal lumen.     

Metabolism of acetylpolyamines by monoamine oxidase,diamine oxidase and polyamine oxidase

N¹-Monoacetylspermine, N¹,N¹²-diacetylspermine and N¹-monoacetylspermidine were found to be good substrates for rat liver polyamine oxidase, but not for rat liver mitochondrial monoamine oxidase. N⁸-Monoacetylspermidine, monoacetylcadaverine, monoacetylputrescine and monoacetyl-1,3-diaminopropane were oxidized by the monoamine oxidase when the substrate concentration was 10.0 mM, but not by the polyamine oxidase. All the acetylpolyamines except N¹,N¹²-diacetylspermine were also oxidized by hog kidney diamine oxidase although their affinities for the oxidase appeared low. The present data suggest that acetylpolyamines are not easily metabolized in vivo by either monoamine oxidase or diamine oxidase in mammalian tissues although N¹-monoacetylspermine, N¹,N¹²-diacetylspermine and N¹-monoacetylspermidine are attacked by polyamine oxidase.     

Catabolism of polyamines

Summary. Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N¹-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects.Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N⁸-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.     

Distribution of newly synthesized amylase in microsomal subfractions of guinea pigs pancreas

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).¹ Amylase appeared in the following positions in the gradient: (a) a light region which contained ∼35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained ∼10% of enzymic activity in a sharp peak but which had very little accompanying OD₂₆₀ absorption; (c) a pellet at the bottom of the centrifuge tube which contained ∼20% of the enzymic activity. After 5 to 20 min'' in vivo labeling with leucine-1-C¹⁴, radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized ∼80% of the total amylase in the fraction from the heavy region of the gradient, but only ∼20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.     

Levels of plasma ceruloplasmin protein are markedly lower following dietary copper deficiency in rodents

Ceruloplasmin (Cp) is a multicopper oxidase and the most abundant copper binding protein in vertebrate plasma. Loss of function mutations in humans or experimental deletion in mice result in iron overload consistent with a putative ferroxidase function. Prior work suggested plasma may contain multiple ferroxidases. Studies were conducted in Holtzman rats (Rattus norvegicus), albino mice (Mus musculus), Cp?/? mice, and adult humans (Homo sapiens) to investigate the copper–iron interaction. Dietary copper-deficient (CuD) rats and mice were produced using a modified AIN-76A diet. Results confirmed that o-dianisidine is a better substrate than paraphenylene diamine (PPD) for assessing diamine oxidase activity of Cp. Plasma from CuD rat dams and pups, and CuD and Cp?/? mice contained no detectable Cp diamine oxidase activity. Importantly, no ferroxidase activity was detectable for CuD rats, mice, or Cp?/? mice compared to robust activity for copper-adequate (CuA) rodent controls using western membrane assay. Immunoblot protocols detected major reductions (60–90%) in Cp protein in plasma of CuD rodents but no alteration in liver mRNA levels by qRT-PCR. Data are consistent with apo-Cp being less stable than holo-Cp. Further research is needed to explain normal plasma iron in CuD mice. Reduction in Cp is a sensitive biomarker for copper deficiency.