Equations of substrate-inhibition kinetics applied to pig kidney diamine oxidase (DAO, E.C. 1.4.3.6).

Pig kidney diamine oxidase (DAO) and other semicarbazide-sensitive amine oxidases (SSAO) show clear substrate-inhibition kinetics and a reaction-scheme mechanism based on two substrate binding sites. We evaluated several reaction scheme mechanisms with a non-linear regression program (NCSS), estimating R2, the constants of the equations and their standard errors and we determined the deviation of experimental data from theoretical equations. The best fit was obtained with a "dead end" mechanism with two binding sites. Based on this scheme, other schemes for a two-substrate reaction and for mechanisms of inhibition were constructed. These reaction schemes, even at low substrate concentration, fitted experimental data better than Michaelis-Menten kinetics, and provided information on the mechanisms of action of inhibitors. The presence of two substrate-binding sites on pig kidney DAO was confirmed by all experimental data.

Biochemical aspects and functional role of the copper-containing amine oxidases.

The copper-containing amine oxidases of the class EC 1.4.3.6 share many biochemical similarities. They contain cupric copper and catalyse the same general reaction. In mammals, diamine oxidase has a role in the metabolism of histamine, some other diamines and spermine oxidase, involved in the metabolism of polyamines. However, the functional role of benzylamine oxidase (BAO) and the tissue semicarbazide-sensitive amine oxidases (SSAO) is still under investigation. Circulating BAO is derived from the tissue SSAO. It has a high affinity for benzylamine and its role in the extra-cellular matrix includes the maturation of pro-elastin and control of the inflammatory process.

Semicarbazide-sensitive amine oxidases in heart and bovine serum.

In guinea pig dorsal skin the semicarbazide-sensitive amine oxidase (SSAO) is localised in fibroblasts. Fibroblasts in culture lose the ability to express this enzymatic activity with doublings, thus suggesting that the SSAO expression needs some factors which are not present in the 10% bovine serum culture medium. Fresh bovine serum of adult animals contains two SSAO activities, one with high affinity for benzylamine and one with lower affinity. The enzyme with lower affinity for benzylamine was identified as spermine oxidase, the oxidation of [14C]-benzylamine was inhibited by semicarbazide, alpha-aminoguanidine and B24, a specific inhibitor of benzylamine oxidase and spermine oxidase, both SSAO enzymes. The enzymatic activity of bovine serum was partially purified, the kinetic properties and sensitivity to inhibitors studied. A mathematical procedure for the analysis of the kinetics resulting from the activity of two enzymes acting on the same substrate seems to give better results than the methods previously described.

The histaminase activity of rat white adipocytes.

OBJECTIVE AND DESIGN: Investigate the reaction conditions which allow the measurement of high affinity histamine oxidation. MATERIAL: Isolated rat white adipocytes. METHODS: The histaminase activity of rat white adipocytes and its inhibition by B24 and MDL 72274 was measured fluorimetrically or radiochemically in different experimental conditions. RESULTS: Histamine oxidation by rat white adipocytes is enhanced by elevated pH and by the presence of bicarbonate ions. Under these conditions the oxidative deamination of histamine by white adipocytes, becomes comparable to that of other histaminases, suggesting that this amine oxidase activity may play a role in the catabolism of histamine in vivo. The specific semicarbazide-sensitive amine oxidase (SSAO) inhibitors MDL 72274 and B24 inhibit the oxidation of both histamine and benzylamine by the adipocyte preparation. CONCLUSIONS: Although there are kinetic differences between the behaviour of these two amine substrates, these results would be consistent with a SSAO being responsible for both activities.

Increased desensitization by picomolar phorbol ester of the endothelium-mediated effect of histamine in the perfused rat mesenteric bed.

The vasodilatatory, endothelium-mediated, effect of histamine (H), through H1 receptor, in the isolated and perfused mesenteric bed of the rat, undergoes strong desensitization during perfusion or repetitive injections of noradrenaline (NA) and H. The mesenteric bed completely desensitized to H is responsive to carbachol (C) and this latter compound does not affect the H desensitization. The homologous desensitization to C effect is very small, attaining less than 10% after 30 min of continuous perfusion. In this work the effect of inhibitors or activators of protein-kinase(s)-C (PKC) are studied during continuous perfusion of H or C in preparations preconstricted by NA. Staurosporine antagonizes the onset of the H desensitization, while the rate of desensitization in increased by phorbol-12-myristate-13-acetate (PMA). PMA, at a concentration from 10(-12) to 10(-10)M, selectively enhances the homologous desensitization of H, while at 10(-8)M it also produces a desensitization to C. At least two different PKC isoenzymes might be involved in the desensitization of the vasodilatatory effect of H and C in the isolated and perfused rat mesenteric bed.

Semicarbazide-sensitive amine oxidase activity in white adipose tissue of the insulin-deficient rat.

We have investigated whether the effects in white adipose tissue due to insulin deficiency might also be related to an alteration of histamine levels which are regulated by semicarbazide-sensitive amine oxidase. The lack of circulating insulin induced by streptozotocin produced, in rat white adipose tissue, a loss of affinity of the semicarbazide-sensitive amine oxidase for histamine oxidation. In parallel, a decrease of cell transformation, measured by glycerol-3-phosphate dehydrogenase activity and an augmented sensitivity to histamine lipolysis were observed. These findings could contribute to the understanding of histamine metabolism and function in diabetic rats and to the knowledge concerning amine oxidases in this animal pathology.

The role of semicarbazide-sensitive amine oxidase with a high affinity for benzylamine (Bz. SSAO) in the catabolism of histamine in the mesenteric arterial bed of the rat.

In the mesenteric arterial bed of the rat the semicarbazide-sensitive amine oxidase with a high affinity for benzylamine (Bz. SSAO) (E.C. 1.4.3.6) is also able to oxidize histamine. In the perfused mesenteric arterial bed of the rat the complete inhibition of the Bz. SSAO obtained with a specific inhibitor, B24, almost completely reduces the efflux of imidazole acetic acid and increases the relaxing effect of histamine. Bz. SSAO appears to be the only enzyme present in these blood vessels able to catabolize histamine.

Semicarbazide-sensitive amine oxidase activity of guinea pig dorsal skin.

A semicarbazide-sensitive amine oxidase activity with a high affinity for benzylamine (Bz.SSAO) (E.C. 1.4.3.6) is present in guinea pig dorsal skin. This enzymic activity oxidized benzylamine, histamine, 1,4-methylhistamine and acetylputrescine and was inhibited by semicarbazide and by B24 (3,5-diethoxy-4-aminomethylpyridine), a selective inhibitor of Bz.SSAO enzymes. It cross reacted with the antibodies raised against pure pig plasma benzylamine oxidase. Immunohistochemistry showed that it was localized in fibroblasts. Bz.SSAO activity of guinea pig dorsal skin increased during the process of skin healing. A treatment of the wounds with 3 micrograms of b-FGF significantly accelerated the process of skin healing and the increase of Bz.SSAO activity.

Histaminase activity of mesenteric artery of the rat.

In rat mesenteric artery homogenates histamine is oxidatively deaminated at high rate whereas putrescine is a poor substrate. The oxidation of histamine appears to be mainly catalyzed by an SSAO enzyme with high affinity for benzylamine (Bz.SSAO). Histamine oxidation is inhibited by B24 (2,5-diethoxy-4-aminomethylpyridine), a selective inhibitor of Bz.SSAO enzymes, and reduced by the presence of benzylamine. The Bz.SSAO enzyme which is present in the rat mesenteric artery is not only able to oxidize histamine, but also methylamine, acetylputrescine and some methylated forms of histamine including 1,4-methylhistamine.

Histamine lipolytic activity and semicarbazide-sensitive amine oxidase (SSAO) of rat white adipose tissue (WAT).

Histamine has previously been described as a possible substrate for the semicarbazide-sensitive amine oxidase activity (SSAO) of rat white adipose tissue (WAT). We report here on a histamine function in this tissue which concerns the activity of this deaminating system distinct from the classical diamine oxidase. Our results show that: (1) histamine plays a role in controlling rat adipose tissue lipolysis with the contribution of H1 and H2 receptors that participate in histamine lipolysis in an opposite way. Both H1 and H2 roles can be differentiated using selective agonists (2- and 4-methyl histamine) and antagonists (pyrilamine and cimetidine); (2) histamine might also control rat lipolysis induced by noradrenergic agonists; (3) the SSAO present in rat WAT controls histamine levels at the receptor sites as shown by the modification of histamine lipolytic potency obtained when inhibitors of this enzyme are used.

Skin wound healing: some biochemical parameters in guinea-pig.

Hairs were removed from the dorsal skin of guinea-pigs and 5-6 wounds (7 x 7 mm) were surgically induced by totally removing the epidermal and part of the dermal surface. They were then allowed to heal. The newly formed wound tissues were dissected at different times during the process and analysed by biochemical and histological methods. Hydroxyproline, proteins, DNA and semicarbazide-sensitive amine oxidase (SSAO) were measured, as were [14C]leucine and [3H]thymidine incorporation in some samples. The peroxidase-like activity of plasma albumin and the histology of wounds stained with haematoxylin-eosin were also studied. It was shown that SSAO enzymes, which are present in normal guinea-pig skin and have a high affinity for benzylamine are localized in fibroblasts. During skin healing in the newly formed tissue there was an increase in protein content which reached a maximum after 4-6 days; DNA content also increased. The rate of incorporation of [3H]thymidine and [14C]leucine paralleled DNA and protein content, respectively. The content of hydroxyproline had greatly decreased with respect to that in normal skin after 2-10 days. SSAO activity increased much less than DNA after 4 days whereas after 10-11 days it increased more than DNA, thus indicating that at this time it was probably produced by fibroblasts. No significant increase in the peroxidase-like activity of albumin was observed 4, 8 or 11 days after surgery. Treatment of the animals with methylprednisolone acetate (20 mg kg-1, i.m.) two days before surgery decreased the rate of skin healing but did not alter the level of albumin peroxidase activity of the plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium modulatory properties of 2,6-dibutylbenzylamine (B25) in rat isolated vas deferens, cardiac and smooth muscle preparations.

1. In rat isolated vas deferens the new compound 2,6-dibutylbenzylamine (B25) evoked a series of repeating rhythmic contractions. Concentration-response curves constructed for this effect were bell-shaped, indicating a biphasic effect for this compound. By contrast, B25 depressed heart contractility without any visible positive inotropic or chronotropic activity. 2. Experiments with tetrodotoxin, reserpine, capsaicin, alpha-adrenoceptor blocking compounds and other agents permit us to exclude a release of neuromediators or a direct stimulation of post-synaptic receptors to account for the rhythmic effect of B25 in the rat vas deferens. 3. In the same tissue, the increase in 45Ca2+ uptake, the voltage-dependency as well as the dependence of the B25-induced rhythmic activity upon the external calcium concentration indicate a direct activation of voltage-sensitive calcium channels (VSCC). 4. Verapamil paradoxically stimulated the rhythmic effect of B25 in the rat vas deferens. La3+ was inactive while nifedipine was a weak inhibitor. By contrast Ni2+ and Mn2+ ions were good inhibitors (IC50 < 10(-4) M), suggesting that a possible opening of T-type VSCC underlies rhythmic effect of B25. 5. In radioligand binding studies competition experiments with [3H]-nitrendipine indicated that only at high concentrations was B25 able to interact with dihydropyridine-sensitive binding sites of heart and vas deferens smooth muscle. 6. B25 (3-30 microM) counteracted the inhibitory effects of omega-conotoxin GVIA in field-stimulated rat vas deferens.

Some problems with the diamine oxidase (DAO) assay using putrescine as substrate in rat liver.

Determination of diamine oxidase (DAO) activity in rat liver preparations by measuring the formation of radioactive delta 1-pyrroline from 14C-putrescine is complicated by the complexity of competing metabolic pathways. This can lead to complete masking of the DAO activity present when rat liver homogenates are used as the enzyme source. However, subcellular fractionation of rat liver homogenates makes it possible to detect some putrescine oxidizing activity in the microsomal fraction when assayed at pH 8.5. When 1 mM putrescine was used as the substrate, over 90% of this activity was inhibited by 6 x 10(-4) M selegiline (deprenyl), indicating that monoamine oxidase (MAO) rather than DAO activity was being measured. The observed activity was also interfered with by agents that reduced acetylation processes and polyamine synthesis. A different picture appears when microM concentrations of putrescine are used: in these conditions all interference is strongly reduced and DAO activity can be measured in rat liver microsomes. Furthermore, kinetic studies on deaminative oxidation of 14C-putrescine at concentrations from 1 microM to 5 mM confirm the existence of two enzymes: one with a high affinity for the substrate and similar to intestinal mucosa DAO in its sensitivity to alpha-aminoguanidine, and the other one with a low affinity and selegiline-sensitive.

Further studies on semicarbazide-sensitive amine oxidase activities (SSAO) of white adipose tissue.

1. White adipose tissue (WAT) from mice, rabbits, pigs and human subjects was investigated for the characterization of the tissue-bound semicarbazide-sensitive benzylamine oxidase activities (SSAO) present in each species. 2. Enzymes from mice, rabbits and pigs shared similar biochemical characteristics: they exerted histaminase activity, oxidized methylamine and acetylputrescine and were completely blocked by carbonyl reagents and by 3,5-ethoxy-4-aminomethylpyridyne (B24), in a dose-dependent fashion. 3. SSAO activity from human WAT had a lower affinity for benzylamine compared with enzymes in the other species and did not show any histaminase activity. 4. These results show that SSAO from human tissues might have different properties from SSAO of other species.

Erythrocyte spermidine levels in IDDM patients.

OBJECTIVES: To evaluate whether erythrocyte levels of polyamines spermidine and spermine (expressed in nmol/ml packed erythrocytes [PRBCs]) are modified in insulin-dependent diabetes mellitus (IDDM) and are associated with the presence of retinopathy or nephropathy. RESEARCH DESIGN AND METHODS: We studied erythrocyte spermidine and spermine levels in 38 IDDM patients with or without persistent microalbuminuria (urinary albumin excretion rate [AER] between 20 and 200 micrograms/min), macroalbuminuria (AER greater than 200 micrograms/min), or retinopathy compared with 60 sex- and age-matched control subjects. RESULTS: Mean +/- SD erythrocyte spermine content was similar in both diabetic (9.7 +/- 5.5 nmol/ml PRBCs) and control (8.8 +/- 3.5 nmol/ml PRBCs) subjects, whereas spermidine was higher in diabetic (19.1 +/- 7.2 nmol/ml PRBCs) than in control (14.5 +/- 4 nmol/ml PRBCs, P = 0.0007) subjects. Moreover, spermidine was significantly higher in the groups with microalbuminuria (n = 11, 22.5 +/- 9.2 nmol/ml PRBCs) and macroalbuminuria (n = 4, 22.2 +/- 5.7 nmol/ml PRBCs) than in both normoalbuminuric (n = 23, 16.9 +/- 5.6 nmol/ml PRBCs) and control (F = 9.78, P = 0.0001) subjects, and correlated with log AER (r = 0.41, P = 0.009). Similarly, proliferative retinopathy was associated with a significant increase in spermidine (n = 5, 20 +/- 7 nmol/ml PRBCs compared with control subjects [P = 0.0009]). CONCLUSIONS: Our data suggest that erythrocyte spermidine content is increased in IDDM patients associated with both diabetic nephropathy and advanced retinopathy.

Histaminase activity in rat lung and its comparison with intestinal mucosal diamine oxidase.

In rat lung microsomes, an enzyme showing high histaminase activity is present. The oxidation of histamine is dependent on the presence of two enzymic activities, both inhibited by alpha-aminoguanidine and by B24, an inhibitor of semicarbazide-sensitive amine oxidases (SSAO) which have benzylamine as preferential substrate. These enzymic activities differ in substrate specificity: one appears to be a classical tissue bound SSAO enzyme with high affinity for benzylamine, the other a diamine oxidase (DAO) with properties that are very different from the classical DAO. This latter enzyme is not inhibition by high histamine concentrations and is more active at pH 8.5 than at pH 7.4.

Semicarbazide-sensitive amine oxidase activity (SSAO) of rat epididymal white adipose tissue.

An amine oxidase activity distinguishable from MAO, which is inhibited by carbonyl reagents is present in rat epididymal WAT. This enzyme, referred to as semicarbazide-sensitive amine oxidase (SSAO), appears concentrated in adipose cells. Close homologies between WAT SSAO and the circulating plasma BAO are discussed.