Effect of phosphate ion on the activity of bovine plasma amine oxidase.

The system bovine plasma amine oxidase-polyamine-phosphate ion was investigated by activity measurements and 31P NMR spectroscopy. Lineweaver-Burk plots showed that phosphate ion, under physiological conditions, is an apparent competitive inhibitor of bovine plasma amine oxidase. While NMR measurements of the T1 of 31P do not suggest the binding of phosphate to/or near the paramagnetic Cu(II) sites of bovine plasma amine oxidase, the chemical shift dependence of 31P on spermidine concentration indicates the formation of a spermidine-phosphate complex. The value of the dissociation constant of this complex was found 18.5 +/- 1.4 mM, at pH 7.2, by NMR, in good agreement with the value 17.0 +/- 0.8 mM calculated from activity measurements, assuming the enzyme activity is proportional to the free amine concentration, under second order conditions. Our data suggest that the decrease of the free spermidine, due to the binding of phosphate ion, is responsible of the observed inhibition of bovine plasma amine oxidase.     

Electron microscopic evidence for endocytosis of superoxide dismutase by hepatocytes using protein-gold adducts

Cu, Zn superoxide dismutase, an intracellular, carbohydrate-free protein, was found to bind to hepatocytes and endothelial cells by electron microscopy of liver treated with enzyme-gold conjugates. In cultured hepatocytes, the free enzyme, but not bovine serum albumin, competed for the binding, and at 37 degrees C, the gold-protein complexes were internalized in a typical process of receptor-mediated endocytosis.     

The role of copper in bovine serum amine oxidase

Summary The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence ofN,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.Abbreviations BSAO bovine serum amine oxidase - DDC N,N-diethyldithiocarbamate - PQQ pyrroloquinoline quinone     

Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases.

The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J. P. (1990) Science 248, 981-987). The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P. G., Evers, M., Waterham, H. R., Kuipers, J., Arnberg, A. C., and Geert, A. B. (1989) Biochim. Biophys. Acta 1008, 157-167). In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H. polymorpha. Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with [14C]phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography. Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor. By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein. In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals.     

Evidence of sex hormone binding globulin binding sites in the medial preoptic area and hypothalamus.

We have demonstrated a high density of both radiolabeled progesterone and estradiol conjugated to bovine serum albumin binding sites in the medial preoptic area and hypothalamus. Infusions of sex hormone binding globulin into the medial preoptic area of rats increased their female sexual receptivity similarly to the effect of estradiol conjugated to bovine serum albumin, suggesting sex hormone binding globulin acts at binding sites for estradiol conjugated to bovine serum albumin. In this study sex hormone binding globulin was used to displace radiolabeled progesterone conjugated to bovine serum albumin from plasma membrane fractions from the medial preoptic area-anterior hypothalamus and medial basal hypothalamus of ovariectomized rats injected with either 5 microg estradiol benzoate or sesame oil vehicle. We found that sex hormone binding displaced radiolabeled progesterone conjugated to bovine serum albumin in both areas and that in vivo estradiol treatment greatly increased the relative displacement by sex hormone binding globulin in the medial preoptic area-anterior hypothalamus. We interpret these data as indicating the presence of sex hormone binding globulin receptors in brain plasma membranes and further suggest that endogenous steroid conditions may alter these receptors.     

Amine Oxidase from Lentil Seedlings: Energetic Domains and Effect of Temperature on Activity

Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).     

Preparation of Amine Oxidase from Bovine Serum by Affinity Chromatography on Aminohexyl-Sepharose

Amine oxidase was purified from bovine serum by affinity chromatography on aminohexyl substituted Sepharose. The enzyme was adsorbed on the chromatographic support in a suspension of aminohexyl Sepharose in diluted serum. After thorough washing with buffer, the gel was packed in a column and the enzyme eluted with 10 mM octylamine. Using this procedure it was possible to obtain apparently homogenous amine oxidase in a single-step procedure. The specific enzyme activity was 0.14 μoles benzaldehyde formed per minute at 25°C per mg enzyme protein. Based on the activity of amine oxidase in serum, the yield of enzyme was 64 %.     

Stereochemical trends in copper amine oxidase reactions

Copper amine oxidases (EC 1.4.3.6) exhibit atypical stereochemical patterns in the reactions they catalyze. Dopamine and tyramine are oxidized with abstraction of the pro-R hydrogen by the porcine plasma amine oxidase, the pro-S hydrogen by pea seedling amine oxidase and a net nonstereospecific proton abstraction by the bovine plasma enzyme. This provides the first example in which a reaction catalyzed by enzymes in the same formal class occurs by all three possible stereochemical routes. To assess the underlying mechanistic significance of this heterogeneity, we have established the stereochemical course of the oxidation of tyramine by five additional copper amine oxidases using 1H NMR spectroscopy. Reactions catalyzed by rabbit and sheep serum amine oxidases are nonstereospecific. These enzymes exhibit rare mirror image binding with differential flux through two opposite and stereospecific reaction pathways. Differential primary kinetic isotope effects are observed for each mode, 8 and 4.6 for pro-S abstraction and 2.6 and 2.7 for pro-R abstraction by the sheep and rabbit amine oxidases, respectively. Tyramine oxidations catalyzed by the soybean and chick pea amine oxidases and porcine kidney diamine oxidase, however, are all stereospecific, occurring with loss of the pro-S hydrogen at C-1. Solvent exchange profiles are consistent within each stereochemical class of enzyme; the pro-R and nonstereospecific enzymes exchange solvent into C-2 of product aldehydes, the pro-S enzymes do not.     

Binding of Progesterone to Nerve Cell Membranes of Rat Brain Using Progesterone Conjugated to 125I-Bovine Serum Albumin as a Ligand

Radioiodinated bovine serum albumin conjugated to progesterone was used as a probe to examine binding parameters of steroids to membrane preparations from rat brain tissue. The binding of 11 alpha-hydroxyprogesterone-11-hemisuccinate-125I-bovine serum albumin conjugate reached saturation after 30 min of incubation at 5 degrees C. Several bovine serum albumin-conjugated steroids were then tested for competition displacement studies. Among these steroid conjugates, the bovine serum albumin conjugate at position 3 of progesterone had the highest affinity, with an estimated inhibition constant of 28.5 +/- 2.1 nM (n = 3), whereas bovine serum albumin itself and the 17 beta-estradiol 6-(O-carboxy-methyl)oxime-bovine serum albumin conjugate showed no specific displacement. In addition, the binding sites were localized in an axolemma-enriched fraction of rat brainstem. Specific binding was obtained in tissues from cerebral cortex, brainstem, cerebellum, corpus striatum, and hypothalamus, but little or no binding occurred in uterus, ovary, liver, and spleen. The present data indicate that progesterone-125I-bovine serum albumin conjugate can be used as a ligand to study progesterone-membrane receptor interactions.     

Effects of adriamycin on respiratory chain activities in mitochondria from rat liver, rat heart and bovine heart. Evidence for a preferential inhibition of complex III and IV

The inhibition of respiratory chain activities in rat liver, rat heart and bovine heart mitochondria by the anthracycline antibiotic adriamycin was measured in order to determine the adriamycin-sensitive sites. It appeared that complex III and IV are efficiently affected such that their activities were reduced to 50% of control values at 175 +/- 25 microM adriamycin. Complex I displayed a minor sensitivity to the drug. Of the complex-I-related activities tested, only duroquinone oxidation appeared sensitive (50% inhibition at approx. 450 microM adriamycin). Electron-transfer activities catalyzed by complex II remained essentially unaltered up to high drug concentrations. Of the activities measured for this complex, only duroquinone oxidation was significantly affected. However, the adriamycin concentration required to reduce this activity to 50% exceeded 1 mM. Mitochondria isolated from rat liver, rat heart and bovine heart behaved essentially identical in their response to adriamycin. These data support the conclusion that, in these three mitochondrial systems, the major drug-sensitive sites lie in complex III and IV. Cytochrome c oxidase and succinate oxidase activity in whole mitochondria exhibited a similar sensitivity towards adriamycin, as inner membrane ghosts, suggesting that the drug has direct access to its inner membrane target sites irrespective of the presence of the outer membrane. By measuring NADH and succinate oxidase activities in the presence of exogenously added cytochrome c, it appeared that adriamycin was less inhibitory under these conditions. This suggests that adriamycin competes with cytochrome c for binding to the same site on the inner membrane, presumably cardiolipin.     

Stereochemical course of tyramine oxidation by semicarbazide-sensitive amine oxidase.

Two semicarbazide-sensitive amine oxidases (SSAO's) from bovine and porcine aortic tissue were partially purified and characterized, and the stereochemical course of amine oxidation was evaluated. The porcine and bovine SSAO's were membrane bound glycoproteins, with Km values for benzylamine of 8 and 16 microM, respectively. The reactivity of SSAO with semicarbazide and phenylhydrazine suggests that the cofactor is a carbonyl type molecule. The stereochemical course of the bovine and porcine aortic semicarbazide-sensitive amine oxidase reaction was investigated using chiral tyramines, deuterated at C-1 and C-2, and 1H-NMR spectroscopy to establish the loss or retention of deuterium in product p-hydroxyphenethyl alcohols. The preferred mode of tyramine oxidation was found to occur with the loss of pro-S proton at C-1, coupled with solvent exchange into C-2, a pattern which has not been observed for any copper amine oxidase examined to date. The solvent exchange reaction also occurred stereospecifically, with loss from and reprotonation to the pro-R position, suggesting that these two processes occur from the same face of the enamine double bond.     

Do mammalian amine oxidases and the mitochondrial polyamine transporter have similar protein structures?

Polyamine transport across the mitochondria membrane occurs by a specific, common uniporter system and appears controlled by electrostatic interactions as for polyamine oxidative deamination by bovine serum and mitochondrial matrix amine oxidases was found. In fact in all the cases, while the catalytic constants or the maximum uptake rate values show little changes with the number of the positive charges of the substrates, Michaelis–Menten constant values demonstrate exponential dependence, confirming that electrostatic forces control the docking of the substrate into the enzyme active site or polyamine channel. By the treatment of the kinetic data in terms of Gibbs equation or Eyring theory, the contribution of each positive charge of the polyamine to the Gibbs energy values for the oxidative deamination of polyamines by two mammalian amine oxidase and for polyamine transport, are obtained. These values were comparable and in good accordance with those reported in literature. Previous studies demonstrated that two negative functional groups in the active site of bovine serum and mitochondrial matrix amine oxidases interact electrostatically with three positive charges of the polyamines in the formation of the enzyme–substrate complex. Remembering the structure–function relationship of proteins, our results suggest analogous interactions in the polyamine transporter and, as a consequence, a partial structural similitude between two proteins. It follows that the primary sequences of the amino oxidases and the mitochondrial transport may, in part, be conserved.     

A purification procedure for the isolation of homogeneous preparations of bovine aorta amine oxidase and a study of its lysyl oxidase activity.

It has been reported that bovine aorta amine oxidase oxidizes lysine residues in tropoelastin to allysine (Rucker, R.B. and O'Dell, B.L. (1971) Biochim. Biophys. Acta 235, 32-43). Pure bovine aorta amine oxidase was isolate by DEAE-cellulose, hydroxylapatite, Bio-Gel A-1.5 m and concanavalin A-Sepharose 4B chromatography. Enzymatic, chromatographic and immunochemical tests disclosed that pure bovine aorta amine oxidase was not a lysyl oxidase capable of oxidizing the lysine residues of tropoelastin to allysine; The bovine aorta amine oxidase preparation used by Rucker and O'Dell appears to have been contaminated with lysyl oxidase which is the emzyme that oxidizes some of the lysine residues in tropoelastin and tropocollagen to allysine.     

Cyanide as a copper-directed inhibitor of amine oxidases: implications for the mechanism of amine oxidation

 The interactions of five copper-containing amine oxidases with substrates and substrate analogues in the presence of the copper ligands cyanide, azide, chloride, and 1,10-phenanthroline have been investigated. While cyanide inhibits, to varying degrees, the reaction of phenylhydrazine with porcine kidney amine oxidase (PKAO), porcine plasma amine oxidase (PPAO), bovine plasma amine oxidase (BPAO), and pea seedling amine oxidase (PSAO), it enhances the reaction of Arthrobacter P1 amine oxidase (APAO) with this substrate analogue. This indicates that cyanide exerts an indirect effect on topa quinone (TPQ) reactivity via coordination to Cu(II) rather than through cyanohydrin formation at the TPQ organic cofactor. Moreover, cyanide binding to the mechanistically relevant TPQ^• semiquinone form of substrate-reduced APAO and PSAO was not observable by EPR or resonance Raman spectroscopy. Hence, cyanide most likely inhibits enzyme reoxidation by binding to Cu(I) and trapping the Cu(I)-TPQ^• form of amine oxidases, and thus preventing the reaction of O₂ with Cu(I). In contrast, ligands such as azide, chloride, and 1,10-phenanthroline, which preferentially bind to Cu(II), inhibit by stabilizing the aminoquinol Cu(II)-TPQ_red redox state, which is in equilibrium with Cu(I)-TPQ^•. Received: 12 December 1996 / Accepted: 20 March 1997     

Amine oxidase activity in commercial preparations of bovine serum albumin

Amine oxidase activity has been identified in commercial samples of bovine serum albumin (BSA). Benzylamine, phenylethylamines and to a lesser extent, indoleamines, were found to be substrates. The amine oxidase activity was inhibited by semicarbazide and was virtually absent in electrophoretically purified samples. Kinetic analysis of benzylamine deamination and experiments utilizing mixed substrates indicate that more than one catalytic activity may be involved. The results show that amine deamination should be considered as a potential source of error in experiments employing high concentrations of commercially available BSA preparations. This would be of particular importance for $\text{in vitro}$ studies with dopamine since this amine was found to be deaminated at a rapid rate.     

Composition and enzyme activities of Spiroplasma citri membranes.

Spiroplasma citri was cultured in three different media that supplied cholesterol and fatty acids from: (i) horse serum, (ii) pleuropneumonia-like organism (PPLO) serum fraction, or (iii) bovine serum albumin-fatty acid-cholesterol. The ability of PPLO serum fraction to support growth varied by lot number. Neither PPLO serum fraction nor the bovine serum albumin medium supported growth as well as the horse serum medium. Analysis of cholesterol, lipid phosphorus, and membrane protein showed the horse serum- and PPLO-grown cells to be indistinguishable, but the bovine serum albumin-grown cells were deficient in lipid phosphorus. The three cultures did not show markedly different fatty acid compositions, but, in all cases, the cultures preferentially incorporated palmitic acid and discriminated against linoleic acid. Cultures grown for different times from logarithmic growth through a degenerative phase showed relatively constant ratios of cholesterol/protein and lipid phosphorus/protein. Fatty acid composition was also relatively constant at the different stages. Adenosine triphosphatase and p-nitrophenyl phosphatase were mainly associated with the membrane, whereas reduced nicotinamide adenine dinucleotide oxidase was either readily removed or not associated with the membrane. The reduced nicotinamide adenine dinucleotide oxidase was inactivated at temperatures above 35 degrees C.     

Evidence for pyrroloquinolinequinone as the carbonyl cofactor in lysyl oxidase by absorption and resonance Raman spectroscopy

The present study investigated the possibility that pyrroloquinolinequinone (PQQ), an aromatic carbonyl recently indicated to be the carbonyl cofactor in bovine plasma amine oxidase, may also be present at the active site of lysyl oxidase. The absorption and resonance Raman spectra of the phenylhydrazones of bovine plasma amine oxidase, of peptides derived from the active site of bovine aorta lysyl oxidase, and of PQQ were very similar, indicating that the carbonyl cofactor of lysyl oxidase is PQQ or a compound which closely resembles PQQ.     

Inactivation of copper-containing amine oxidases by turnover products.

For bovine serum amine oxidase, two different mechanisms of substrate-induced inactivation have been proposed. One consists of a slow oxidation by H2O2 of a conserved residue in the reduced enzyme after the fast turnover phase [Pietrangeli, P., Nocera, S., Fattibene, P., Wang, X.T., Mondovì, B. & Morpurgo, L. (2000) Biochem. Biophys. Res. Commun.267, 174-178] and the other of the oxidation by H2O2 of the dihydrobenzoxazole in equilibrium with the product Schiff base, during the catalytic cycle [Lee, Y., Shepard, E., Smith, J., Dooley, D.M. & Sayre, L.M. (2001) Biochemistry40, 822-829]. To discriminate between the two mechanisms, the inactivation was studied using Lathyrus cicera (red vetchling) amine oxidase. This, in contrast to bovine serum amine oxidase, formed the Cu+-semiquinolamine radical with a characteristic UV-vis spectrum when oxygen was exhausted by an excess of any tested amine in a closed cuvette. The inactivation, lasting about 90 min, was simultaneous with the radical decay and with the formation of a broad band (shoulder) at 350 nm. No inactivation occurred when a thousand-fold excess of amine was rapidly oxidized in an L. cicera amine oxidase solution stirred in open air. Thus, the inactivation is a slow reaction of the reduced enzyme with H2O2, following the turnover phase. Catalase protected L. cicera amine oxidase from inactivation. This effect was substrate-dependent, varying from full protection (benzylamine) to no protection (putrescine). In the absence of H2O2, a specific inactivating reaction, without formation of the 350 nm band, was induced by some aldehydes, notably putrescine. Some mechanisms of inactivation are proposed.     

Inhibition of lymphocyte growth by spermidine in medium containing fetal bovine serum

The effect of spermidine and fetal bovine serum on DNA, RNA, and protein synthesis in phytohemagglutinin-stimulated human lymphocytes was investigated. At 10(-4) M spermidine, DNA, RNA, and protein synthesis ceased and 70% of the original cell population died within 62 hr. Lower spermidine concentrations had no significant effect on DNA and protein synthesis, but caused an early, unexplained increase in the rate of RNA synthesis. Heating at 56 degrees C for 30 min had no effect on the plasma amine oxidase activity in fetal bovine and horse sera but abolished the activity in human plasma. It is concluded that low amounts of aminoaldehydes and acrolein produced by plasma amine oxidase at spermidine concentrations below 10(-4) M do not noticeably alter lymphocyte metabolism. However, the aminoaldehydes and acrolein produced become abruptly cytotoxic at 10(-4) M spermidine.     

Inhibition of bovine plasma semicarbazide-sensitive amine oxidase by caffeine

Semicarbazide-sensitive amine oxidase (SSAO) is a copper-containing enzyme that catalyzes the oxidative deamination of endogenous and exogenous primary amines. SSAO exists in mammals both as a plasma-soluble and as a membrane-bound form, and its active site is able to come into contact with numerous xenobiotic, amine-containing compounds. The kinetic studies performed in this work showed that caffeine inhibition of bovine serum amine oxidase was noncompetitive when benzylamine was used as substrate and mixed when the substrate used was methylamine. Since caffeine contains an imidazole ring, it cannot be excluded that it might bind to an inhibitory imidazoline-binding site on SSAO.