©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Localization of I-Imidazoline Binding Sites on Monoamine Oxidases (*)

Frédérique Tesson (1), Isabelle Limon-Boulez (1), Philippe Urban (2), Magda Puype (3), Jol Vandekerckhove (3), Isabelle Coupry (1), Denis Pompon (2), Angelo Parini (1)(§)

From the (1) Institut National de la Santé et de la Recherche Médicale (INSERM U 388), Institut Louis Bugnard, CHU Rangueil, 31054 Toulouse Cedex, France, (2) Centre National de la Recherche Scientifique (CNRS UPR 2420), Centre de Génétique Moléculaire, 91198 Gif Sur Yvette, France, and (3) Laboratory of Physiological Chemistry, State University of Gent, B-9000 Gent, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Imidazoline binding sites (IBS) were proposed to be responsible for some of the pharmacological and therapeutic activities of imidazoline and related compounds and have been classified into two subtypes, IBS and IBS. Convergent studies attribute a role in central blood pressure regulation to the IBS. In contrast, the function of IBS remains unknown.

In the present study, by combining biochemical and molecular biology approaches, we show that 1) microsequencing of IBS purified from rabbit kidney mitochondria allowed the recovery of four peptide sequence stretches displaying up to 85.7% similarity with human, rat, and bovine monoamine oxidases (MAO)-A and -B; 2) IBS and MAO displayed identical biophysical characteristics as their activities, measured by [H]idazoxan binding and [C]tyramine oxidation, respectively, could not be separated using various chromatographic procedures; and 3) heterologous expression of human placenta MAO-A and human liver MAO-B in yeast, inherently devoid of IBS and MAO activities, led to the co-expression of [H]idazoxan binding sites displaying ligand-recognition properties typical of IBS.

These results show definitely that IBS is located on both MAO-A and -B. The fact that IBS ligands inhibited MAO activity independently of the interaction with the catalytic region suggests that IBS might be a previously unknown MAO regulatory site.


INTRODUCTION

The pharmacological and therapeutic effects of several imidazoline and guanidinium derivatives, such as clonidine, guanabenz, and idazoxan, have been related to their specific interaction with -adrenergic receptors (1, 2, 3) . However, during the last 10 years, several studies showed that some of these effects, including the centrally mediated decrease in blood pressure (4) and the proconvulsant (5) and the anxiogenic-like (6) activities, were partially independent of the stimulation of -adrenergic receptors. This suggested the involvement of putative imidazoline receptors in mediating the ``nonadrenergic'' activities of the imidazoline and guanidinium derivatives. The existence of imidazoline binding sites (IBS)() has been further supported by numerous binding and autoradiography studies showing that three imidazoline -adrenergic tritiated ligands, clonidine, p-aminoclonidine, and idazoxan, label binding sites that display a high affinity for imidazoline derivatives and are not recognized by catecholamines (7, 8, 9, 10) . To date, two major subclasses of IBS have been identified based on their high (I) or low (I) affinity for clonidine (11) . Convergent studies (4, 12) attribute a role in blood pressure regulation to the IBS. In contrast, the function of IBS remains largely unknown.

The identification of the functional activity of IBS has been hindered mostly by the lack of a purified endogenous ligand as well as by the difficulty to define the agonist or antagonist properties of their synthetic ligands. To address the functional role of IBS we used an alternative approach consisting of the characterization of their subcellular localization and structural properties. By this strategy, we showed that IBS is a 60-kDa protein (13) located in the outer membrane of mitochondria (14) and selectively regulated by Kand Hin vitro (13) . Thereafter, the major mitochondrial localization of IBS has been confirmed in different tissues and species (15, 16) . By comparing the structural and regulatory properties of IBS with those of other outer membrane mitochondrial proteins, we observed that IBS share with monoamine oxidases (MAO), enzymes metabolizing endogenous active substances ( i.e. adrenaline, noradrenaline, serotonin, and dopamine) and exogenous amines, the same molecular weight, subcellular localization, and ion regulation. This prompted us to investigate the structural and functional relationships between IBS and MAO. In the present study, we demonstrate that IBS is located on both MAO-A and MAO-B. In addition, the fact that IBS ligands inhibited MAO activity independently of the interaction with the catalytic region suggests that IBS might be involved in the regulation of the enzyme activity.


EXPERIMENTAL PROCEDURES

Materials

[H]Idazoxan (40-60 Ci/mmol) was obtained from Amersham International. [C]Tyramine (50-60 mCi/mmol) was from DuPont NEN. [C]Phenylethylamine was purchased from Isotopchim (Ganagobie, France). (±)-Idazoxan was supplied by Dr. Malen from I.D.R. Servier (Suresnes, France). Cirazoline was a gift from Synthelabo (Paris, France). Clonidine was obtained from Boehringer-Ingelheim (Ridgefield, CT). Rauwolscine was purchased from Roth (Karlsruhe, Germany). Moxonidine was a gift from Laboratoire Therapeutique Moderne. Hydroxylapatite-agarose Ultrogel was obtained from I.B.F. (Paris, France). Superose-12 and phenyl-Sepharose 4B gels were purchased from Pharmacia Biotech Europe (St. Quentin en Yvelynes, France). All remaining drugs were obtained from Sigma (Paris, France).

Microsequencing

The IBS was purified from rabbit kidney mitochondrial membranes to apparent homogeneity by using sequential chromatofocusing and hydroxylapatite chromatographies (13) . The purified fraction was further analyzed by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluroride membrane. The 60-kDa band corresponding to IBS was detected by staining with Amido Black, excised, and subjected to on-membrane digestion with trypsin. The generated peptides were separated by narrow-bore reversed-phase high performance liquid chromatography, as described previously (17, 18) . Eluting peptides were collected manually and stored at 20 °C until further processing. Amino acid sequencing was carried out with an Applied Biosystems Sequencer (model 477A), on-line connected with a phenylthiohydantoin amino acid analyzer (model 120A).

Yeast Transformation

The Saccharomyces cerevisiae W303 cells ( MAT a, leu2, his3, ura3, ade2-1, can, cyr+) were transformed by pYeDP60 plasmid (control yeast), or the expression plasmids pHMAOAV60 and pHMAOBV60 constituted, respectively, by human placental MAO-A and human liver MAO-B open reading frames cloned into pYeDP60 expression plasmid, as described previously (19) . Spheroplasts, obtained by enzymatic digestion of yeast cell walls, and crude mitochondrial fractions were prepared as previously described (20) .

Chromatographic Procedures

Preparations and solubilization of mitochondrial membranes from rabbit renal cortex were performed as described previously (13) . Hydrophobic chromatography was carried out in a glass chromatography column (1 30 cm) filled with 25 ml of phenyl-Sepharose 4-B gel and equilibrated with 10 column volumes of the following buffer: 0.1% digitonin, 300 mM NaCl, 50 mM Tris-HCl, pH 7.4 at 20 °C. Thirteen ml of solubilized extract were diluted in 10 volumes of a buffer containing 300 mM NaCl, 50 mM Tris-HCl, pH 7.4, at 20 °C to adjust the digitonin concentration at 0.1%. Solubilized material was applied to the phenyl-Sepharose 4-B column at a constant flow rate of 0.5 ml/min, at 20 °C. The column was then washed with 3 volumes of buffer containing 0.1% digitonin, 50 mM sodium phosphate, pH 7.4. Elution was performed by applying a linear gradient (0-2 mM) of CHAPS, and 1-2-ml fractions were collected.

Hydroxylapatite-agarose chromatography was performed in a 1 30-cm chromatography column filled with 25 ml of hydroxylapatite-agarose-Ultrogel. The gel was equilibrated with 10 column volumes of buffer A containing 0.1% digitonin, 100 mM NaCl, 50 mM Tris-HCl, pH 7.4, at 20 °C. Thirty ml of solubilized material (protein concentration, 1-2 mg/ml) were applied to the column at a constant flow rate of 0.5 ml/min, at 20 °C. Washing and elution were performed as described previously (13) . Fractions of 0.8 ml were collected and tested for sodium phosphate concentration. Prior to MAO and IBS activities assays, eluted fractions from hydroxylapatite-agarose columns were desalted by centrifugation on UNISEP Ultracent-30 (Bio-Rad) to adjust the sodium phosphate concentration at 10 mM.

Size exclusion chromatography was performed on a Superose 12 fast protein liquid chromatography column. Fractions from hydroxylapatite-agarose column displaying [H]idazoxan binding and MAO activities were pooled, desalted, concentrated to 200 µl, and applied to the Superose 12 column. The elution was performed using buffer A at a flow rate of 0.3 ml/min. Fractions of 0.5 ml were collected.

Binding Studies

Mitochondrial membranes from rabbit renal cortex or transformed yeast cells (50 µg of protein) were incubated at 20 °C for 45 min in a final volume of 250 µl of 50 mM Tris-HCl, pH 7.4, 2 mM EGTA, 1 mM MgCl, unless otherwise mentioned, with the required concentrations of [H]idazoxan in the presence of 10 M rauwolscine to mask any -adrenergic receptor. Nonspecific binding was defined in the presence of 1 µM cirazoline. At the end of the incubation, bound radioligand was separated from free by vacuum filtration as described previously (13) . Filters were placed in 5 ml of Ready-Safe scintillation fluid (Beckman), and the radioactivity was counted in a liquid scintillation spectrometer (Packard, model Tri-Carb 300) at 48% efficiency. [H]Idazoxan binding on solubilized extracts was performed in the same experimental conditions except that incubation was ended by precipitation with bovine -globulin/polyethylene glycol followed by vacuum filtration over glass fiber filters (Whatman GF/B). Binding data were analyzed using a nonlinear least-square curve fitting procedure (PRISM, GraphPAD Software, San Diego, CA).

Monoamine Oxidase Activity Measurement

Membranes (50 µg of protein) or solubilized extracts were incubated at 37 °C for 20 min, unless otherwise mentioned, in a final volume of 250 µl of sodium phosphate buffer, 50 mM, pH 7.5, with the required concentrations of [C]tyramine or [C]phenylethylamine. Pargyline (10 M) was used to define monoamine oxidase-specific activity. The reaction was ended by addition of 1 ml of HCl, 2 N, 4 °C. The reaction product was extracted by addition of 2 ml of ethyl acetate/toluene (v/v), and the radioactivity contained in the organic phase was counted in a liquid scintillation spectrometer at 97% efficiency. Steady-state kinetic parameters were calculated using a nonlinear least-square curve fitting procedure (PRISM).


RESULTS AND DISCUSSION

The IBS was purified from rabbit kidney mitochondrial membranes to apparent homogeneity by using sequential chromatofocusing and hydroxylapatite chromatographies (13) . Microsequencing of selected tryptic peptides allowed for the recovery of four peptide sequence stretches. Amino acid sequences of such peptides displayed 50-85.7% similarity with human, rat, and bovine MAO-A and MAO-B (Fig. 1). These data, along with other studies showing that IBS and MAO are co-localized in different areas of rabbit and human brain (20, 21) , further supported the hypothesis of structural relationship between IBS and MAOs.


Figure 1: Partial amino acid sequences of rabbit IBS: comparison with the sequences of human, rat, and bovine MAOs. Partial sequences of purified IBS were obtained by microsequencing, as described under ``Experimental Procedures.'' Amino acid sequence portions of human, rat, and bovine MAO-A and MAO-B were retrieved from the SwissProt data base and compared with IBS partial sequences using the FASTA search program. Percentages of identity are given in parentheses. Identical residues are indicated by ``:'' and similar residues by ``.'' marks, respectively. X, nonidentified amino acid residues.



To address this issue, we combined biochemical and molecular biology approaches. First, we investigated whether IBS and MAO could be discriminated through their biophysical properties. Treatment of mitochondria from rabbit kidney with increasing digitonin concentrations allowed a dose-dependent solubilization of both IBS and MAO activities. The recoveries of [H]idazoxan binding and [C]tyramine degradation rate at the different digitonin concentrations were significantly correlated ( r = 0.987, p < 0.01), suggesting a similar insertion of IBS and MAO in the mitochondrial membrane matrix. Using hydrophobic, hydroxylapatite, and size exclusion chromatography, IBS and MAO activities were systematically eluted in the same fractions (Fig. 2), and the recovery of [H]idazoxan binding and [C]tyramine degrading activity were almost identical after each chromatographic procedure (data not shown). These data indicate that IBS and MAO share structural properties preventing their separation by purification procedures.


Figure 2: Elution profiles of IBS and MAO activities after separation of solubilized rabbit renal cortex mitochondrial membranes by phenyl-Sepharose 4-B ( A), hydroxylapatite-agarose ( B), and Superose 12 ( C) chromatography. Chromatographic procedures were performed as described under ``Experimental Procedures.'' For each chromatography, the elution profile was recorded by a UV detector set at 280 nm. Fractions were tested for IBS and MAO activities in a final volume of 125 µl. The IBS density () was measured by binding studies with 20 nM [H]idazoxan using 10 M cirazoline to define nonspecific binding. MAO activity () was defined by measuring the specific oxidation of 1 mM [C]tyramine (isotopic dilution, 1:100) for 20 min, in presence or absence of 10 M pargyline. Solid lines, optical density.



To assess whether IBS and MAO are a single entity or two co-purified, tightly associated proteins, we transformed yeast with recombinant cDNA encoding for human placenta MAO-A or human liver MAO-B (22) . Then, we verified if IBS could be co-expressed with the enzyme activity. As shown in Fig. 3, IBS and MAO were not expressed in wild-type and control plasmid transformed yeast. In contrast, expression of MAO-A or MAO-B gave rise simultaneously to [H]idazoxan binding in yeast mitochondria. [H]Idazoxan binding was saturable, of high affinity and reversible. Indeed, Scatchard analysis of saturation experiments showed that [H]idazoxan interacted with a homogeneous population of high affinity binding sites in yeast cells expressing MAO-A ( B1.81 ± 0.41 pmol/mg of protein, K94.4 ± 13.3 nM) or MAO-B ( B1.39 ± 0.13 pmol/mg of protein, K25.7 ± 1.7 nM). In addition, [H]idazoxan-specific binding was completely dissociated after the addition of a large excess of cold ligand to the mitochondrial preparations ( t= 2.32 min). The [H]idazoxan binding sites co-expressed with MAO-A and MAO-B display ligand recognition properties consistent with those described for IBS. Indeed, competition studies showed that, in yeast expressing MAO-A or MAO-B, [H]idazoxan binding was inhibited in a dose-dependent manner and with similar affinities by the IBS ligands cirazoline ( K5.08 ± 1.73 nM; 2.97 ± 1.09 nM for MAO-A and -B, respectively) and guanabenz ( K35.2 ± 9.5 nM; 41.8 ± 8.2 nM for MAO-A and -B) and poorly affected by clonidine or moxonidine, selective ligands for IBS, and by the antagonist rauwolscine ( K> 10 µM). Taken together, these data clearly show that IBS is located on both MAO-A and MAO-B.


Figure 3: Expression of MAOs and IBS in yeast cells. IBS density and MAO activity in wild-type, control, and transformed yeast cells expressing MAO-A ( upper panel) or MAO-B ( lower panel). IBS densities ( white bars) and MAO activities ( black bars) in mitochondrial membranes were evaluated on 50 µg of protein by saturation studies of [H]idazoxan binding (using [H]idazoxan concentration from 1 to 150 nM) and by applying the radiochemical assay with [C]tyramine for MAO-A ( upper panel) or [C]phenylethylamine for MAO-B ( lower panel), respectively. Results were the mean ± S.E. of four experiments.



Next, to define the localization of IBS with respect to the binding sites of classical MAO inhibitors, we tested the effect of clorgyline, deprenyl, pargyline, Ro 411049, and Ro 196327 on [H]idazoxan binding. In yeast expressing MAO-A or MAO-B, MAO inhibitors failed to inhibit [H]idazoxan binding at concentration up to 5 µM. These findings, in addition to previous results obtained from [H]idazoxan binding studies in rabbit cerebral (20) and renal cortex() and rat cerebral cortex (23) , indicate that IBS are not associated to MAO regions interacting with classical MAO inhibitors.

To investigate the involvement of IBS in the regulation of MAO activity, we studied the effect of imidazoline and related compounds on MAO-dependent [C]tyramine oxidation in mitochondria from rabbit kidney and transformed yeast cells. As observed for [H]idazoxan binding, MAO activity was inhibited dose-dependently by cirazoline and guanabenz but not by clonidine, moxonidine, or rauwolscine (Fig. 4 A). The mechanism of MAO inhibition by cirazoline and guanabenz was distinct from the ``suicide inhibition'' previously reported for various MAO inhibitors (24) . Indeed, MAO activity was fully recovered after removing of cirazoline or guanabenz by extensive membrane washing (98 ± 2% versus control). To determine the type of MAO inhibition by imidazoline derivatives, we studied the effect of cirazoline on [C]tyramine oxidation in mitochondria from rabbit kidney. The double reciprocal plot of MAO velocity versus substrate concentration curve showed that cirazoline acts as a noncompetitive MAO inhibitor as it decreased Vand did not affect K(Fig. 4 B). Thus, inhibition of MAO activity by imidazoline derivatives occurs by a mechanism distinct from the direct interaction with the MAO catalytic site. Comparison of results from radioligand and enzyme assays showed that inhibition constants of imidazoline derivatives were lower for [H]idazoxan binding to IBS than for MAO activities. Multiple factors may account for discrepancy. First, in competition studies, we found that [H]idazoxan binding to mitochondrial IBS was inhibited by the MAO substrates tyramine, serotonin, and phenylethylamine, at concentrations close to the lowest required for the measure of MAO activity (Fig. 5). Thus, competition of ligand binding to IBS by the MAO substrates may be responsible, in part, for the decrease in the potency of imidazoline derivatives to inhibit the enzyme activity. Second, previous studies have shown that IBS and MAO activities are regulated by different factors including K(25) and H(13) , protein(s) and various endogenous ligands isolated in the central nervous system and in the periphery (13, 24, 26, 27) . It is conceivable that the presence or the absence of one of these regulatory factors could modulate the effects of IBS on MAO activity. This is particularly true considering that IBS ligands bind to a MAO regulatory domain and do not interact directly with the catalytic site. Finally, two observations indicate that the high affinity state of IBS may be associated to a small population of MAOs: 1) in different human tissues and transformed yeast cells, the density of IBS is much lower than the number of MAO molecules (14, 15, 22, 28, 29) and 2) within the same tissue, various conformational states of MAOs can be expressed depending on their assembling in the mitochondrial membranes and their interaction with membrane or cytosolic factors (30) . Thus, the effects of IBS ligands on a subpopulation of MAOs may not be precisely quantified by the classical enzyme assay, which measures the total MAO activity. At present, further studies are necessary to define the role of IBS in the inhibition of MAO activity. However, the involvement of IBS in the regulation of MAO activity is supported by biochemical and functional studies: first, the order of potency for inhibition of [H]idazoxan binding and MAO activities are identical; second, both ligand binding to IBS and inhibition of MAO activity by imidazoline derivatives are fully reversible processes; finally, IBS ligands, independently of their interaction with -adrenergic receptors, regulate centrally mediated activities where MAO are implied (5, 6, 31) .


Figure 4: A, inhibition of MAO activity by imidazoline and related compounds. Mitochondrial membranes (50 µg) from yeast expressing MAO-A ( left panel) or from rabbit kidney ( right panel) were incubated in 50 mM sodium phosphate buffer, pH 7.5, for 20 min at 37 °C, in presence of increasing concentrations of cirazoline (), guanabenz (), clonidine (), moxonidine (), or rauwolscine (). MAO activity was tested in presence of [C]tyramine at K values around 150-200 µM. The results shown are the average of duplicate determination and are representative of three to four separate experiments. B, double reciprocal plot of MAO velocity versus substrate concentration curve in the presence or in absence of cirazoline. Rabbit kidney mitochondrial membranes (50 µg) were incubated in 50 mM sodium phosphate buffer, pH 7.5, for 20 min at 37 °C, in the absence () or in the presence of 10() or 10 M () cirazoline prior to enzyme assay. MAO activity was tested in presence of 5-1000 µM [C]tyramine. Pargyline (10 M) was used to define monoamine oxidase specific activity. (Control values: V= 7.3 ± 1.5 nmol/min/mg of protein; K = 119.9 ± 12.5 µM). The figure is representative of four independent experiments.




Figure 5: Inhibition of [H]idazoxan binding by the MAO substrate tyramine, serotonin, and phenylethylamine. Mitochondrial membranes from rabbit kidney were incubated with [H]idazoxan (1.5 nM) and increasing concentrations of tyramine, serotonin, or phenylethylamine. The plot is representative of four separate experiments.



In conclusion, our results definitively show that IBS is located on both MAO-A and MAO-B. Indirect evidence for a relationship between IBS and MAOs has been previously suggested by other studies showing that their density increases simultaneously in human brain in the elderly (21) and Alzheimer's disease (32) , and the IBS density is down-regulated by rat chronic treatment with MAO inhibitors clorgyline and pargyline (23) .

The molecular relationship between IBS and MAOs being clearly defined, our results should contribute to characterize definitively the functional activity of IBS and to identify a novel mechanism of MAO regulation.


FOOTNOTES

*
This work was partially supported by the CNRS, the Contrat de Recherche Externe INSERM 910205, and grants from Biopharma (France) and I.R.I. Servier (France). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: INSERM U 388, Institut Louis Bugnard, CHU Rangueil, 1, Av. Jean Poulhès, 31054 Toulouse Cedex, France. Tel.: 33-61-32-26-22; Fax: 33-62-17-25-54.

The abbreviations used are: IBS, imidazoline binding sites; MAO, monoamine oxidase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

F. Tesson, I. Limon-Boulez, P. Urban, M. Puype, J. Vandekerckhove, I. Coupry, D. Pompon, and A. Parini, unpublished results.


ACKNOWLEDGEMENTS

We thank C. Bouchier and her colleagues from Généthon (France) for their contribution in sequence analysis.


REFERENCES
  1. Timmermans, P. B. M. W. M., and Van Zwieten, P. A. (1982) J. Med. Chem. 25, 1389-1401 [Medline] [Order article via Infotrieve]
  2. Wendt, R. L. (1980) in Pharmacology of Antihypertensive Drugs (Scribabine, A., ed). pp. 99-111, Raven Press, New York
  3. Doxey, J. C., Roach, A. G., and Smith, C. F. C. (1983) Br. J. Pharmacol. 78, 489-505 [Abstract]
  4. Bousquet, P., Feldman, J., and Schwartz, J. (1984) J. Pharmacol. Exp. Ther. 230, 232-236 [Abstract]
  5. Jackson, H. C., Dickinson, S. L., and Nutt, D. J. (1991) Psychopharmacology 105, 558-562 [Medline] [Order article via Infotrieve]
  6. Venault, P., Jacquot, F., Save, E., Sara, S., and Chapouthier, G. (1993) Life Sci. 52, 639-645 [Medline] [Order article via Infotrieve]
  7. Bricca, G., Greney, H., Zhang, J., Dontenwill, M., Stutzmann, J., Belcourt, A., and Bousquet, P. (1994) Eur. J. Pharmacol. 266, 25-33 [CrossRef][Medline] [Order article via Infotrieve]
  8. Ernsberger, P., Meeley, M. P., Mann, J. J., and Reis, D. J. (1987) Eur. J. Pharmacol. 134, 1-13 [CrossRef][Medline] [Order article via Infotrieve]
  9. Coupry, I., Podevin, R. A., Dausse, J. P., and Parini, A. (1987) Biochem. Biophys. Res. Commun. 147, 1055-1060 [Medline] [Order article via Infotrieve]
  10. Mallard, N. J., Hudson, A. L., and Nutt, D. J. (1992) Br. J. Pharmacol. 106, 1019-1027 [Abstract]
  11. Michel, M. C., and Ernsberger, P. (1992) Trends Pharmacol. Sci. 13, 369-370 [CrossRef][Medline] [Order article via Infotrieve]
  12. Ernsberger, P., Giuliano, R., Willette, R. N., and Reis, D. J. (1990) J. Pharmacol. Exp. Ther. 253, 408-418 [Abstract]
  13. Limon, I., Coupry, I., Lanier, S. M., and Parini, A. (1992) J. Biol. Chem. 267, 30, 21645-21649
  14. Tesson, F., Prip-Buus, C., Lemoine, A., Pegorier, J. P., and Parini, A. (1991) J. Biol. Chem. 266, 155-160 [Abstract/Free Full Text]
  15. Tesson, F., Limon, I., and Parini, A. (1992) Eur. J. Pharmacol. 219, 335-338 [Medline] [Order article via Infotrieve]
  16. Regunathan, S., Meeley, M. P., and Reis, D. J. (1993) Biochem. Pharmacol. 45, 1667-1675 [Medline] [Order article via Infotrieve]
  17. Bauw, G., De Loose, M., Inze, D., Van Montagu, M., and Vandekerckhove, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4806-4810 [Abstract]
  18. Bauw, G., Van Den Bulcke, M., Van Damme, J., Puype, M., Van Montagu, M., and Vandekerckhove, J. (1988) J. Protein Chem. 7, 194-196
  19. Urban, P., Cullin, C., and Pompon, D. (1990) Biochimie ( Paris) 72, 463-472 [CrossRef][Medline] [Order article via Infotrieve]
  20. Renouard, A., Widdowson, P. S., and Cordi, A. (1993) Br. J. Pharmacol. 109, 625-631 [Abstract]
  21. Sastre, M., and Garcia-Sevilla, J. A. (1993) J. Neurochem. 61, 881-889 [Medline] [Order article via Infotrieve]
  22. Urban, P., Andersen, J. L., Hsu, H. P. P., and Pompon, D. (1991) FEBS Lett. 286, 142-146 [CrossRef][Medline] [Order article via Infotrieve]
  23. Olmos, G., Gabilondo, A. M., Miralles, A., Escriba, P. V., and Garcia-Sevilla, J. A. (1993) Br. J. Pharmacol. 108, 597-603 [Abstract]
  24. Singer, T. P., and Ramsay, R. R. (1993) in Monoamine Oxidase: Basic and Clinical Aspects (Yasuhara, H., Parvez, S. H., Oguchi, K., Sandler, M., and Nagatsu, T., eds) pp. 23-43, VSP, Utrecht
  25. Coupry, I., Atlas, D., Podevin, R. A., Uzielli, I., and Parini, A. (1990) J. Pharmacol. Exp. Ther. 252, 293-299 [Abstract]
  26. Li, G., Regunathan, S., Barrow, C. J., Eshraghi, J., Cooper, R., and Reis, D. J. (1994) Science 263, 966-969 [Medline] [Order article via Infotrieve]
  27. Atlas, D. (1994) Science 266, 462-463 [Medline] [Order article via Infotrieve]
  28. Diamant, S., Eldar-Geva, T., and Atlas, D. (1992) Br. J. Pharmacol. 106, 101-108 [Abstract]
  29. Riley, L. A., and Denney, R. M. (1991) Biochem. Pharmacol. 42, 1953-1959 [Medline] [Order article via Infotrieve]
  30. Weyler, W., Hsu, Y. P. P., and Breakefield, X. O. (1989) Pharmacol. Ther. 47, 391-417
  31. Ghika, J., Tennis, M., Hoffman, E., Schoenfeld, D., and Growdon, J. (1991) Neurology 41, 986-991 [Abstract]
  32. Ruiz, J., Martin, I., Callado, L. F., Meana, J. J., Barturen, F., and Garcia-Sevilla, J. A. (1993) Neurosci. Lett. 160, 109-112 [CrossRef][Medline] [Order article via Infotrieve]

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