©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Imidazoline/Guanidinium Binding Domains on Monoamine Oxidases
RELATIONSHIP TO SUBTYPES OF IMIDAZOLINE-BINDING PROTEINS AND TISSUE-SPECIFIC INTERACTION OF IMIDAZOLINE LIGANDS WITH MONOAMINE OXIDASE B (*)

(Received for publication, June 15, 1995; and in revised form, August 8, 1995)

Rita Raddatz (§) Angelo Parini (1) Stephen M. Lanier (¶)

From the Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina 29425 and INSERM U 388, Pharmacologie Moleculaire et Physiopathologie Renale, Institut Louis Bugnard, Toulouse 31054, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Pharmacologically active compounds with an imidazoline and/or guanidinium moiety are recognized with high affinity by a family of membrane-bound proteins collectively known as imidazoline binding sites or imidazoline/guanidinium receptive sites. Two such receptive sites may correspond to imidazoline binding domains identified on the A and B isoforms of monoamine oxidase (MAO), but the detection of monoamine oxidase isoforms in multiple tissues contrasts with the restricted expression of imidazoline-binding proteins.

To address these issues, we determined the relationship between monoamine oxidase isoforms and subtypes of imidazoline-binding proteins in human tissues known to express one or both isoforms of MAO. 2-(3-Azido-4-[I]iodophenoxy)methylimidazoline ([I]AZIPI), a photoaffinity adduct that selectively labels imidazoline-binding proteins, photolabeled an M(r) = 59,000 peptide in liver and an M(r) = 63,000 peptide in placenta, consistent with the M(r) of the MAO isoforms identified by immunoblots in these tissues. The photolabeled species in liver was immunoprecipitated with MAO-B selective antibodies, whereas the photolabeled species in placenta was immunoprecipitated by MAO-A selective antibodies consistent with the isoform of MAO predominantly expressed in these tissues. The imidazoline/guanidinium ligands interact with the enzyme at a site distinct from the substrate recognition domain, and the immunoprecipitated peptides in liver and placenta display distinct ligand recognition properties consistent with those reported for subtypes of imidazoline binding sites.

However, the imidazoline binding domain was not detected in platelet membrane preparations containing amounts of MAO-B equivalent to those in the photolabeled liver membranes indicating that recognition of this domain is tissue-restricted. Restricted access to the imidazoline binding domain on platelet MAO-B was not altered by membrane washing with 500 mM KCl or by solubilization and partial purification of the enzyme suggesting that there are distinct subpopulations of MAO. Identification of a binding domain on MAO that recognizes this class of pharmacologically active compounds suggests a novel mechanism for regulation of substrate oxidation/selectivity or that the enzyme may subserve an as yet undefined function.


INTRODUCTION

Imidazoline/guanidinium receptive sites or imidazoline binding sites are defined as the nonadrenergic receptor binding sites for a group of structurally related compounds containing imidazoline or guanidinium moieties. Although many of these compounds interact with adrenergic receptors, they also produce ill-defined effects on ion transport, insulin secretion, and blood pressure regulation that are mediated by interactions with multiple, pharmacologically distinct imidazoline-binding proteins (1, 2, and references therein). (^1)The imidazoline/guanidinium receptive sites also recognize endogenous substances that mimic some of these effects(3, 4, 5, 6, 7) .

The imidazoline-binding protein in liver is predominantly localized to mitochondrial membranes and cannot be separated from the mitochondrial enzyme monoamine oxidase (MAO, (^2)EC 1.4.3.4) during purification, suggesting a potential relationship between these two entities(8) . Partial amino acid sequencing of a purified rabbit kidney imidazoline binding protein revealed high sequence similarity to monoamine oxidases of other species whose sequences are known, and heterologous expression of human MAO-A or MAO-B indicates that both isoforms recognize the imidazoline, [^3H]idazoxan, a ligand commonly used to identify imidazoline binding sites in various tissues(9) . However, the affinity of the enzymes for [^3H]idazoxan following expression in Saccharomyces cerevisiae is 10-50-fold lower than that expected for the imidazoline binding site (9) , and some members of the imidazoline binding protein family do not recognize this radioligand when evaluated in their natural environment within the cell(10) .

If indeed MAO and subtypes of imidazoline-binding proteins are identical, then one must also explain the apparent discrepancy that exists in the tissue localization of the two entities. Whereas MAO is widely distributed, members of the family of imidazoline-binding proteins exhibit a more restricted expression. Definition of the relationship between MAO and imidazoline-binding proteins is also complicated by differences in the stoichiometry of the two entities in tissues where both proteins are apparently expressed suggesting that either there are subpopulations of monoamine oxidase that do not bind imidazoline/guanidinium ligands or that the imidazoline binding domain is not accessible in all tissues. In addition, it is difficult to explain the functional effects of these compounds based solely upon their interaction with monoamine oxidases suggesting that there are imidazoline-binding proteins that are not monoamine oxidases.

To address these issues, we utilized a photoaffinity probe ([I]AZIPI) to label the imidazoline-binding proteins in different tissues and monoclonal antibodies that selectively recognize the MAO isoforms to immunoprecipitate the labeled membrane proteins. We report that the [I]AZIPI-labeled species in human liver mitochondria and placenta are identical with the MAO isoforms expressed in the two tissues. The imidazoline/guanidinium ligands interact with MAO isoforms at a site distinct from the substrate binding domain and may represent a novel site for MAO regulation. The interaction of such ligands at the imidazoline binding domain of the B isoform of MAO was not observed in platelets suggesting that availability of this site is tissue-selective. The differential photolabeling of liver and platelet MAO-B was maintained following washing of the membranes in high salt buffer or solubilization and partial purification of the enzymes from the two tissues.


EXPERIMENTAL PROCEDURES

Materials

Renaissance Western blot Chemiluminescence Reagent kit, [I]sodium iodide, and [^3H]pargyline (36 Ci/mmol) were purchased from DuPont NEN, and [^3H]idazoxan (41 Ci/mmol) was purchased from Amersham. Idazoxan, clonidine, guanabenz, clorgyline, deprenyl, and pargyline were provided by Research Biochemicals Int. (Natick, MA), and cirazoline was a gift from Synthelabo (Paris, France). Ascites fluids containing monoclonal antibodies to human MAO-A and -B (11, 12) were kindly provided by Dr. Richard Denney (University of Texas Medical Branch, Galveston, TX). Horseradish peroxidase-conjugated secondary antibodies were purchased from Chemicon (Temecula, CA). GammaBind G-Sepharose brand protein G-Sepharose beads and Polybuffer exchanger 94 resin were purchased from Pharmacia Biotech Inc. Polyvinylidene difluoride membranes were purchased from Gelman Sciences (Ann Arbor, MI). Kodak XAR 5 film was purchased from Chesapeake X-Ray (Florence, SC). Acrylamide, bisacrylamide, SDS, Tween 20, and prestained midrange molecular weight protein standards were purchased from Bio-Rad. Ecoscint A scintillation fluid was purchased from National Diagnostics (Atlanta, GA). Centricon-10 centrifugal concentrators were purchased from Amicon Inc. (Beverly, MA).

Membrane Preparation

Full-term human placenta were collected within 2 h of delivery and washed in 100 mM Tris-HCl, pH 7.4, at 4 °C. Tissue samples were homogenized in lysis buffer (5 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 4 °C) and filtered through two layers of gauze mesh. A low speed pellet was generated by centrifugation at 1000 times g for 10 min at 4 °C. The resulting supernatant was centrifuged at 35,000 times g for 10 min at 4 °C, and this high speed pellet was washed twice in membrane buffer (50 mM Tris, pH 7.5, 0.6 mM EDTA, 5 mM MgCl(2), 0.1 mM PMSF), rapidly frozen, and stored at -70 °C for up to 2 months. A fraction of human liver enriched in mitochondria was obtained as described previously(13) . Outdated human platelets were collected by centrifugation at 800 times g for 30 min at 4 °C in the presence of 2 mM EDTA and 10 µM indomethacin, resuspended, and frozen in 5 mM Tris-HCl, pH 7.4 containing 2 mM EDTA, 10 µM indomethacin, and 75 µg/ml PMSF. Platelets were lysed by freeze-thawing twice in lysis buffer and homogenized using a glass Dounce. Membranes were pelleted by centrifugation at 35,000 times g for 10 min at 4 °C, washed, and resuspended in membrane buffer.

Photoaffinity Labeling

The cirazoline derivative, 2-(3-amino-4-iodophenoxy)methylimidazoline was synthesized, iodinated, and converted to the photolabile azide ([I]-AZIPI) for use as a photoaffinity adduct as described previously(2, 10) . Membranes were incubated in reduced light with 1-2 nM [I]AZIPI for 30 min at 24 °C, chilled on ice, and diluted 10-fold with 4 °C membrane buffer containing 2 mM dithiothreitol immediately prior to photolyzing at 4 °C for 5 min in a Ray-O-Vac photolysis chamber (320 nm). Membranes were pelleted in a microcentrifuge, solubilized in loading buffer (100 mM Tris-HCl, pH 6.8, containing 1% SDS, 50% glycerol, 25% beta-mercaptoethanol, and bromphenol blue) at 100 °C for 5 min and subjected to SDS-polyacrylamide gel electrophoresis(14) . Competing ligands were preincubated with the membranes for 5-10 min at room temperature. Membrane protein was measured by the method of Lowry et al.(15) .

Immunoblots

Membrane proteins were solubilized in loading buffer as above and subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes with a semidry electroblotter (Integrated Separation Systems, Hyde Park, MA) for 1 h at 200 mA. The blots were blocked with 5% nonfat dried milk in wash buffer (20 mM Tris-HCl, pH 7.6, 165 mM NaCl, and 0.2% Tween 20) for 1 h, washed twice, and incubated for 1 h at 24 °C with primary antibodies diluted in wash buffer. After washing, blots were incubated with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody diluted 1:10,000 in wash buffer for 20 min at 24 °C. The horseradish peroxidase reaction was visualized by Renaissance Western blot Chemiluminescence Reagent (DuPont NEN) and exposure to Kodak XAR-5 film.

Immunoprecipitation

Membranes were photolabeled and resuspended in 100 µl of solubilization buffer (membrane buffer with 1% Triton X-100 and 0.05% SDS). The soluble extract was precleared with 10 µl (packed volume) of Sepharose G beads and incubated with the indicated ascites fluid (1:100 final dilution) containing antibodies to MAO-A or MAO-B for 12-16 h at 4 °C. The antibody-antigen complexes were precipitated by the addition of 10 µl (packed volume) of Sepharose G beads and brief centrifugation in a microcentrifuge. The resulting pellet was washed in 500 µl of membrane buffer, solubilized in loading buffer at 100 °C for 10 min, and subjected to SDS-polyacrylamide gel electrophoresis.

Radioligand Binding

Membranes were diluted in membrane buffer and preincubated with competing ligands for 10 min at 24 °C. [^3H]Pargyline (100 nM) or [^3H]idazoxan (3-150 nM) was added, and the samples were incubated with shaking at 24 °C for 30 min. [^3H]Idazoxan binding was performed in the presence of 10 µM rauwolscine, and nonspecific binding was determined in the presence of 10 µM cirazoline. In [^3H]pargyline binding studies, nonspecific binding was determined in the presence of 100 µM pargyline. Membranes were collected by vacuum filtration on glass fiber filters (No. 32, Schleicher & Schuell) and washed in 4 times 3 ml of 100 mM Tris, pH 7.4, 4 °C. Radioactivity retained on the filters was determined by liquid scintillation spectrometry with approximately 50% efficiency.

Solubilization and Partial Purification of MAO-B by Chromatofocusing

Membranes were solubilized and the enzyme was partially purified using a chromatofocusing matrix as described previously(16) . Briefly, human liver (9 mg of protein) and platelet membranes (300 mg of protein) were resuspended in solubilization buffer containing MOPS (25 mM), pH 7, EGTA (2 mM), PMSF (1 mM), and 1% digitonin at a detergent:protein ratio of 3:1. The mixture was incubated at 4 °C for 30 min, and the soluble fraction was collected following centrifugation at 100,000 times g for 60 min at 4 °C. The chromatofocusing matrix (Polybuffer Exchanger 94) was equilibrated with 0.1% digitonin in MOPS (25 mM), pH 7, EGTA (2 mM), PMSF (1 mM), and solubilized material was applied at 0.4 ml/min. Proteins were eluted with Polybuffer 74 (diluted 1:8), pH 4.0, containing digitonin (0.1%), EGTA (2 mM), and PMSF (0.1 mM). Fractions were collected and protein was monitored by optical density at 280 nm. The pH of fractions containing MAO-B was adjusted to 7.4 prior to concentration by Centricon centrifugal concentrators and subsequent photolabeling. To remove free [I]AZIPI from the solubilized preparations, the samples were desalted on Sephadex G-25 gel filtration columns prior to photolysis. The eluate was photolyzed in the presence of 2 mM dithiothreitol and concentrated by lyophilization prior to addition of loading buffer and gel electrophoresis.


RESULTS

Identification of MAO Isoforms and Photoaffinity Labeling of Imidazoline-binding Proteins in Human Liver Mitochondria and Human Placenta

As a first step to define the relationship between MAO and imidazoline binding sites, we characterized the proteins in human liver and placenta, tissues that express both the MAO isoforms and imidazoline-binding proteins. As described previously, human placenta expressed predominantly MAO-A(11) , and human liver mitochondria expressed high levels of the MAO-B isoform (17) (Fig. 1A). Immunoblots using antibodies selective for human MAO-A (MAO-A-4D3 ascites)(11) , or MAO-B (MAO-B-1C2 ascites) (12) , identified a single protein with apparent molecular weight of 63,000 in human placental membranes and a peptide with apparent molecular weight of 59,000 in human liver mitochondrial membranes, respectively. (^3)The apparent molecular weights of the MAO isoforms were similar to previously reported estimates based on gel mobility and the calculated molecular weights of 59,700 and 58,800 determined from the nucleotide sequences (18) .


Figure 1: Monoamine oxidase isoforms and imidazoline/guanidinium receptive sites in human placental and liver mitochondrial membranes. Monoamine oxidase isoforms and imidazoline/guanidinium receptive sites in human placental and human liver mitochondrial membranes were identified by immunoblotting or photoaffinity labeling, respectively. In each experiment, membranes were solubilized and electrophoresed under denaturing conditions on 8% SDS-polyacrylamide gels. Autoradiographs were obtained by exposing the dried gels at -70 °C for 5-7 days. The migration of midrange molecular weight standards is indicated by the numbers to the left of the immunoblot or autoradiographs (M(r) times 10). The arrows indicate the migration of proteins with apparent molecular weights of 63,000 and 59,000. A, identification of MAO-A and MAO-B by immunoblotting. Nitrocellulose transfers of polyacrylamide gels of human and rat liver mitochondria (200 µg of membrane protein) and human placenta (350 µg of membrane protein) were immunoblotted with MAO-A-4D3 (1:200) or MAO-B-1C2 ascites (1:1000) as described under ``Experimental Procedures.'' Immunoreactive proteins were identified by a chemiluminescent reaction with horseradish peroxidase-conjugated secondary antibodies and subsequent exposure to film. Similar results were obtained in three experiments. B, autoradiograph of photoaffinity-labeled human placental and human liver mitochondrial membranes. Human placental membranes (400 µg of membrane protein) and human liver mitochondrial membranes (50 µg of membrane protein) were photolabeled with [I]AZIPI (1.4 nM) in the presence or absence of 10 µM cirazoline. Similar results were obtained in three experiments.



Peptides with apparent molecular weights similar to that of the MAO isoforms were also covalently labeled in the two human tissues with the photoaffinity adduct, [I]AZIPI, that selectively labels imidazoline-binding proteins (Fig. 1B). Photolabeling of the M(r) = 59,000 and 63,000 peptides was blocked by cirazoline, an imidazoline that exhibits high affinity for members of the family of imidazoline-binding proteins. Thus, the apparent molecular weight of the imidazoline-binding protein in human placental and human liver mitochondrial membranes corresponds to that of the MAO-A or -B isoform predominantly expressed in these tissues.

Immunoprecipitation of [I]AZIPI-labeled Proteins from Liver Mitochondrial and Placental Membranes

To determine whether the ligand binding subunit of imidazoline-binding proteins in human liver mitochondrial and placental membranes was identical to MAO, we attempted to immunoprecipitate the [I]AZIPI-labeled species with monoclonal antibodies selective for the MAO-A and -B isoforms (MAO-A-4D3 and MAO-B-1C2 ascites fluids)(19) . The species labeled by [I]AZIPI in placenta (M(r) = 63,000) or liver mitochondria (M(r) = 59,000) were immunoprecipitated by MAO-A-4D3 and MAO-B-1C2 ascites, respectively (Fig. 2A). The selectivity of immunoprecipitation of the labeled species was tested by immunoprecipitating aliquots of [I]AZIPI-labeled membranes from the tissues with both ascites fluids (Fig. 2B). The photolabeled species in the placental membranes was not immunoprecipitated with the MAO-B-1C2 ascites, nor was the photolabeled species in liver mitochondrial membranes immunoprecipitated by the MAO-A-4D3 ascites (Fig. 2B). The efficiency of immunoprecipitation for monoamine oxidase A or B was 95% and 50%, respectively, and paralleled the relative distribution of photolabeled peptides in the supernatant and immunoprecipitation pellet. (^4)Thus, the [I]AZIPI-labeled imidazoline-binding proteins were selectively immunoprecipitated by antibodies recognizing the isoform of MAO predominantly expressed in each tissue. The apparent molecular weight of the immunoprecipitated photolabeled peptide was directly related to that of the MAO isoform present in the tissue, suggesting that the photolabeled species are indeed MAO and not a co-immunoprecipitated protein. The ligand binding subunits of imidazoline-binding proteins in human placental and in human liver mitochondrial membranes were therefore identified as the isoforms of the mitochondrial enzyme, MAO.


Figure 2: Immunoprecipitation of photoaffinity-labeled human placental and human liver mitochondrial membranes. Human liver mitochondrial (400 µg of membrane protein) and human placental (250 µg of membrane protein) membranes were photolabeled with [I]AZIPI (0.9 nM). In A, the MAO isoform present in the membranes was immunoprecipitated with MAO-A-4D3 ascites (placenta) or MAO-B-1C2 ascites (liver mitochondria) both at 1:100 final dilution. These results are representative of 2-3 experiments. In separate experiments (B), both MAO-A-4D3 ascites and MAO-B-1C2 ascites (1:100) were used to immunoprecipitate the MAO isoforms present in each tissue.



Identification of Multiple Ligand Binding Domains on MAO

Various imidazoline/guanidinium ligands interact poorly with the MAO active site(20, 21, 22, 23) . To determine whether [I]AZIPI photoincorporates at the enzyme active site, membranes were photolabeled with [I]AZIPI in the presence of the mechanism-based, irreversible MAO inhibitors pargyline, clorgyline, and deprenyl (Fig. 3, A and B). Saturating concentrations of the MAO inhibitors did not eliminate photoaffinity labeling of the imidazoline-binding proteins in placental or liver mitochondrial membranes. Similarly, imidazoline or guanidinium ligands did not compete for binding of pargyline, an inhibitor of both MAO-A and -B (Fig. 3C). [^3H]Pargyline binding in placental membranes was inhibited by the MAO-A selective ligand, clorgyline, but not by the MAO-B selective ligand, deprenyl. In liver mitochondrial membranes, [^3H]pargyline binding was inhibited by deprenyl, but not by clorgyline as expected for a tissue expressing predominantly MAO-B (Fig. 3C). However, the imidazoline ligand cirazoline (1 µM), which effectively blocks [I]AZIPI photoincorporation, did not inhibit [^3H]pargyline binding in any of the tissues examined (Fig. 3C). These results indicate that [I]AZIPI incorporates into a site on MAO distinct from the binding site of the acetylenic MAO inhibitors.


Figure 3: Relationship between binding sites for imidazoline or guanidinium ligands and monoamine oxidase inhibitors. Placental membranes (500 µg of membrane protein) (A) and liver mitochondrial membranes (100 µg of membrane protein) (B) were photolabeled with [I]AZIPI (2 nM) in the presence of buffer, 1 µM cirazoline, 1 µM idazoxan, 10 µM amiloride, 10 µM clonidine, 10 µM rauwolscine, 100 µM pargyline, 0.1 µM clorgyline, or 1 µM deprenyl. Following electrophoresis on an 8% polyacrylamide gel under denaturing conditions, autoradiographs were obtained by exposing the dried gels at -70 °C for 1 (A) or 7 days (B). The migration of midrange molecular weight standards are indicated by the numbers to the left of the autoradiographs (M(r) times 10). The arrows indicate the migration of the photolabeled proteins with apparent molecular weights of 63,000 (A) and 59,000 (B). C, identification of MAO-A in placental membranes and MAO-B in liver mitochondrial membranes using the MAO inhibitor [^3H]pargyline. Binding of [^3H]pargyline (100 nM) was determined in 100 µg of liver mitochondrial membrane protein or 400 µg of placental membrane protein in the presence of buffer (TB), 100 µM pargyline (parg), 0.1 µM clorgyline (clor), 1 µM deprenyl (depr), or 1 µM cirazoline (ciraz) in duplicate as described under ``Experimental Procedures.'' Total bound radioactivity for liver and placenta was 4,900 cpm and 15,600 cpm, respectively, and was measured with approximately 50% efficiency. The data are expressed as percent total binding (TB) and are representative of the results of two experiments.



The ligand recognition properties of the [I]AZIPI photoincorporation sites on MAO-A and -B were examined to determine the relationship between these sites and imidazoline-binding proteins identified in other tissues (Fig. 3, A and B). Photolabeling in both liver mitochondrial and placental membranes was equally sensitive to inhibition by cirazoline; however, the recognition of idazoxan and amiloride varied in the two tissues. Photoaffinity labeling of the imidazoline-binding protein in human placenta was more sensitive to competition by the guanidinium ligand, amiloride, while the imidazoline compound, idazoxan, competed more effectively in liver (Fig. 3). These results indicate that the pharmacological heterogeneity of imidazoline-binding proteins may reflect the presence of different MAO isoforms.

Photolabeling of MAO-B from Human Liver Mitochondria and Human Platelets

Although the preceding results indicate the identity of MAO and subtypes of imidazoline-binding proteins, there is an apparent discrepancy between the tissue distribution of the two entities. The quantitative relationship between MAO and imidazoline-binding proteins was thus further investigated in two tissues that express high levels of MAO-B, liver mitochondria and platelets. The relative amounts of MAO-B present in liver mitochondrial, and platelet membranes were determined by immunoblotting using MAO-B-selective monoclonal antibodies (MAO-B-1C2 ascites). Similar levels of MAO-B immunoreactivity were identified in 50 µg of membrane protein from liver mitochondrial membranes and 300 µg of membrane protein from platelet membranes (Fig. 4A). Photolabeling was therefore compared in aliquots of liver mitochondrial and platelet membranes containing similar amounts of MAO-B. [I]AZIPI photoincorporated into a M(r) = 59,000 peptide in liver mitochondrial membranes but not in platelet membranes (Fig. 4B). The relative amounts of MAO-B in the two tissues were also determined using the MAO radioligand, [^3H]pargyline (Fig. 4C). Binding of this mechanism-based inhibitor indicated the presence of active enzyme in both platelet and liver mitochondrial membranes suggesting that the site of [I]AZIPI photoincorporation on MAO-B is differentially accessible in these tissues.


Figure 4: Relationship between imidazoline/guanidinium receptive sites and MAO-B in platelet and liver mitochondrial membranes. A, immunoblot of MAO-B in platelet and liver mitochondrial membranes. Platelet membranes (300 µg of membrane protein) and liver mitochondrial membranes (50 µg of membrane protein) were solubilized, electrophoresed on a 10% SDS-polyacrylamide gel, and transferred to membranes. The blot was probed with the anti-MAO-B monoclonal antibody MAO-B-1C2 (1:1000), and immunoreactive proteins were identified as described under ``Experimental Procedures.'' The arrow indicates the migration of the immunoreactive protein with an apparent molecular weight of 59,000 in both platelet and liver mitochondrial membranes. B, autoradiograph of [I]AZIPI-labeled species in liver mitochondrial and platelet membranes. Platelet membranes (300 µg of total protein) and liver mitochondrial membranes (50 µg of total protein) were photolabeled with [I]AZIPI (1.4 nM) in the presence or absence of 10 µM cirazoline. Following electrophoresis on a 10% SDS-polyacrylamide gel, autoradiographs were obtained by exposing the dried gels at -70 °C for 11 days. A photolabeled species in platelet membranes was detected with longer exposure. The arrow indicates the migration of the photolabeled protein with M(r) of 59,000. In A and B, the migration of midrange molecular weight standards are indicated by the numbers to the left of the immunoblot or autoradiograph (M(r) times 10). C, [^3H]pargyline binding in liver mitochondrial and platelet membranes. [^3H]Pargyline (100 nM) was incubated with aliquots of liver mitochondrial (100 µg of membrane protein) or platelet membranes (600 µg of membrane protein) in the presence of buffer (total binding) or 100 µM pargyline (nonspecific binding). Specific binding = total binding minus nonspecific binding. Nonspecific binding represented 4% and 6% of total binding for liver and platelets, respectively. Bound radioactivity was measured with approximately 50% efficiency. Data represent the average ± range for two experiments. D, [^3H]idazoxan binding in liver mitochondrial and platelet membranes. Increasing concentrations of [^3H]idazoxan (3-150 nM) were incubated with aliquots of liver mitochondrial (, 150 µg of membrane protein) or platelet (bullet, 500 µg of membrane protein) membranes in the presence of 10 µM rauwolscine. Nonspecific binding was determined in the presence of 10 µM cirazoline. At radioligand concentrations near the K, specific binding represented 90% of total binding in liver and 32% of total binding in platelets. Data are representative of two experiments performed in duplicate.



A similar lack of availability of the imidazoline binding domain on MAO-B in platelet membranes was observed using the imidazoline ligand [^3H]idazoxan (Fig. 4D). The relative amounts of MAO-B in liver mitochondria and platelet membranes were determined by both immunoblotting and [^3H]pargyline binding, and membrane aliquots containing similar amounts of MAO-B were incubated with increasing concentrations of [^3H]idazoxan. The alpha(2)-adrenergic receptor antagonist rauwolscine (10 µM) was included in the incubation buffer to prevent ligand interaction with alpha(2)-adrenergic receptors. [^3H]Idazoxan exhibited similar affinities for the imidazoline binding sites in these two preparations (K(d) 10-20 nM); however, the binding capacity of the liver membranes was greater than that of the platelet membranes. These data indicate that the lack of photoaffinity labeling of MAO-B in platelet membranes is not due to a difference in affinity for imidazoline ligands, but rather to a lower binding capacity.

As an initial approach to determine the cause of the limited access to the imidazoline binding domain on platelet MAO-B, experiments were performed to address the possibility that this domain is masked in the platelet membrane environment. In the first series of experiments, liver mitochondria and platelet membranes were mixed and incubated for 10 min at 24 °C prior to [I]AZIPI labeling to determine whether the imidazoline binding domain on platelet MAO-B was masked by a diffusable substance. The presence of platelet membranes did not inhibit the photolabeling of liver mitochondria (Fig. 5A). Next, we removed peripheral membrane proteins and small molecular weight substances present in the crude membrane preparations by washing the membranes in buffer containing 500 mM KCl, a cation that allosterically increases the dissociation rate of imidazoline/guanidinium ligands from imidazoline binding sites(24, 25) . However, the availability of the imidazoline binding domain did not increase following high salt washes (data not shown). The differential photolabeling of liver and platelet MAO-B was also maintained after detergent solubilization of the enzymes from the membrane (Fig. 5B).


Figure 5: Accessibility of the imidazoline binding domain in liver mitochondria and platelet membranes. A, autoradiograph of [I]AZIPI-labeled species in liver mitochondria, platelet membranes, or a mixture of both. Liver membranes (50 µg of protein), platelet membranes (300 µg of protein), or a mixture (50 µg of liver mitochondria protein and 300 µg of platelet membrane protein) were incubated at 24 °C for 10 min prior to photolabeling with [I]AZIPI (0.7 nM) in the presence or absence of cirazoline (10 µM). Following electrophoresis on a 10% SDS-polyacrylamide gel, autoradiographs were obtained by exposing the dried gels at -70 °C for 10 days. The arrow indicates the migration of the photolabeled protein with M(r) of 59,000. B, immunoblot and autoradiograph of [I]AZIPI-labeled MAO-B in detergent-solubilized preparations of liver and platelet membranes. Approximately 35% of membrane proteins and 40% of MAO-B detectable by immunoblotting were solubilized by extraction of platelet and liver membranes with 1% digitonin. Left, aliquots of solubilized liver (25 µg of protein) and platelet (100 µg of protein) membranes were electrophoresed on a 10% SDS-polyacrylamide gel and transferred to membranes. The blot was probed with the anti-MAO-B monoclonal antibody MAO-B-1C2 (1:1000), and immunoreactive proteins were identified as described under ``Experimental Procedures.'' Right, aliquots of solubilized liver (25 µg of protein) and platelet (125 µg of protein) membranes containing similar amounts of MAO-B immunoreactivity, as indicated in the left panel, were photolabeled with [I]AZIPI (1.4 nM) in the presence or absence of 10 µM cirazoline. Following electrophoresis on a 10% SDS-polyacrylamide gel, autoradiographs were obtained by exposing the dried gels at -70 °C for 8 days. The migration of midrange molecular weight standards are indicated by the numbers to the left of the autoradiographs or immunoblot (M(r) times 10). The arrows indicate the migration of photolabeled or immunoreactive species with M(r) of 59,000.



In a second approach to address this issue, solubilized membrane proteins from liver and platelet were fractionated by chromatofocusing. Proteins were eluted with a pH gradient from pH 7 to 4, and the peak of MAO-B immunoreactivity was detected in the pH 5.4-5.8 fractions for both the liver and platelet preparations (Fig. 6, A and B). This pH range is similar to the previously determined pI of 5.5 for the imidazoline-binding protein purified from rabbit kidney(2, 16) . Fractions from liver (pH 5.4-5.5) and platelet (pH 5.6) were concentrated, and aliquots containing similar amounts of MAO-B were photolabeled (Fig. 6C). The imidazoline binding domain on platelet MAO-B remained inaccessible after fractionation of the solubilized material using the chromatofocusing matrix.


Figure 6: Partial purification and photolabeling of liver and platelet MAO-B. Solubilized extract from liver membranes (3.5 mg of protein in 2.5 ml) (A) or platelet membranes (100 mg in 100 ml) (B) was fractionated using a chromatofocusing matrix (bed volumes of 5 ml and 17 ml, respectively). Absorbed proteins were eluted with a pH gradient of pH 7-4, and protein was monitored by absorbance at 280 nm (bullet-bullet). MAO-B content was determined by immunoblotting (insets). Aliquots of collected fractions from liver (50 µl) and platelet (100 µl) were electrophoresed on 10% SDS-polyacrylamide gels and transferred to membranes. The blots were probed with the anti-MAO-B monoclonal antibody MAO-B-1C2 (1:1000), and immunoreactive proteins were identified as described under ``Experimental Procedures.'' The fraction numbers are indicated under each lane of the immunoblots. The arrows indicate the migration of the immunoreactive protein with an apparent molecular weight of 59,000 in both liver mitochondria and platelet fractions. Fraction size: 1 ml (liver) and 4 ml (platelet). C, immunoblot and autoradiograph of [I]AZIPI-labeled fractions eluted from the chromatofocusing matrix. Aliquots of the eluted fractions enriched in MAO-B (fraction 23 from platelet and fractions 23-25 from liver) were concentrated, and similar amounts of enzyme (left panel) were photoaffinity-labeled using [I]AZIPI (right panel). Blots were probed with the anti-MAO-B monoclonal antibody MAO-B-1C2 (1:1000), and immunoreactive proteins were identified as described under ``Experimental Procedures.'' The arrow indicates the migration of the immunoreactive protein with an apparent molecular weight of 59,000 in both liver and platelet fractions (left panel). Autoradiographs of the photolabeled samples were obtained by exposing the dried gels at -70 °C for 8 days. In the right panel, the arrow indicates the migration of a photolabeled species with M(r) of 59,000. The migration of midrange molecular weight standards are indicated by the numbers to the left of the autoradiograph or immunoblot (M(r) times 10).




DISCUSSION

Although many of the cellular effects of imidazoline/guanidinium compounds are mediated by known neurotransmitter receptor systems (i.e. adrenergic), some of their effects apparently involve interaction with a family of imidazoline binding sites of unknown identity. The present study indicates that two members of this protein family identified in human placenta and liver are identical with the A and B isoforms of the enzyme monoamine oxidase, respectively. The identities of additional imidazoline binding proteins that differ in their ligand recognition properties and subcellular distribution have yet to be determined(8, 26, 27, 28, 29) .

Members of the family of imidazoline binding proteins are subtyped as I(1) and I(2) based on their ability to recognize various imidazoline or guanidinium ligands. Differences in ligand recognition properties of the I(1) and I(2) sites include selective recognition of the imidazoline clonidine by I(1) sites(30) . The relative insensitivity of [I]AZIPI photoincorporation in placenta or liver mitochondria to competition by clonidine indicates that both of the imidazoline-binding proteins identified in these tissues belong to the I(2) subgroup of imidazoline-binding proteins which are localized to the outer mitochondrial membrane. The I(2) subtype can be further subclassified based upon differential recognition of the guanidinium compound amiloride (30) . The imidazoline binding domain on MAO-A in placental membranes exemplifies the amiloride-sensitive I(2) subtype while photolabeling of the imidazoline binding domain on MAO-B in liver mitochondrial membranes is relatively insensitive to amiloride. Similarly, imidazoline binding sites identified in human placenta and liver by radioligand binding with [^3H]idazoxan differentially recognize amiloride(8, 28) . Thus, two pharmacologically defined I(2) subtypes in these tissues are identical with the A and B isoforms of MAO.

MAO isoforms are identified in a wide variety of tissues by enzyme activity, immunoreactivity, and the use of radiolabeled enzyme inhibitors(31) . In contrast, the tissue distribution of imidazoline binding sites is more restricted. Such a discrepancy exists in human platelet membranes, which express both immunoreactive and functional MAO-B that is poorly recognized by the photoaffinity adduct [I]AZIPI. There are several possible explanations for the observation that the imidazoline binding domain is not equally detected in all tissues expressing MAO: 1) the existence of additional isoforms of monoamine oxidase, generated perhaps by alternative splicing, that differ in the enzyme domain that recognizes imidazoline/guanidinium ligands; 2) cell-specific post-translational modification of the enzyme such that the binding domain for imidazoline/guanidinium ligands is selectively masked; 3) the existence of tissue-specific protein(s) that allosterically influence accessibility to the imidazoline binding domain; or 4) occupation of the imidazoline binding domain by an endogenous substance that is present in selected tissues.

Although analysis of cDNA clones encoding monoamine oxidase B isolated from various tissues do not indicate additional sequence diversity(18, 32) , the enzyme genes are complex and consist of multiple exons. Alternative splicing could result in tissue-specific expression of MAO subtypes containing the imidazoline binding domain. Thus, in tissues where detection of the imidazoline binding site is limited, such as platelets, the MAO population may consist predominantly of enzyme subtypes lacking this domain. MAO also undergoes several post-translational modifications including the covalent attachment of the flavin cofactor and the formation of several disulfide bonds which are required for enzyme activity. Additionally, there is a consensus site for N-linked glycosylation at amino acids 181 and 145 of MAO-A and -B, respectively, but the proteins appear not to be glycosylated(18, 33) .

Possibilities 3 and 4 are of note for several reasons, particularly the large gain in detectable imidazoline binding sites observed during two chromatographic steps used for the purification of rabbit kidney imidazoline-binding protein(16) . Such an observation may be due to the separation of the imidazoline-binding protein from an associated protein or a small, endogenous organic ligand. The latter possibility is in line with the demonstration of endogenous substances such as clonidine-displacing substance or agmatine which are postulated to be endogenous ligands for members of the family of imidazoline-binding proteins(3, 4, 5, 6, 7) . This point also parallels several reports indicating the existence of endogenous substances that regulate MAO activity (34, 35, 36, 37) . However, the imidazoline binding domain on platelet MAO-B remains inaccessible following attempts to remove associated substances by high salt washes, removal of the enzyme from its membrane environment by detergent solubilization, and partial purification of the enzyme by chromatofocusing. These data suggest that the differential recognition of the imidazoline binding domain on MAO-B in liver and platelet is due to structural differences in the enzyme itself.

Imidazoline/guanidinium compounds are not apparent substrates for MAO and do not compete for binding of radiolabeled inhibitors to the enzyme (21) . Similarly, MAO inhibitors that bind to the enzyme active site do not inhibit labeling of the enzyme in human liver and placenta by the photoaffinity adduct [I]AZIPI and do not inhibit [^3H]idazoxan binding to the heterologously expressed enzymes(9) . Thus the binding of these compounds likely involves a site on the enzymes distinct from the substrate recognition site. In addition, in rat liver and rabbit cerebral cortex, various MAO inhibitors exhibit K(i) values in the micromolar range for imidazoline binding sites identified with [^3H]idazoxan(20, 22) . However, in rat brain membranes, the MAO-A isoform selective inhibitor clorgyline exhibits picomolar affinity for a subpopulation of imidazoline-binding proteins and the interaction of clorgyline with these sites is irreversible, as is its ability to inhibit substrate oxidation(23) . Thus, the relationship between these two enzyme domains and the actual structure of the functional enzyme complex remain unclear.

Although there is clearly an imidazoline binding domain on the enzyme, the consequences of occupation of this site on enzyme activity or substrate selectivity are not clear. Relatively high concentrations of imidazoline/guanidiniums are required to observe an effect on enzyme activity(9, 20) . Enzyme activity is noncompetitively inhibited by certain imidazolines at ligand concentrations 100-1000-fold higher than their K(i) determined in radioligand binding studies corresponding to a concentration that is 10-100-fold higher than the estimated concentration required to saturate available imidazoline binding sites(9, 20) . It is thus difficult to correlate all of the various cellular effects of imidazoline or guanidinium compounds with alterations in MAO-induced neurotransmitter metabolism, suggesting the involvement of other imidazoline-binding proteins. Alternatively, perhaps MAO is a multifunctional enzyme that possesses as yet unknown actions initiated by occupation of the imidazoline binding domain. Demonstration of a binding site on MAO for this class of pharmacologically active compounds that is detected in a cell-type-specific manner is of particular significance given the putative role of the enzyme in the etiology and/or therapeutic management of various neurodegenerative diseases(31) .


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant RO1-NS24821 (to S. M. L.), Council for Tobacco Research Grant 2235 (to S. M. L.), and Contrat de Recherche Externe INSERM Grant 910205 (to A. P.). 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.

§
Supported in part by Training to Improve Cardiovascular Drug Therapy Grant 5-T32-HL07260-18 awarded by the National Institutes of Health.

To whom correspondence should be addressed: Dept. of Pharmacology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 803-792-2574; Fax: 803-792-2475.

(^1)
The group of imidazoline-binding proteins that recognize both imidazoline and guanidinium compounds with high affinity is referred to in the literature by various terms including nonadrenergic imidazoline-preferring binding sites, I(2) imidazoline binding sites (I(2)BS), imidazoline/guanidinium receptive sites, or I receptors.

(^2)
The abbreviations used are: MAO, monoamine oxidase; [I]AZIPI, 2-(3-azido-4[I]iodophenoxy)methylimidazoline; PMSF, phenylmethylsulfonyl fluoride; MOPS, 3-(N-morpholino)propanesulfonic acid.

(^3)
The MAO-A isoform is also present in liver of several species. Immunoblotting indicates that the relative proportion of the two isoforms in liver varies from sample to sample. Relative comparison of the immunogenerated signals is also complicated by differences in the efficiency of recognition of denatured enzyme on nitrocellulose blots by the MAO-A-4D3 and MAO-B-1C2 ascites fluids (11) .

(^4)
The efficiency of immunoprecipitation of MAO-A and MAO-B from placental and liver mitochondrial membranes was determined in separate experiments by immunoblots using a polyclonal antiserum that recognizes both MAO isoforms. Approximately 95% of MAO-A present in placental membranes was found in the immunoprecipitation pellet compared to 90% of the photolabeled species. MAO-B was immunoprecipitated from liver mitochondrial membranes with lower efficiency (45% in immunoprecipitation pellet), and, correspondingly, 50% of the photolabeled species was present in the immunoprecipitation pellet.


ACKNOWLEDGEMENTS

We express our appreciation to Drs. V. Bakthavachalam and J. L. Neumeyer of Research Biochemicals for providing 2-(3-aminophenoxy)methylimidazoline used as a precursor to AZIPI. We thank Dr. Richard M. Denney of the University of Texas Medical Branch, Galveston, TX for the generous gift of MAO monoclonal antibodies, Dr. Singh (Division of Pediatrics, Medical University of South Carolina) for the human liver mitochondria, and Dr. P. Halushka (Dept. of Pharmacology, Medical University of South Carolina) for human platelet concentrate. We also thank Mark Dole for technical assistance.


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