Raddatz R, Parini A, Lanier S M
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425, USA.
J Biol Chem. 1995 Nov 17;270(46):27961-8. doi: 10.1074/jbc.270.46.27961.
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-[125I]iodophenoxy)methylimidazoline ([125I]A-ZIPI), a photoaffinity adduct that selectively labels imidazoline-binding proteins, photolabeled an M(r) = approximately 59,000 peptide in liver and an M(r) = approximately 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.
带有咪唑啉和/或胍基部分的药理活性化合物能被一类统称为咪唑啉结合位点或咪唑啉/胍基受体位点的膜结合蛋白以高亲和力识别。两个这样的受体位点可能对应于在单胺氧化酶（MAO）的A和B同工型上鉴定出的咪唑啉结合结构域，但在多个组织中检测到的单胺氧化酶同工型与咪唑啉结合蛋白的有限表达形成对比。为了解决这些问题，我们确定了已知表达一种或两种MAO同工型的人体组织中，单胺氧化酶同工型与咪唑啉结合蛋白亚型之间的关系。2-(3-叠氮基-4-[¹²⁵I]碘苯氧基)甲基咪唑啉([¹²⁵I]A-ZIPI)，一种选择性标记咪唑啉结合蛋白的光亲和加合物，在肝脏中光标记了一个Mr约为59,000的肽段，在胎盘中光标记了一个Mr约为63,000的肽段，这与通过免疫印迹在这些组织中鉴定出的MAO同工型的Mr一致。肝脏中的光标记物种用MAO-B选择性抗体进行免疫沉淀，而胎盘中光标记物种则用MAO-A选择性抗体进行免疫沉淀，这与这些组织中主要表达的MAO同工型一致。咪唑啉/胍基配体在一个与底物识别结构域不同的位点与该酶相互作用，肝脏和胎盘中免疫沉淀的肽段表现出与报道的咪唑啉结合位点亚型一致的独特配体识别特性。然而，在含有与光标记肝膜中MAO-B量相当的血小板膜制剂中未检测到咪唑啉结合结构域，这表明该结构域的识别具有组织限制性。用500 mM KCl洗涤膜或对酶进行溶解和部分纯化，均未改变血小板MAO-B上对咪唑啉结合结构域的有限可及性，这表明存在不同的MAO亚群。在MAO上鉴定出一个识别这类药理活性化合物的结合结构域，提示了一种调节底物氧化/选择性的新机制，或者该酶可能具有尚未明确的功能。
