A Study of the Primary Inactivating Enzymes of Thyroliberin in the Synaptosomal Membranes of Bovine Brain

Over the past twenty years, there has been an accumulation of evidence indicating that certain peptides that exert biological effects outside the CNS, may also possess neurotransmitter or neuromodulator functions in brain. Thyroliberin (Thyrotropin-Releasing Hormone) was the first of these such peptides displaying a dual role as a hormone and as a neurotransmitter. Its ubiquitous distribution in the hypothalamus and in the extra-hypothalamic regions, and its diverse pharmacological and physiological effects are all features of its dual functions. The study of brain peptidases that hydrolyse thyroliberin is of importance in securing an understanding of the possible mechanisms for termination of its neurotransmitter or neuromodulator actions and the possible enzymic biotransformation of the the original peptide to cleavage products that themselves may exhibit biological activity. The brain peptidases involved in the primary degradation of thyroliberin studied to date are three forms of pyroglutamate aminopeptidase (PAP), a cytosolic, particulate and serum form, and a cytosolic prolyl endopeptidase (PE) activity. This work studied the primary degradation of thyroliberin by the enzymes found in the particulate fraction of bovine brain. It began by investigating the methods available for the quantitation of the particulate PAP activity, and the development of two new assays for its detection, the first a coupled enzyme fluorimetric assay, and the second, a spontaneous cyclisation assay. Using these assays, PAP activity from the synaptosomal membranes of bovine brain was purified and characterised for the first time. This enzyme proved to have many of the characteristics of the previously studied particulate PAP activities, with some notable differences. The particulate PAP activity was shown to be 230kDa in size, had a narrow substrate specificity, cleaving only thyroliberin or very closely related peptides, and displayed a high affinity for thyroliberin. It was inhibited by thiol protease inhibitors, had a requirement for DTT and was unaffected by EDTA. By combining these features, the particulate PAP from bovine brain appears to be a ‘hybrid’ form of the cytosolic and the particulate PAP activities previously studied. Prolyl endopeptidase is widely distributed, has a broad substrate specificty and has been previously characterised in the cytosolic fractions of many species. It is involved in the primary degradation of thyroliberin, producing the bioactive peptide, acid thyroliberin. Prior to this study, PE had only been characterised as a cytosolic activity, and despite references to the possibility of there being a particulate activity, it was never identified. PE activity was located on the synaptosomal membranes of bovine brain, and following vigorous salt-washing, osmotic shock and solubilisation with detergents, has been identified conclusively as a membraneassociated activity. It was purified from the synaptosomal membranes, and characterised as having a broad substrate specificity and a high affinity for thyroliberin (in fact a higher affinity than the particulate PAP). It was inhibited by some of the thiol protease inhibitors and some of the metal chelators, suggesting that it may be a thimet protease. It also is inhibited by the specific prolyl endopeptidase inhibitor, Z-Pro-prolinal. List o f A b b r e v ia t io n s ACE: Angiotensin Converting Enzyme ACTH: Adrenocorticotrophin AMP: Adenosine Monophosphate CCK: Cholecystokinin cDNA: Complementary DNA cGMP: 3’, 5’-Cyclic Guanosine Monophosphate CNS: Central Nervous System CoA: Coenzyme A CRS: Cold Restraint Stress CSF: CerebroSpinal Fluid Cydo(His-Pro): His-Pro Diketopiperazine DAP IV: Dipeptidyl Aminopeptidase IV DFP: Diisopropyl Fluorophosphate DMSO: Dimethylsulphoxide DTT: Dithiothreitol EDTA: Ethylenediaminetetra Acetic Acid GABA: Gamma Aminobutyric Acid GH: Growth Hormone Gly-ProMCA: Glycylproline-7-amino-4-methyl Coumarin Z-Gly-ProMCA: N-Benzyloxycarbonyl-glycylproline-7-amino-4-methyl Coumarin <Glu: Pyroglutamic Acid (5-pyrrolidone-2-carboxylic acid) <Glu-His: Pyroglutamyl Histidine <Glu-His-Pro: Acid Thyroliberin <Glu-His-ProNH2 : Thyroliberin <Glu-His-ProOH: Acid Thyroliberin <Glu-MCA: Pyroglutamate-7-amino-4-methyl Coumarin <Glu-(Me)His-ProNH2 : Pyroglutamyl methyl-histidine proline 7-amino-4-methyl Coumarin (a Thyroliberin Analogue) GPCR: G-Protein Coupled Receptors <Glu-ProNH2 : Pyroglutamyl Proline Amide (a Thyroliberin Analogue) 5-HT: Seretonin Ki: Inhibitor Constant Km: Michaelis Menten Constant LDH: Lactate Dehydrogenase LHRH: Luteinising Hormone Releasing Hormone MCA: 7-Amino-4-Methyl Coumarin ME: Median Eminence mRNA: Messenger Ribonucleic Acid MSH: Melanocyte-Stimulating Hormone NA: Naphthyl Amide PAP: Pyroglutamate Aminopeptidase PCMB: p-Chloromercuribenzoate PDMK: Pyroglutamyl Diazomethyl Ketone PE: Prolyl Endopeptidase PIT: Pituitary PKC: Protein Kinase C PMSF: Phenylmethane Sulphonyl Fluoride PPCE: Post Proline Cleaving Enzyme PRL: Prolactin Ps4: Preprothyroliberin PTU: Propylthiouracil SM: Sulphamethoxazole T3 : Triiodothyronine T4 : Tetraiodothyronine (thyroxine) TEMED: N-| N-| N’.N’.-Tetramethyl Sulphonic Acid TPA:12-o-Tetra-Decanyl-Phorbal-13-Acetate TRH: Thyrotropin Releasing Hormone TSH: Thyrotropin VIP: Vasoactive Intestinal Peptide T a b le o f C o n t e n t s 1. Thyrotropin Releasing Hormone 1 1.1. Physiological Actions of Thyroliberin at the Level of the Adenohypophysis 2 1.2. Distribution of Thyroliberin 4 1.3. Thyroliberin as a Possible Neuroregulator 7 1.3.1. Action of thyroliberin on neurons 8 1.3.2. Release and reuptake of thyroliberin at synaptic terminals 8 1.3.3. Behavioural effects of thyroliberin 8 1.4. Thyroliberin Receptors 9 1.4.1. Cloning and functional characterisation of the thyroliberin receptor 11 1 .4.2. Internalisation of the thyroliberin receptor follows binding 11 1.4.3. Localisation of thyroliberin receptor mRNA 12 1 .4.4. Regulation of thyroliberin receptors 13 1.5. Thyroliberin Biosynthesis 14 1 .5.1. Distribution of the thyroliberin prohormone 15 1.5.2. Processing of the thyroliberin prohormone 16 1.6. Degradation of Thyroliberin 18 1.6.1. Pyroglutamate aminopeptidase 22 1.6.1.1. Inhibitors of the soluble PAP 23 1.6.2. The particulate PAP 24 1 .6 .2 .1 . The distribution of PAP II activity 26 1.6.3. Serum PAP 27 1 .6.4. PAP regulation 28 1.6.4.1. PAP regulation by thyroid hormones 28 1 .6.4.2. Significance of regulation studies in establishing the biochemical role of PAP I, PAP II and the serum thyroliberinase 30 1 .6.4.3. Regulation of pyroglutamyl aminopeptidase by steroid hormones 31 1 .6.4.4. Possible mechanisms of PAP enzyme regulation 33 1 .6.5. Prolyl endopeptidase activity 34 1.6.5.1. Tissue distribution of prolyl endopeptidase 38 1 .6.5.2. Characterisation of prolyl endopeptidase 39 1.6.5.3. Substrate specificity of prolyl endopeptidase 39 1 .6.5.4. Prolyl endopeptidase inhibitors 42 1.6.5.5. Prolyl endopeptidase actions on neuropeptides 47 1 .6.5.6. Regulation of prolyl endopeptidase activity 49 Section