The fibrillogenic properties of Alzheimer's A~3 peptides corresponding to residues 1 -40 of the normal human sequence and to two mutant forms containing the replacement Ala21 to Gly or Glu22 to GIn were compared. At pH 7.4 and 37°Cthe GIn22 peptide was found to aggregate and precipitate from solution faster than the normal A/3, whereas the G1y21 peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and GIn22 sequences was associated with spectral changes consistent with a transformation from random coil to//sheet, whereas the spectrum of the G1y21 peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope 65Zn2~.Of several competing metal ions, tested at 20x the concentration of Zn2~, Cu2~displaced >95% of the radioactivity from all three peptides and Ni2~produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe2~and Al35 were without effect. In a saturation binding assay, a value of 3.2 p~M was obtained for the binding of Zn2~to A~3but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the G1y21 mutation is not due to enhanced fibrillogenic or different metal-binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.
Evidence indicates that neuronally released thyrotropin-releasing hormone (TRH) is selectively inactivated by TRH-degrading ectoenzyme (TRH-DE) (EC 3.4.19.6). TRH-DE inhibitors may be used to enhance the therapeutic actions of TRH and to investigate the functions of TRH and TRH-DE in the central nervous system.Although TRH-DE appears to exhibit a high degree of specificity toward TRH, systematic specificity studies, which would facilitate inhibitor design, have not been previously conducted for this enzyme. In this paper we present the first description of TRH-DE specificity across a directed peptide library in which the histidyl (P 1 ) residue of TRH was replaced by a series of amino acids. Peptides were synthesized using standard solid phase chemistry. Kinetic parameters were measured either by continuous or discontinuous fluorometric assays or by quantitative high pressure liquid chromatography. The P 1 residue was found to influence significantly both the ability of the peptides to bind to TRH-DE, as measured by their K i values, and the ability of TRH-DE to catalyze their hydrolysis. Moderately bulky, uncharged P 1 residues were found to bind preferentially to TRH-DE. Results from this screen provide valuable information for the development of TRH-DE inhibitors and have led to the identification of two potent, reversible TRH-DE inhibitors, L-pyroglutamyl-L-asparaginyl-L-prolineamide (K i ؍ 17.5 M) and Glp-AsnPro-7-amido-4-methyl coumarin (K i ؍ 0.97 M).
Thyrotropin-releasinghormone-degrading ectoenzyme (TRH-DE) 1 (EC 3.4.19.6) is a type II cell surface peptidase located on synaptosomal membranes in the central nervous system (1Ϫ4). TRH-DE catalyzes the hydrolysis of the Glp-His bond in thyrotropin-releasing hormone (TRH), a tripeptide with the amino acid sequence L-pyroglutamyl-L-histidyl-L-prolineamide (Glp-His-ProNH 2 ) (5Ϫ10). This enzyme is strategically placed to play a significant role in extracellular inactivation of TRH, and current evidence strongly indicates that TRH-DE is the principal enzyme responsible for terminating the actions of neuronally released TRH (11Ϫ14).Although first recognized as a hypothalamic regulatory hormone, TRH is now believed to function as a neurotransmitter and/or neuromodulator within the central nervous system (15, 16) where it displays a broad spectrum of stimulatory actions independent of its neuroendocrine functions (15Ϫ17). Based on its central nervous system effects, TRH has been found to have potential use in the treatment of brain and spinal injury (18,19) and several central nervous system disorders, including spinocerebellar degeneration, cognitive deficits, and spinal cord pain transmission (16,17). The mechanisms by which TRH improves these conditions are not fully elucidated but appear to involve the potentiation by TRH of other neurotransmitter systems. Despite its promise, the use of TRH as a therapeutic agent is critically undermined by its susceptibility to proteolytic degradation (20).Compounds that potently and selectively inhibit TRH-DE may be use...
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