Purification of pyroglutamate aminopeptidase and development of assay methods employing its antibody

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Thyrotrotropin-releasing hormone(TRH) may play a role in regulating eating behavior, body temperature and conscious status. On the other hand, Histidyl-proline diketopiperazine, Cyclo(His-Pro), is known as endogenous metabolite of TRH.An Attempt was made to elucidate the nature of pyroglutamate aminopeptidase (pGlup) which converted TRH to Cyclo(His-Pro) endogenously.The results obtained were as follows:1. pGlup activitiy was determined by using pyroglutamy 1-4-methy lcoumarinyl amide as the substrate. 2. The purification of pGlup was performed by applying bovine amd rat brain homogenate on Sephacryl S-200 and GF-250 (HPLC). The molecular weight of pGlup protein was approximately 380000. 3. Radioimmunoassay of pGlup protein was established in our laboratory, using antibody to purified enzyme protein. 4. The distribution of pGlup in the rat brain showed that this enzyme protein was particularly rich in cerebellum and hypothalamus. 5. The positive correlation between brain pGlup concentration and Cyclo (His-Pro) during the developmental process of the rat was observed. 6. There was no difference in the circulating pGlup between patients with thyroid dysfunctio and normal subjects. 7. Some of patients with amyotrophic lateral sclerosis showed elevated pGlup level in the cerebrospinal fluid.The present results suggest that pGlup may be one of the important factor in regulating brain Cyclo(His-Pro) content. In addition, pGlup determination in the blood and cerebrospinal fluid of the patients may be an indicator of some pathological conditions of the diseases.