Tetramethylbenzidineを用いた培養甲状腺濾胞細胞のPeroxidase活性の測定

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Thyroid peroxidase (TPO) is well known to be an essential enzyme for the biosynthesis of thyroid hormone. The changes of TPO activities in thyroid tissue have already been reported in pathophysiological and experimental conditions by several assay methods. Most of these assay methods, however, need relatively large amounts of tissue (over 10mg wet tissue) to obtain enzyme fraction for assay by homogenization and fractionation of the tissue. Therefore, it is difficult to apply these methods to relatively small numbers (less than 106 cells) of thyroid cells. In the present study, we attempted to develop a new and simple method for the assay of TPO activity using tetramethylbenzidine (TMB) as substrate. The reaction mixture were composed of 250μl of commercially available tetramethylbenzidine solution containing H2O2 (TM-Blue®, TSI-CDP) and 250μl of cell lysate obtained by freeze-thawing in 0.1M citrate buffer (pH 4.8). Various doses of known amounts of horseradish peroxidase (HRP; 0-1000μU) were assayed as a standard at the same time. TPO activity in cell lysate was expressed as the activity corresponding to HRP activity. In this assay method, TPO activity of sonicated-cell lysate was higher than that of nonsonicated-cell lysate, and the activity in sonicated cell lysate was linearly correlated with cell numbers. Next, the effects on the TPO activity of the direct addition of various agents into the reaction mixture were also examined. Both methylmercaptoimidazole (MMI) and NaN3 strongly inhibited TPO activity in sonicated-cell lysate as well as in mitochondria-microsomal fraction of thyroid tissue with the respective IC50 value of less than 5μM and less than 0.1mM. In the present method, the authors could demonstrate the following: 1) After 4 days of suspension culture with TSH (0.5mU/ml), TPO activity of the follicles increased 3.2-5.6 fold when compared with that cultured in the absence of TSH. 2) (Bu)2cAMP (DBC; 1mM) and forskolin (20μM) also increased TPO activity of the follicles by 2.9-5.2 and by 2.9-6.2 fold. 3) The addition of NaI (0-100μM) into medium dose-dependently inhibited the induction of TPO activity by TSH. 4) EGF (10-8M) and PMA (10-6M) as well as NaI (100μM) also inhibited the induction of TPO activity in the follicles by TSH, DBC and forskolin during culture for 4 days. Accordingly, it is indicated that these agents may inhibit an induction of TPO activity at least in part at the step of post-cAMP. The present assay method was simple and convenient for assay of TPO activity in a relatively small number of cultured thyroid cells. However, further improvement was needed to obtain a full activity of TPO in the cells because intracellular reducing agents may affect TPO activity in the present method.
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Tetramethylbenzidineを用いた培養甲状腺濾胞細胞のPeroxidase活性の測定

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