甲状腺穎粒系ヘム蛋白の分光学的研究

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This study was performed to examine the novel mechanisms of thyroid hormone biosynthesis, by which the electron transport systems in this gland is coupled with thyroid iodide peroxidase and for this purpose, first of all, the spectrophotometric nature of the membrane-bound thyroid peroxidase was elaborated. After a component that formed a cyanide difference spectrum at (KCN) = 5μM with a peak at 445 nm and a small trough at 410 nm had been removed by centrifugal procedure, the washed hog thyroid microsomes with sufficient peroxidase activity were used for the experiments, of which the final preparations contained 0.095 nmoles cytochrome b5 and 0.090 nmoles oxyhemglobin per mg of proteins. The reference and sample cuvettes contained 2.5 ml of thyroid microsomal suspensions (6.7 mg of protein per ml) in 0.1 M potassium phosphate buffer, pH 7.4 and the difference spectra were measured with a Shimadzu Model 5000 spectrophotometer and Union Giken Model SM 401 spectrophotometer at 25°. Since the peroxidase in the thyroid microsomes was specifically inactivated by NADPH under aerobic experimental conditions, it became possible to obtain spectral data on the enzyme bound to the microsomes. And the spectral characteristics of hog thyroid peroxidase bound to microsomes were compared with those of the peroxidase-inactivated microsomes, solubilized thyroid peroxidase and some other purified hemoproteins. Spectrophotometric titrations of hog thyroid microsomes with cyanide and azide gave monophasic dissociation curves having dissociation constants of 17 and 27 μM, respectively, at pH 7.4. These values were approximately equal to the concentrations of cyanide and azide that caused half-maximal inhibition to the peroxidase activity of the microsomes. Upon the addition of NADPH to an aerobic suspension of the microsomes, the Soret bands of oxidized and reduced thyroid peroxidase decreased with a concomitant decrease in the peroxidase activity. From the difference spectra between the intact and peroxidase-inactivated microsomes, the Soret band of the enzyme was assumed to be at 410 nm in the ferric form and at 421 nm in the "stable" ferrous form. Though it has so far been generally regarded that thyroid peroxidase is similar to horseradish peroxidase, the following proterties of the membrane-bound thyroid peroxidase obtained from the present study were markedly different from those of hemoglobin, myoglobin and horseradish peroxidase, but rather similar to those of lactoperoxidase. The affinity of the reduced enzyme for cyanide was relatively high and a cyanide difference spectrum was observed in the CO saturated suspension of reduced thyroid microsomes. This difference spectrum which represents cyanide complex of the reduced form of thyroid peroxidase in sample cuvette and CO complex of the reduced form in reference cuvette can be the most suitable and characteristic one to detect the spectrum of thyroid peroxidase present in thyroid microsomes, especially for clinical materials. Because the amounts of thyroid microsomes prepared from patients are usually extremely limited and even if methemoglobin is present or generated through autoxidation in thyroid microsomes, for example, in the case of aging and so forth, this difference spectrum can not be affected by the presence of methemoglobin. Furthermore, the reduced enzyme as well as the oxidized enzyme forms a spectrophotometrically detectable complex with azide at neutral pH under aerobic condition in the presence of CO, differently from horseradish peroxidase. From these data in addition to the labile property of thyroid peroxidase to H2O2, it appears that this enzyme is an anomalous type of peroxidase compared with the other known enzymes. The nature of thyroid peroxidase prepared by non-proteolytic method,
一般社団法人日本内分泌学会の論文

甲状腺穎粒系ヘム蛋白の分光学的研究

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