Inhibition of thyroid iodide peroxidase by hydrazines and NSD-1055 (4-bromo-3-hydroxybenzyl-oxyamine).

Abstract

Hydrazine (1 X10~M) and phenylhydrazine (1X10~M) inhibit thyroid peroxidase completely. As with horse-radish peroxidase (HRP), thyroid peroxidase is inhibited irreversibly by these compounds. NSD-1055 (4-bromo3-hydroxybenzyloxyamine), which is an inhibitor of histidine decarboxylase and other pyridoxal enzymes, also inhibits thyroid iodide peroxidase as it does horse-radish peroxidase, but NSD-1055 inhibits the peroxidase reversibly. {Endocrinology 88: 1264, 1971) A NDREJEW et al. (1) demonstrated that iso-£»niazid (INH) and hydrazine competitively inhibited horse-radish peroxidase. Later, Altschuler et al. (2) found that INH competitively inhibited peroxidase activity of dog thyroid gland homogenate. We have recently observed that, when horse-radish peroxidase is inhibited by iproniazid (isonicotyl-isopropylhydrazine) and other hydrazines, the inhibitor is bound to the nonheme portion of the enzyme irreversibly and stoichiometrically (3). Thyroid iodide peroxidase is also irreversibly inhibited in vivo and in vitro by iproniazid (4). Iproniazid has been shown to yield isopropylhydrazine, which then interacts with a carbonyl function in monoamine oxidase (5). Thyroid iodide peroxidase is also thought to interact with isopropylhydrazine moiety and thus might be inhibited by other hydrazine compounds. In this paper, we have studied the mechanism of inhibition of purified thyroid iodide peroxidase by hydrazine (H2N-NH2) and phenylhydrazine (CCH5NH-NH2). We also studied the effect of NSD-1055 (4-bromo-hydroxyphenzyloxyamine) on purified thyroid iodide peroxidase and HRP. Materials and Methods 1. Thyroid iodide peroxidase. Soluble enzyme (iodide peroxidase-tyrosine iodinase) from calf thyroid gland was prepared as described previously (5) and further purified by Sephadex G-200 column chromatography (7). The enzyme assay system contained, in 1.0 ml 0.05 ml enzyme (2.5-5 jug protein), 0.5 ml KrebsRinger phosphate buffer (pH 7.5), 11 nmoles KI, 1 /xCi m I , 1 jumole tyrosine, 1 nmo\e glucose and 0.1 rag glucose oxidase (Worthington Biochemical Received December 8, 1970. Supported in part bv USPHS Grant AM-13,377. 1 Division of Neurology, University of Colorado Medical Center, Denver, Colorado. 2 Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Illinois 60637. Co.). The ratio A4i0 mju/A2so m^ was 0.14. Incubation was carried out at 37 C for 20 min. Following incubation, enzymatically formed iodinated tyrosine was isolated on a cation exchange resin, and radioactivity was measured as described previously (8). Enzymatic activities are expressed as per cent incorporation of I into tyrosine. Inhibition studies by hydrazine, phenylhydrazine and NSD1055 were carried out in 2 different ways: a) Enzyme (5 mg protein) was incubated with each inhibitor (2X1O~M phenylhydrazine, 10~ hydrazine and 2 X10~M NSD-1055) in 0.5 ml of KrebsRinger buffer (0.02M, pH 7.4), at the periods of time indicated in the legend of Fig. 2; 0.1 ml of this solution was diluted 10-fold, and aliquots were used for checking residual enzyme activity. The final concentration of each reagent was 10~M phenylhydrazine, 5X10~M hydrazine and 10~M NSD-1055, respectively. Each reagent at the final concentration noted above has no effect on the enzyme activity if added to the incubation mixture directly without preincubation. b) Inhibitor was added at increasing concentrations to the standard assay mixture and inhibition of enzyme by each inhibitor was examined. 2. Horse-radish peroxidase. Horse-radish peroxidase, purified by electrophoresis, was obtained from Worthington Company (Grade A, RZ 2.8). Purity of the enzyme preparation was checked by ultracentrifugation and disc gel electrophoresis. Horse-radish peroxidase was assayed by a method described in the Worthington Biochemical Corporation Handbook. The rate of decomposition of H2O2 by peroxidase with o-dianisidine as hydrogen donor was determined by measuring the rate of color development at 460 m/n. One unit of peroxidase activity is the amount of enzyme which decomposes 1 jtimole of HaCh/min at 25 C. A 0.003% solution of H2O2 in 0.01M phosphate buffer, pH 6.0, and 1% o-dianisidine in methyl alcohol were prepared fresh daily. To 6.0 ml of the H2O2 solution was added 0.05 ml of o-dianisidine solution; one 2.9 ml aliquot of the mixture was transferred to a test cuvette, and a second aliquot to a control cuvette. At zero time, 0.1 ml of diluted enzyme was added to the test cuvette. Pre-incubation of HRP (0.33 mg/ml) with NSD-