Eur. J. Biochem. 72, 259-263 (1977)
Role of Thyroid Gland on the Peroxidase and Iodinating Enzymes of Submaxillary Gland Tarasankar CHANDRA, Rakhahari DAS, and Asoke G. DATTA Department of Physiology, Indian Institute of Experimental Medicine, Calcutta (Received July 12/0ctober 18, 1976)
The peroxidase (EC 1.11.1.7) and iodinase (EC 1.11.1.8) activities of rat submaxillary gland were found to be increased after thyroidectomy. The enzyme activities were maximal on the seventh day after operation and then decreased slightly. However, the enzyme activities were still more than 100% even 28 days following operation. Administration of thyroxine (10 pg/lOO g body weight) prevented the increase. Puromycin, cycloheximide, and actinomycin D, the inhibitors of protein synthesis, as well as thiouracil partially abolished the increase of activities. These results suggest that thyroxine acts as a regulator of the iodinase and peroxidase enzyme(s) of submaxillary gland.
The formation of iodotyrosines in the thyroid gland [1,2] and the presence of tyrosine iodinase in beef salivary gland [3,4] and rat submaxillary gland [5,6] are now well established. Alexander [7-9] demonstrated the presence of iodide peroxidase in salivary and thyroid extracts and clarified to a great extent the mechanism of formation of iodotyrosines. Recently Hati and Datta [10,11] and Hati et a[. [12] reported the presence of a new type of iodinating enzyme and an active peroxidase (iodide stimulated) in the microsomal preparation of goat submaxillary gland. The presence of the iodinating enzyme(s) in the submaxillary gland is of interest because a special organ, the thyroid, is already present to perform these functions. The possibility may exist that the physiological requirement is satisfied by the submaxillary gland, at least to some extent, when the thyroid gland fails. To test this idea some experiments with thyroidectomized rats were performed. Although we have not been able to demonstrate the formation of thyroxine in the submaxillary gland of either normal or thyroidectomized animals, some interesting results were obtained on the iodinase and peroxidase activities of this gland and are reported in the present communication.
sine, diiodotyrosine, glucose oxidase, tris(hydr0xymethyI)aminomethane, diiodothyronine, triiodothyronine, thyroxine, puromycin, cycloheximide, actinomycin D and and thiouracil. Na13'I was supplied by the Bhabha Atomic Research Centre (Trombay, Bombay). Tyrosine was purchased from E. Merck.
Preparation of Enzymcj Male rats weighing 100- 150 g were purchased from Haffkine Institute, Bombay, and fasted for 6 h before the operation. The animals were supplied with low-iodine diet containing 0.9 calcium gluconate 4 h after the removal of thyroid glands. The thyroidectomized rats were divided into groups for sacrifice on various days after thyroidectomy, as indicated. The submaxillary glands from both normal and thyroidectomized rats were collected and a homogenates were prepared in 0.05 M Tris buffer, pH 7.6, at 0 "C in a glass Potter-Elvehjem homogenizer. The homogenates were centrifuged at 105000 x g for 1 h in a Spinco Model L preparative ultracentrifuge and the supernatant solutions were collected for enzyme assay.
Assay.for Iodinating Activity MATERIALS A N D METHODS
Materials The following materials were obtained from the Sigma Chemical Co. (St Louis, Mo.) : monoiodotyro.-
Er~nzyrnr.~. Peroxidase (EC 1.11.1.7); iodmase (EC 1.11.1.8)
The enzyme was assayed by measuring the incorporation of 1 3 1 T into tyrosine to form monoiodotyrosine and diiodotyrosine or monoiodotyrosine to form diiodotyrosine [ll]. The incubation mixture in the basic system contained 1 ml 105000xg supernatant solution, 300 pmol Tris buffer, pH 7.6,40 pmol KI, 20 pmol glucose, 200 pg glucose oxidase, 20 pCi
260
Thyrodcontrolled Iodination in Submaxillary Gland
carrier-free NaI3' 1, 4 pmol tyrosine or monoiodotyrosine in a final volume of 3.0 ml. The procedure was otherwise as described earlier [13] and in these assays glucose and glucose oxidase were used as H202 generator instead of C u 2 + ,which was used earlier in Kirkwood's laboratory [S,6]. A.ssuy,fbr cr-Amyluse Activity The method of Street and Close [14] was followed for the determination of x-amylase activity. A s s u y , f o ~Pero.uidase Activity The peroxidase activity of the enzyme preparation was assayed according to Bal et ul. [lS] by measuring the change of absorbance at 460 nm in a Zeiss spectrophotometer PMQ I1 using glass cuvettes of 1 cm light path. The reaction mixture contained the following reagents in a final volume of 3 ml : 1SO pmol sodium phosphate buffer, pH 6.0, 2 pmol o-dianisidine dissolved in methanol, 20 pmol H202 and 0.5ml soluble supernatant containing 1.S- 2 mg protein.
thyroidectomy (Table 1). There was no appreciable change in either the weight or the protein content of the submaxillary glands after thyroidectomy and very little change in the protein content of the soluble supernatant fraction (IOSOOO x g ) used for measurement of enzyme activities. The enzyme activity in the thyroidectomized animals decreased slowly after the 7th day to about twice the control levels and then remained relatively constant. Examination of the operated animals at the time of sacrifice did not indicate the presence of any residual thyroid tissue. Experiments were also performed under similar conditions with tyrosine as substrate with similar results to those obtained with monoiodotyrosine as substrate.
Table I . Ioriinasr activity of suhmu riliusy glu~7d of' tiornirrl r r r d ~li?,roidectotni~rd sots All values are means f S.E. from seven animals and inonoiodotyrosine was used as substrate in these experiments. The incorporation of radioactive iodide into diiodotyrosine was measured by the procedure described earlier [I 31. P values calculated using Student's f-test for comparison between normal and thyroidectomized animals. n.s. = not signifcant Days
P
Diiodotyrosine formed
Protein Estimation normal
Protein was measured according to the method of Lowry et a/. [I61 using bovine albumin as the standard.
thyroidectomized ~
nmoljmg protein 26.0 5 3.10 98.0 k 6.63 99.0 f 6.71 74.2 f 4.51 59 64
25.6 & 1.60 27.6 & 0.79 25.0 f 2. I 5 26.2 f 2.30 27 26
Effect of Thyroidectomy on the lodinuse Activity of Suhmu.uillury G1und.v
0 4 7 14 21 * 28
Monoiodotyrosine was iodinated by the soluble supernatant fraction of submaxillary gland and this activity was increased by about 280 % following
Data presented in these two experiments are taken from a single experiment but the results were verified in two other experiments.
RESULTS
I1.b.
< 0.00 I
Role of Thyroid Gland on the Peroxidase and Iodinating Enzymes of Submaxillary Gland Tarasankar CHANDRA, Rakhahari DAS, and Asoke G. DATTA Department of Physiology, Indian Institute of Experimental Medicine, Calcutta (Received July 12/0ctober 18, 1976)
The peroxidase (EC 1.11.1.7) and iodinase (EC 1.11.1.8) activities of rat submaxillary gland were found to be increased after thyroidectomy. The enzyme activities were maximal on the seventh day after operation and then decreased slightly. However, the enzyme activities were still more than 100% even 28 days following operation. Administration of thyroxine (10 pg/lOO g body weight) prevented the increase. Puromycin, cycloheximide, and actinomycin D, the inhibitors of protein synthesis, as well as thiouracil partially abolished the increase of activities. These results suggest that thyroxine acts as a regulator of the iodinase and peroxidase enzyme(s) of submaxillary gland.
The formation of iodotyrosines in the thyroid gland [1,2] and the presence of tyrosine iodinase in beef salivary gland [3,4] and rat submaxillary gland [5,6] are now well established. Alexander [7-9] demonstrated the presence of iodide peroxidase in salivary and thyroid extracts and clarified to a great extent the mechanism of formation of iodotyrosines. Recently Hati and Datta [10,11] and Hati et a[. [12] reported the presence of a new type of iodinating enzyme and an active peroxidase (iodide stimulated) in the microsomal preparation of goat submaxillary gland. The presence of the iodinating enzyme(s) in the submaxillary gland is of interest because a special organ, the thyroid, is already present to perform these functions. The possibility may exist that the physiological requirement is satisfied by the submaxillary gland, at least to some extent, when the thyroid gland fails. To test this idea some experiments with thyroidectomized rats were performed. Although we have not been able to demonstrate the formation of thyroxine in the submaxillary gland of either normal or thyroidectomized animals, some interesting results were obtained on the iodinase and peroxidase activities of this gland and are reported in the present communication.
sine, diiodotyrosine, glucose oxidase, tris(hydr0xymethyI)aminomethane, diiodothyronine, triiodothyronine, thyroxine, puromycin, cycloheximide, actinomycin D and and thiouracil. Na13'I was supplied by the Bhabha Atomic Research Centre (Trombay, Bombay). Tyrosine was purchased from E. Merck.
Preparation of Enzymcj Male rats weighing 100- 150 g were purchased from Haffkine Institute, Bombay, and fasted for 6 h before the operation. The animals were supplied with low-iodine diet containing 0.9 calcium gluconate 4 h after the removal of thyroid glands. The thyroidectomized rats were divided into groups for sacrifice on various days after thyroidectomy, as indicated. The submaxillary glands from both normal and thyroidectomized rats were collected and a homogenates were prepared in 0.05 M Tris buffer, pH 7.6, at 0 "C in a glass Potter-Elvehjem homogenizer. The homogenates were centrifuged at 105000 x g for 1 h in a Spinco Model L preparative ultracentrifuge and the supernatant solutions were collected for enzyme assay.
Assay.for Iodinating Activity MATERIALS A N D METHODS
Materials The following materials were obtained from the Sigma Chemical Co. (St Louis, Mo.) : monoiodotyro.-
Er~nzyrnr.~. Peroxidase (EC 1.11.1.7); iodmase (EC 1.11.1.8)
The enzyme was assayed by measuring the incorporation of 1 3 1 T into tyrosine to form monoiodotyrosine and diiodotyrosine or monoiodotyrosine to form diiodotyrosine [ll]. The incubation mixture in the basic system contained 1 ml 105000xg supernatant solution, 300 pmol Tris buffer, pH 7.6,40 pmol KI, 20 pmol glucose, 200 pg glucose oxidase, 20 pCi
260
Thyrodcontrolled Iodination in Submaxillary Gland
carrier-free NaI3' 1, 4 pmol tyrosine or monoiodotyrosine in a final volume of 3.0 ml. The procedure was otherwise as described earlier [13] and in these assays glucose and glucose oxidase were used as H202 generator instead of C u 2 + ,which was used earlier in Kirkwood's laboratory [S,6]. A.ssuy,fbr cr-Amyluse Activity The method of Street and Close [14] was followed for the determination of x-amylase activity. A s s u y , f o ~Pero.uidase Activity The peroxidase activity of the enzyme preparation was assayed according to Bal et ul. [lS] by measuring the change of absorbance at 460 nm in a Zeiss spectrophotometer PMQ I1 using glass cuvettes of 1 cm light path. The reaction mixture contained the following reagents in a final volume of 3 ml : 1SO pmol sodium phosphate buffer, pH 6.0, 2 pmol o-dianisidine dissolved in methanol, 20 pmol H202 and 0.5ml soluble supernatant containing 1.S- 2 mg protein.
thyroidectomy (Table 1). There was no appreciable change in either the weight or the protein content of the submaxillary glands after thyroidectomy and very little change in the protein content of the soluble supernatant fraction (IOSOOO x g ) used for measurement of enzyme activities. The enzyme activity in the thyroidectomized animals decreased slowly after the 7th day to about twice the control levels and then remained relatively constant. Examination of the operated animals at the time of sacrifice did not indicate the presence of any residual thyroid tissue. Experiments were also performed under similar conditions with tyrosine as substrate with similar results to those obtained with monoiodotyrosine as substrate.
Table I . Ioriinasr activity of suhmu riliusy glu~7d of' tiornirrl r r r d ~li?,roidectotni~rd sots All values are means f S.E. from seven animals and inonoiodotyrosine was used as substrate in these experiments. The incorporation of radioactive iodide into diiodotyrosine was measured by the procedure described earlier [I 31. P values calculated using Student's f-test for comparison between normal and thyroidectomized animals. n.s. = not signifcant Days
P
Diiodotyrosine formed
Protein Estimation normal
Protein was measured according to the method of Lowry et a/. [I61 using bovine albumin as the standard.
thyroidectomized ~
nmoljmg protein 26.0 5 3.10 98.0 k 6.63 99.0 f 6.71 74.2 f 4.51 59 64
25.6 & 1.60 27.6 & 0.79 25.0 f 2. I 5 26.2 f 2.30 27 26
Effect of Thyroidectomy on the lodinuse Activity of Suhmu.uillury G1und.v
0 4 7 14 21 * 28
Monoiodotyrosine was iodinated by the soluble supernatant fraction of submaxillary gland and this activity was increased by about 280 % following
Data presented in these two experiments are taken from a single experiment but the results were verified in two other experiments.
RESULTS
I1.b.
< 0.00 I
