Pergamon Press
Life Sciences, Vol . 21, pp . 1143-1148 Printed in the U.S .A .
STIMULATION OF ORNITHINE DECARBOXYLASE SYNTHESIS IN THE RAT THYROID Steven J . Scheinman, Gerard N. Burrow*,
Theoharis C . Theoharides,
and Zoe N . Canellakis + Department of Internal Medicine
(SJS,GNB) ; Department of Pharmacology (TCT,ZNC)
Yale University School of Medicine, New Haven, *Present address :
Connecticut
06510
University of Toronto, Toronto, Ontario, Canada
+ To whom reprint requests should be submitted. (Received in final form September 6, 1977) Summary High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme " monospecific antibody complex as the immunizing antigen . This new antiserum preparation was successfully labeled with ~25I and was found to retain its specific immune prop erties . Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone 125 I-Labeled antibody and methyl xanthine in rat thyroids in vitro . incorporation into the enzyme antiboTy- complex after induction in vitro showed an increase which paralleled the increase in enzymatic acvity and thus suggested _de novo synthesis of thyroid enzyme protein . L-Ornithine decarboxylase (EC 4 .1 .1 .17 ; L-ornithine carboxy-lyase), the rate-limiting enzyme in the biosynthesis of the polyamines spermidine and spermine in animal tissues (1-3), has been induced by various agents which affect growth processes and particularly by various steroid and polypeptide hormones in their target organs ; the role of these hormones and certain other aspects of ornithine decarboxylase induction have been reviewed recently (4-6) . Recent reports have shown that exogenous TSH and TSH-releasing hormone administration produce elevated levels of thyroid ornithine decarboxylase activity (7-11) . This induction appears to be specific for TSH (9) and suggestive evidence indicates that prostaglandin synthesis might be involved (9, 10) . In order to further characterize TSH-induction of ornithine decarboxylase it seemed appropriate to correlate increases in enzymatic activity with increases in specific immunoprecipitable protein. High titer antiserum specific to rat hepatic ornithine decarboxylase was prepared in rabbits using hepatic enzyme-monospecific antibody complex as the antigen. The antibody used to obtain the immunoprecipitate which has been em ployed as antigen has been described previously and defined as monospecific according to the criteria of immunodiffusion, immunoelectrophoresis, and resolution of the antigen antibody complex using SDS gel electrophoresis (12) . The immunologic properties of this new preparation of antiserum were similar to those reported (12,13) for the previously described monospecific antiserum. The anti1143
114 4
Biosynthesis of Ornithine Decarboxylase
Vol . 21, No . 8, 1977
serum preparation was labeled with 1251 by lactoperoxidase catalyzed iodination (14,15) and the iodinated antiserum was subsequently used to precipitate induced and non-induced thyroid ornithine decarboxylase . Results thus obtained suggest de novo synthesis of thyroid ornithine decarboxylase protein . Experimental Procedure DL-1- 14 C ornithine Compounds were obtained from the following sources : (5 .8 mCi/mmol), 125, sodium carrier-free solution in 0 .1 N NaOH, and aquasol, New England Nuclear Corp . ; ammonium sulfate (extra-fine enzyme grade), Schwarz/ Mann ; complete Freund's adjuvant, Difco ; thyroid stimulating hormone (TSH-NIHB6), kindly supplied by the National Institutes of Health) ; 3-isobutyl-l-methyl xanthine (IBMX), Aldrich Chemical Co . ; lactoperoxidase and glucose oxidase, Sigma Chemical Co . ; all other reagents were of'analytical grade . Antiserum was prepared by injection of enzyme- monospecific antibody complex . For this purpose, immunoprecipitates from six antigen/antibody reactions (1200 u.e ./2 .0 mg antibody) were pooled, emulsified in complete Freund's adju vant and injected into two female rabbits . This procedure was repeated four weeks later with half the amount of immunizing material . Both the hepatic ornithine decarboxylase and the monospecific antiserum used to obtain the enzymeantibody complexes were partially purified by ammonium sulfate fractionation (12) . Control antisera obtained from rabbits receiving injection of Freund's adjuvant alone were handled in a similar manner . All sera were stored at -20 ° . Iodination of the antibody preparation was carried out using lactoperoxidase and glucose oxidase according to the method of Tsai et al . (15) . The iodinated antibody used in routine experiments contained approximately 1 .5 x 106 cpm/mg . Thyroids from male Sprague-Dawley rats (200 g) were pooled and distributed in containers with 4 lobes per flask . The lobes were equilibrated in 5 ml Krebs bicarbonate buffer for 15 min . at 37 ° and then incubated for 1-7 hours subse quent to the addition of 50 mU/ml TSH and 0 .5 mM IBMX . Incubation was carried out in a Dubnoff shaker (in an atmosphere of 95% 02 and 5% C02) . Bacterial counts of the incubation medium which were performed at intervals up to seven hours showed no contamination . For assay of ornithine decarboxylase, 4 lobes, representing 40-60 mg of thyroid tissue, were homogenized in 1 .4 ml of Krebs bicarbonate buffer containing 0 .25 M sucrose, 0 .5 mM EDTA and 10 mM 2-mercaptoethanol . Homogenization was carried out with a Potter-Elvehjem homogenizer with a Teflon pestle ; after centrifugation at 100,000 x g for 30 min ., the supernatant fluid was used for both enzymatic assay and immunoprecipitation studies . Immunoprecipitin assays were performed as described previously (12), with the exception that the entire reaction was carried out in a total volume of 0 .5 ml which overlayed a cushion of 0 .025 ml 2 .0 M sucrose . Enzyme activity was assayed by a slight modification of the method described by Chen et al . (16) . Protein was measured by the procedure of Lowry et al . (17) . One enzymeunit (u .e .) is defined as that amount of enzyme which catalyzes the production of 1 pmol of C02 from ornithine in 60 min . under standard assay conditions . Results and Discussion We have shown that an immunoprecipitate itself is a convenient way of producing high titer antiserum to the enzyme protein contained in the immunizing enzyme-antibody complex . This procedure of using an antigen-antibody complex as the immunogen was used to obtain rabbit antiserum specific for those rat immunoglobulins which had been precipitated with Nippostrongylus brasiliensis extracts
Vol . 21, No . 8, 1977
Biosynthesis of Ornithine Decarboxylase
1145
(18) . This technique has also been successfully used for the production of high titer antisera to murine glucuronidase and mouse liver glycerol-3-phosphate dehydrogenase .1 Seventy-five milligrams of specific antiserum (0 .5 pmol, assuming an average immunoglobulin molecular weight of 150,000) were iodinated . Quantitative consumption of the iodide resulted in the incorporation of approximately 2 .0 atoms of 125I/mol of 6-globulin. This extent of iodination did not change the immunological specificity of the antibody (see Table II), a finding which is in agreement with previous studies (12) . Enzyme from rat thyroid glands induced by a mixture of TSH and IBMX was titrated with antiserum. Residual enzyme activity was measured in the resultant As the amount of specific antiserum added increased the supernatant fluids . ornithine decarboxylase activity decreased to basal levels . Quantitative precipitation of ornithine decarboxylase was achieved when 0.75 mg of antibody was added to 1250 u.e . of ornithine decarboxylase . Addition of non-imune (normal) sera to similar samples of induced enzyme activity resulted in no decrease in ornithine decarboxylase activity . A typical experiment depicting the time course of induction of rat thyroid ornithine decarboxylase as a mixture of TSH and MIX is presented in Table I . TABLE I Time Course of Ornithine Decarboxylase Induction by Thyroid Stimulating Hormone and Methyl Xanthine in Rat Thyroid Glands In Vitro . TIME 0 1 2 3 4 5 6
ENZYME ACTIVITY (pmol/mg/hr) 55 + 22 45 + 18 132+2 265 + 16 568 + 38 1000 _+ 20 313 + 62
The thyroid lobes were handled as described under "Experimental Procedure" and the results represent the average of three separate determinations .
Generally, the mixture of TSH and IBMX caused a 10 to 20-fold enhancement of enzyme activity which reached a peak at about 5-6 hours and declined to base levels by 7 hours . Specific enzyme precipitation by iodinated antibody was performed on both induced and non-induced thyroid extracts taken after 5 hours of incubation under standard conditions (Table II) . TSH stimulation was reflected by an 1 The technique using goat anti-beef liver glucuronidase-mouse liver glucuronidase complex for the production of high titer antisera to marine glucuronidase was communicated by Dr . Roger E . Ganschow, Institute for Developmental Research, Cincinnati, Ohio, and has also been successfully used by K. Paigen, Roswell Park Memorial Institute, Buffalo, New York . It has also been used to obtain antibody to mouse liver glycerol-phosphate dehydrogenase by Lana S . Rittman, Department of Pharmacology, Yale University, New Haven, Connecticut.
114 6
Biosynthesis of Ornithine Decarboxylase
Vol . 21, No . 8, 1977
increase in immunoprecipitable ornithine decarboxylase protein as indicated by the immunoprecipitate of those samples reacted with radio-iodinated antiserum. The antibody used in the current study was similar to an earlier antibody whose specificity was demonstrated by a single precipitin line with immunodiffusion, immunoelectrophoresis and resolution of the antigen antibody complex by SDS gel electrophoresis .
TABLE II Thyroid 125,-Antibody Immunoprecipitable Ornithine Decarboxylase Residual Enzyme Activity (pmol/mg/hr) Induced enzyme + control antibody Induced enzyme + specific 125,-antibody Induced enzyme + specific antibody
1129 + 54 85 + 15 79 +' 23 Total cpm 1 25, Precipitated
125 Non-induced enzyme + specific I-antibody
15,400 ± 3,000
125,-antibody Induced enzyme + specific
31,800 + 1,990
P
Rat thyroid glands were assayed 5 hours after induction as described under "Experimental Procedure." Supernatant samples of homogenized tissue (1 .4 mg protein per ml) of either non-induced (15 u.e .) or induced (1250 u.e .) ornithine decarboxylase were precipitated with 0 .75 mg of iodinated ornithine decarboxylase antiserum. In addition, similar samples of induced ornithine decarboxylase were precipitated with 0.75 mg of either non-immune (normal) serum or specific The results represent the average of four separate determinations . antiserum.
In this report we have used iodinated antiserum to demonstrate that the rise in thyroid ornithine decarboxylase activity following in vitro induction with TSH and IBMX is accompanied by increased amounts of immuno-precipitates protein which reflects de novo synthesis. Use of 125 I-labeled antiserum, as employed in this study,has been shown to provide a true measure of ornithine decarboxylase protein (12) . This is the first report that TSH is able to induce a specific protein . Although the mechanism of induction remains to be elucidated, there is evidence that thyroid cyclic AMP dependent protein kinase might be implicated at the transcriptional level (19-23) ; for a review see Jungmann and Russell (24) . The present results are also in agreement with reports that cycloheximide administration prevents the induction of thyroid enzyme activity (7,9), which is an indication that protein synthesis is necessary. In addition, it has also been reported that the increase in thyroid enzyme activity following TSH administration is not a consequence of a possible decrease in the activity of an orniThe existence of a non-crossthine decarboxylase inhibitor induced by TSH (9) . reacting inactive precursor form of ornithine decarboxylase has not been excluded . However, the transformation of a cross-reacting enzymatically inactive precursor form of hepatic ornithine decarboxylase as a source of induced enzyme activity These has been shown to be unlikely according to previous studies (13,25,26) . studies have also shown that induction of ornithine decarboxylase in regenera-
Vol . 21, No . 8, 1977
Biosynthesis of Ornithine Decarboxylase
114 7
ting rat liver and cultured hepatoma cells results in _de novo enzyme synthesis (25-27) . The current study shows that the maximum of 1251 present in the antiserum which can be precipitated by the sample containing the induced enzyme represents approximately 1% of all cpm 1251 added. It has already been reported that 4% of immunoprecipitable cpm 12 I represent approximately 0.4% of regenerating soluble rat liver protein (12) ; consequently, 1% cpm 1251 might indicate that ornithine decarboxylase protein represents approximately O .lT of induced soluble rat thyroid protein. This measure of nascent ornithine decarboxylase synthesis is in close agreement with studies of Obenrader and Prouty who reported that newly synthesized ornithine decarboxylase following thioacetamide induction is 0 .16% of soluble rat liver protein (27) . Our antiserum, prepared against liver ornithine decarboxylase, exhibits cross-specificity with the thyroid enzyme . In agreement with these results, different preparations of antiserum to liver ornithine decarboxylase were shown to cross-react with ornithine decarboxylase from rat kidney, testes, thymus, spleen (26), and prostate (26,27) . References H. TABOR, S . M. ROSENTHAL, and C . W . TABOR, J. Biol . Chem . 233, 907-914 (1958) . 2 . A. E. PEGG, and H. G. WILLIAMS-ASHMAN, Biochem. J. 108, 533-539 (1968) . 3. A. RAINA and J. ANNE : Acta Chem . Scand- -n.3379 (1968) . 75-2 4 . H. G. WILLIAMS-ASHMAN J . ANNE, G. L. COPPOC, M. E . GERSCH, and S . A. SCHENONE, Adv. Enzyme Re l . 10, 225-245 (1972) . 5. D. R. MORRIS an R. H. FILLINGAME, Annu . Rev . Biochem. _ 43, 303-325 (1974) . 6 . A. RAINA and J. ANNE, Med. Biol . 5_3_,1_2_1-_147__T1975) . 7. S . MATSUZAKI and M. SUZUKI Endocrinol . Japan 21, 529-537 (1974) . 8. S . MATSUZAKI and M. SUZUKI, En ocrinol . Japan 22, 339-345 (1975) . 9. R. RICHMAN, S. PARK, M. AKBAR, S . YU, and G. BURKE, Endocrinology _96, 1403-1412 (1975) . P . R. ZUSMAN and G. N. BURROW, Endocrinology 97, 1089-1095 (1975) . 10 . 11 . S . YU, Y . FRIEDMAN, R. RICHMAN, and G. BURKE, J. Clin . Invest . _57, 745-755 (1976) . 12 . T. C. THEOHARIDES and Z . N. CANELLAKIS, J . Biol . Chem . 251, 1781-1784 (1976) . 13 . T. C. THEOHARIDES and Z. N. CANELLAKIS, Folia Bioch. et Graeca _13, 11-23 (1976) . 14 . M. MORRISON, G. S. BAYSE, and R. G . WEBSTER, Immunochemistry _8, 289-297 (1971) . 15 . C. M. TSAI, C. C . HUANG, and E . S. CANELLAKIS, Biochim. Biophys . Acta _332, 47-58 (1973) . 16 . K. CHEN, J. HELLER, and E . S . CANELLAKIS, Biochem. Biophys . Res . Commun . 68, 401-408 (1976) . 17 . H. LOWRY, N. J. ROSEBROUGH, A. L . FARR, and R . J . RANDALL, J . Biol . Chem . 193, 265-275 (1951) . K. J. BLOCK and R. J. M. WILSON, J . Immunol . 100, 629-636 (1968) . 18 . 19 . C . V. BYUS and D . H . RUSSELL, Science 7, 650-652 (1975) . 20 . S . W. SPAULDING and G. N. BURROW,B ohem. Biophys . Res . Commun . _59, 386391 (1975) . 21 . M. COSTA, E . R. COSTA, C-A . MANEN, I . G. SIPES, and D. H. RUSSELL, Molec . Pharmacol . 12 : 871-878 (1976) . 22 . C. V. BYUS, A . HEDGE, and D. H . RUSSELL, Biochim. Biophys . Acta 498, 3945 (1977) . 23 . S . J. SCHEINMAN and G. N. BURROW, Endocrinology, in press . 0, 1787-1798 (1977) . 24 . - R. A. JUNGMANN and D. H. RUSSELL, Life ci . 25 . Z .N . CANELLAKIS and T.C . THEOHARIDE , Chem . 251, 4436-4441 (1976) . 26 . E. HbLTTX, Biochim. Biophys . Acta 399, 20-427 (1975) _ . 27 . M. F. OBENRADER and W. F. PROUTY, . J Biol . Chem . 252, 2866-2872 (1977) . 1.
7
Biol .
0.
G.
sol.
Life Sciences, Vol . 21, pp . 1143-1148 Printed in the U.S .A .
STIMULATION OF ORNITHINE DECARBOXYLASE SYNTHESIS IN THE RAT THYROID Steven J . Scheinman, Gerard N. Burrow*,
Theoharis C . Theoharides,
and Zoe N . Canellakis + Department of Internal Medicine
(SJS,GNB) ; Department of Pharmacology (TCT,ZNC)
Yale University School of Medicine, New Haven, *Present address :
Connecticut
06510
University of Toronto, Toronto, Ontario, Canada
+ To whom reprint requests should be submitted. (Received in final form September 6, 1977) Summary High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme " monospecific antibody complex as the immunizing antigen . This new antiserum preparation was successfully labeled with ~25I and was found to retain its specific immune prop erties . Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone 125 I-Labeled antibody and methyl xanthine in rat thyroids in vitro . incorporation into the enzyme antiboTy- complex after induction in vitro showed an increase which paralleled the increase in enzymatic acvity and thus suggested _de novo synthesis of thyroid enzyme protein . L-Ornithine decarboxylase (EC 4 .1 .1 .17 ; L-ornithine carboxy-lyase), the rate-limiting enzyme in the biosynthesis of the polyamines spermidine and spermine in animal tissues (1-3), has been induced by various agents which affect growth processes and particularly by various steroid and polypeptide hormones in their target organs ; the role of these hormones and certain other aspects of ornithine decarboxylase induction have been reviewed recently (4-6) . Recent reports have shown that exogenous TSH and TSH-releasing hormone administration produce elevated levels of thyroid ornithine decarboxylase activity (7-11) . This induction appears to be specific for TSH (9) and suggestive evidence indicates that prostaglandin synthesis might be involved (9, 10) . In order to further characterize TSH-induction of ornithine decarboxylase it seemed appropriate to correlate increases in enzymatic activity with increases in specific immunoprecipitable protein. High titer antiserum specific to rat hepatic ornithine decarboxylase was prepared in rabbits using hepatic enzyme-monospecific antibody complex as the antigen. The antibody used to obtain the immunoprecipitate which has been em ployed as antigen has been described previously and defined as monospecific according to the criteria of immunodiffusion, immunoelectrophoresis, and resolution of the antigen antibody complex using SDS gel electrophoresis (12) . The immunologic properties of this new preparation of antiserum were similar to those reported (12,13) for the previously described monospecific antiserum. The anti1143
114 4
Biosynthesis of Ornithine Decarboxylase
Vol . 21, No . 8, 1977
serum preparation was labeled with 1251 by lactoperoxidase catalyzed iodination (14,15) and the iodinated antiserum was subsequently used to precipitate induced and non-induced thyroid ornithine decarboxylase . Results thus obtained suggest de novo synthesis of thyroid ornithine decarboxylase protein . Experimental Procedure DL-1- 14 C ornithine Compounds were obtained from the following sources : (5 .8 mCi/mmol), 125, sodium carrier-free solution in 0 .1 N NaOH, and aquasol, New England Nuclear Corp . ; ammonium sulfate (extra-fine enzyme grade), Schwarz/ Mann ; complete Freund's adjuvant, Difco ; thyroid stimulating hormone (TSH-NIHB6), kindly supplied by the National Institutes of Health) ; 3-isobutyl-l-methyl xanthine (IBMX), Aldrich Chemical Co . ; lactoperoxidase and glucose oxidase, Sigma Chemical Co . ; all other reagents were of'analytical grade . Antiserum was prepared by injection of enzyme- monospecific antibody complex . For this purpose, immunoprecipitates from six antigen/antibody reactions (1200 u.e ./2 .0 mg antibody) were pooled, emulsified in complete Freund's adju vant and injected into two female rabbits . This procedure was repeated four weeks later with half the amount of immunizing material . Both the hepatic ornithine decarboxylase and the monospecific antiserum used to obtain the enzymeantibody complexes were partially purified by ammonium sulfate fractionation (12) . Control antisera obtained from rabbits receiving injection of Freund's adjuvant alone were handled in a similar manner . All sera were stored at -20 ° . Iodination of the antibody preparation was carried out using lactoperoxidase and glucose oxidase according to the method of Tsai et al . (15) . The iodinated antibody used in routine experiments contained approximately 1 .5 x 106 cpm/mg . Thyroids from male Sprague-Dawley rats (200 g) were pooled and distributed in containers with 4 lobes per flask . The lobes were equilibrated in 5 ml Krebs bicarbonate buffer for 15 min . at 37 ° and then incubated for 1-7 hours subse quent to the addition of 50 mU/ml TSH and 0 .5 mM IBMX . Incubation was carried out in a Dubnoff shaker (in an atmosphere of 95% 02 and 5% C02) . Bacterial counts of the incubation medium which were performed at intervals up to seven hours showed no contamination . For assay of ornithine decarboxylase, 4 lobes, representing 40-60 mg of thyroid tissue, were homogenized in 1 .4 ml of Krebs bicarbonate buffer containing 0 .25 M sucrose, 0 .5 mM EDTA and 10 mM 2-mercaptoethanol . Homogenization was carried out with a Potter-Elvehjem homogenizer with a Teflon pestle ; after centrifugation at 100,000 x g for 30 min ., the supernatant fluid was used for both enzymatic assay and immunoprecipitation studies . Immunoprecipitin assays were performed as described previously (12), with the exception that the entire reaction was carried out in a total volume of 0 .5 ml which overlayed a cushion of 0 .025 ml 2 .0 M sucrose . Enzyme activity was assayed by a slight modification of the method described by Chen et al . (16) . Protein was measured by the procedure of Lowry et al . (17) . One enzymeunit (u .e .) is defined as that amount of enzyme which catalyzes the production of 1 pmol of C02 from ornithine in 60 min . under standard assay conditions . Results and Discussion We have shown that an immunoprecipitate itself is a convenient way of producing high titer antiserum to the enzyme protein contained in the immunizing enzyme-antibody complex . This procedure of using an antigen-antibody complex as the immunogen was used to obtain rabbit antiserum specific for those rat immunoglobulins which had been precipitated with Nippostrongylus brasiliensis extracts
Vol . 21, No . 8, 1977
Biosynthesis of Ornithine Decarboxylase
1145
(18) . This technique has also been successfully used for the production of high titer antisera to murine glucuronidase and mouse liver glycerol-3-phosphate dehydrogenase .1 Seventy-five milligrams of specific antiserum (0 .5 pmol, assuming an average immunoglobulin molecular weight of 150,000) were iodinated . Quantitative consumption of the iodide resulted in the incorporation of approximately 2 .0 atoms of 125I/mol of 6-globulin. This extent of iodination did not change the immunological specificity of the antibody (see Table II), a finding which is in agreement with previous studies (12) . Enzyme from rat thyroid glands induced by a mixture of TSH and IBMX was titrated with antiserum. Residual enzyme activity was measured in the resultant As the amount of specific antiserum added increased the supernatant fluids . ornithine decarboxylase activity decreased to basal levels . Quantitative precipitation of ornithine decarboxylase was achieved when 0.75 mg of antibody was added to 1250 u.e . of ornithine decarboxylase . Addition of non-imune (normal) sera to similar samples of induced enzyme activity resulted in no decrease in ornithine decarboxylase activity . A typical experiment depicting the time course of induction of rat thyroid ornithine decarboxylase as a mixture of TSH and MIX is presented in Table I . TABLE I Time Course of Ornithine Decarboxylase Induction by Thyroid Stimulating Hormone and Methyl Xanthine in Rat Thyroid Glands In Vitro . TIME 0 1 2 3 4 5 6
ENZYME ACTIVITY (pmol/mg/hr) 55 + 22 45 + 18 132+2 265 + 16 568 + 38 1000 _+ 20 313 + 62
The thyroid lobes were handled as described under "Experimental Procedure" and the results represent the average of three separate determinations .
Generally, the mixture of TSH and IBMX caused a 10 to 20-fold enhancement of enzyme activity which reached a peak at about 5-6 hours and declined to base levels by 7 hours . Specific enzyme precipitation by iodinated antibody was performed on both induced and non-induced thyroid extracts taken after 5 hours of incubation under standard conditions (Table II) . TSH stimulation was reflected by an 1 The technique using goat anti-beef liver glucuronidase-mouse liver glucuronidase complex for the production of high titer antisera to marine glucuronidase was communicated by Dr . Roger E . Ganschow, Institute for Developmental Research, Cincinnati, Ohio, and has also been successfully used by K. Paigen, Roswell Park Memorial Institute, Buffalo, New York . It has also been used to obtain antibody to mouse liver glycerol-phosphate dehydrogenase by Lana S . Rittman, Department of Pharmacology, Yale University, New Haven, Connecticut.
114 6
Biosynthesis of Ornithine Decarboxylase
Vol . 21, No . 8, 1977
increase in immunoprecipitable ornithine decarboxylase protein as indicated by the immunoprecipitate of those samples reacted with radio-iodinated antiserum. The antibody used in the current study was similar to an earlier antibody whose specificity was demonstrated by a single precipitin line with immunodiffusion, immunoelectrophoresis and resolution of the antigen antibody complex by SDS gel electrophoresis .
TABLE II Thyroid 125,-Antibody Immunoprecipitable Ornithine Decarboxylase Residual Enzyme Activity (pmol/mg/hr) Induced enzyme + control antibody Induced enzyme + specific 125,-antibody Induced enzyme + specific antibody
1129 + 54 85 + 15 79 +' 23 Total cpm 1 25, Precipitated
125 Non-induced enzyme + specific I-antibody
15,400 ± 3,000
125,-antibody Induced enzyme + specific
31,800 + 1,990
P
Rat thyroid glands were assayed 5 hours after induction as described under "Experimental Procedure." Supernatant samples of homogenized tissue (1 .4 mg protein per ml) of either non-induced (15 u.e .) or induced (1250 u.e .) ornithine decarboxylase were precipitated with 0 .75 mg of iodinated ornithine decarboxylase antiserum. In addition, similar samples of induced ornithine decarboxylase were precipitated with 0.75 mg of either non-immune (normal) serum or specific The results represent the average of four separate determinations . antiserum.
In this report we have used iodinated antiserum to demonstrate that the rise in thyroid ornithine decarboxylase activity following in vitro induction with TSH and IBMX is accompanied by increased amounts of immuno-precipitates protein which reflects de novo synthesis. Use of 125 I-labeled antiserum, as employed in this study,has been shown to provide a true measure of ornithine decarboxylase protein (12) . This is the first report that TSH is able to induce a specific protein . Although the mechanism of induction remains to be elucidated, there is evidence that thyroid cyclic AMP dependent protein kinase might be implicated at the transcriptional level (19-23) ; for a review see Jungmann and Russell (24) . The present results are also in agreement with reports that cycloheximide administration prevents the induction of thyroid enzyme activity (7,9), which is an indication that protein synthesis is necessary. In addition, it has also been reported that the increase in thyroid enzyme activity following TSH administration is not a consequence of a possible decrease in the activity of an orniThe existence of a non-crossthine decarboxylase inhibitor induced by TSH (9) . reacting inactive precursor form of ornithine decarboxylase has not been excluded . However, the transformation of a cross-reacting enzymatically inactive precursor form of hepatic ornithine decarboxylase as a source of induced enzyme activity These has been shown to be unlikely according to previous studies (13,25,26) . studies have also shown that induction of ornithine decarboxylase in regenera-
Vol . 21, No . 8, 1977
Biosynthesis of Ornithine Decarboxylase
114 7
ting rat liver and cultured hepatoma cells results in _de novo enzyme synthesis (25-27) . The current study shows that the maximum of 1251 present in the antiserum which can be precipitated by the sample containing the induced enzyme represents approximately 1% of all cpm 1251 added. It has already been reported that 4% of immunoprecipitable cpm 12 I represent approximately 0.4% of regenerating soluble rat liver protein (12) ; consequently, 1% cpm 1251 might indicate that ornithine decarboxylase protein represents approximately O .lT of induced soluble rat thyroid protein. This measure of nascent ornithine decarboxylase synthesis is in close agreement with studies of Obenrader and Prouty who reported that newly synthesized ornithine decarboxylase following thioacetamide induction is 0 .16% of soluble rat liver protein (27) . Our antiserum, prepared against liver ornithine decarboxylase, exhibits cross-specificity with the thyroid enzyme . In agreement with these results, different preparations of antiserum to liver ornithine decarboxylase were shown to cross-react with ornithine decarboxylase from rat kidney, testes, thymus, spleen (26), and prostate (26,27) . References H. TABOR, S . M. ROSENTHAL, and C . W . TABOR, J. Biol . Chem . 233, 907-914 (1958) . 2 . A. E. PEGG, and H. G. WILLIAMS-ASHMAN, Biochem. J. 108, 533-539 (1968) . 3. A. RAINA and J. ANNE : Acta Chem . Scand- -n.3379 (1968) . 75-2 4 . H. G. WILLIAMS-ASHMAN J . ANNE, G. L. COPPOC, M. E . GERSCH, and S . A. SCHENONE, Adv. Enzyme Re l . 10, 225-245 (1972) . 5. D. R. MORRIS an R. H. FILLINGAME, Annu . Rev . Biochem. _ 43, 303-325 (1974) . 6 . A. RAINA and J. ANNE, Med. Biol . 5_3_,1_2_1-_147__T1975) . 7. S . MATSUZAKI and M. SUZUKI Endocrinol . Japan 21, 529-537 (1974) . 8. S . MATSUZAKI and M. SUZUKI, En ocrinol . Japan 22, 339-345 (1975) . 9. R. RICHMAN, S. PARK, M. AKBAR, S . YU, and G. BURKE, Endocrinology _96, 1403-1412 (1975) . P . R. ZUSMAN and G. N. BURROW, Endocrinology 97, 1089-1095 (1975) . 10 . 11 . S . YU, Y . FRIEDMAN, R. RICHMAN, and G. BURKE, J. Clin . Invest . _57, 745-755 (1976) . 12 . T. C. THEOHARIDES and Z . N. CANELLAKIS, J . Biol . Chem . 251, 1781-1784 (1976) . 13 . T. C. THEOHARIDES and Z. N. CANELLAKIS, Folia Bioch. et Graeca _13, 11-23 (1976) . 14 . M. MORRISON, G. S. BAYSE, and R. G . WEBSTER, Immunochemistry _8, 289-297 (1971) . 15 . C. M. TSAI, C. C . HUANG, and E . S. CANELLAKIS, Biochim. Biophys . Acta _332, 47-58 (1973) . 16 . K. CHEN, J. HELLER, and E . S . CANELLAKIS, Biochem. Biophys . Res . Commun . 68, 401-408 (1976) . 17 . H. LOWRY, N. J. ROSEBROUGH, A. L . FARR, and R . J . RANDALL, J . Biol . Chem . 193, 265-275 (1951) . K. J. BLOCK and R. J. M. WILSON, J . Immunol . 100, 629-636 (1968) . 18 . 19 . C . V. BYUS and D . H . RUSSELL, Science 7, 650-652 (1975) . 20 . S . W. SPAULDING and G. N. BURROW,B ohem. Biophys . Res . Commun . _59, 386391 (1975) . 21 . M. COSTA, E . R. COSTA, C-A . MANEN, I . G. SIPES, and D. H. RUSSELL, Molec . Pharmacol . 12 : 871-878 (1976) . 22 . C. V. BYUS, A . HEDGE, and D. H . RUSSELL, Biochim. Biophys . Acta 498, 3945 (1977) . 23 . S . J. SCHEINMAN and G. N. BURROW, Endocrinology, in press . 0, 1787-1798 (1977) . 24 . - R. A. JUNGMANN and D. H. RUSSELL, Life ci . 25 . Z .N . CANELLAKIS and T.C . THEOHARIDE , Chem . 251, 4436-4441 (1976) . 26 . E. HbLTTX, Biochim. Biophys . Acta 399, 20-427 (1975) _ . 27 . M. F. OBENRADER and W. F. PROUTY, . J Biol . Chem . 252, 2866-2872 (1977) . 1.
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