Effect of Thyrotropin on Ornithine Decarboxylase and of Polyamines on RNA Polymerase in the Thyroid STEPHEN W. SPAULDING Department of Medicine, Buffalo General Hospital, State University of Neiv York at Buffalo, Buffalo, New York 14203 ABSTRACT. Two major forms of RNA polymerase were partially purified from calf thyroid nuclei. Type I showed greatest activity in 30 mM (NH4)2SO4, transcribed native DNA best and was resistant to theinhibitoralpha-amanitin.Typell showed greatest activity in 100 mM (NH4)2SO4, transcribed denatured DNA template best and was sensitive to low concentrations of alpha-amanitin. Polyamines have been reported to affect the RNA polymerase reaction, and ornithine decarboxylase (ODC), the enzyme which controls polyamine synthesis, shows increased activity in many instances when there is increased RNA synthesis. To see whether TSH affected ODC activity in the thyroid in vitro, its activity was measured in homogenates
K
NOWLEDGE concerning the control of RNA synthesis is important in understanding the regulation of protein synthesis. In the thyroid, TSH reportedly causes a rapid increase in RNA synthesis both in vivo and in vitro (1,2) and longer TSH stimulation increases the RNA content per cell (3). Some evidence suggests that new RNA synthesis is necessary for the expression of several important metabolic responses to TSH (4-6). Clearly, one effect of TSH is simply the stimulation of nucleotide biosynthesis (7,8) but there are data suggesting that as in other tissues, the rate-limiting process in RNA synthesis involves the polymerization of the nucleotides. TSH has been reported to increase nuclear RNA synthesis (9) and to increase the amount of heterogeneous rapidly-labelled high molecular weight RNA in thyroid slices (10). RNA synthesis occurs by the transcription of DNA template by the enzyme RNA polymerase. RNA polymerase is a large Received August 25, 1976. Supported in part by the Margaret Duffy and Robert Cameron Troup Fund, Buffalo General Hospital, West Haven Conn. V.A. Hospital and NIH Grants AM 16878 and 17060.
from slices that had been incubated with 50 mU/ml TSH for various lengths of time. ODC activity was 380% of control in calf thyroid slices that had been incubated with TSH for 5 h. The addition of exogenous polyamine to the RNA polymerase II preparation enhanced the transcription of highly polymerized DNA: 3 mM spermine increased activity by 800%, while spermidine and putrescine were somewhat less stimulatory. Low concentrations of spermine slightly enhanced the activity of RNA polymerase I preparations. Polyamine synthesis could thus play a role in the longterm effects of TSH on RNA synthesis. (Endocrinology 100: 1039, 1977)
labile molecule made of multiple sub-units, and is predominantly associated with chromatin in eukaryotic cells (11). Three classes of RNA polymerase have been described in eukaryotic cells: Type I is apparently responsible for ribosomal RNA synthesis, Type II for heterogeneous nuclear RNA, mRNA precursors, and Type III for cytoplasmic tRNA precursors. The three forms display differential sensitivities to the inhibitory octapeptide alpha-amanitin (11). In purifying RNA polymerase, both stimulatory and inhibitory factors are removed, thus affecting the apparent activity, the degree of purification and the recovery obtained (11). One widespread class of compounds that can affect RNA polymerase activity is the polyamines (12,13). If physiological levels of polyamines alter thyroidal RNA polymerases, the control of polyamines could be important in the regulation of RNA synthesis in the thyroid. The enzyme ornithine decarboxylase (ODC), is thought to be rate-limiting in the synthesis of polyamines. It has one of the shortest half-lives of mammalian enzymes and is induced by hormones in many tissues, including TSH in the thyroid in vivo (14,15). It therefore
1039
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 23:43 For personal use only. No other uses without permission. . All rights reserved.
SPAULDING
1040
seemed of interest to determine whether ODC activity could be shown to increase in thyroid slices exposed to TSH and to see whether polyamines affect the activity of RNA polymerases in the thyroid. Materials and Methods Ornithine decarboxylase Bovine thyroid slices were incubated in triplicate in Krebs-Ringer bicarbonate buffer (KRB) with glucose (2 mg/ml) at 0 C for 30 min, then transferred to 10 ml fresh KRB with glucose containing 25 /xg/ml cephalexin (for bacteriostasis) with or without 50 mU/ml TSH (Armour Thytropar). After incubation at 37 C for various periods, the slices were removed, homogenized in 2 ml of iced 0.25M sucrose containing 0.5 mM ethylenediamine tetraacetic acid (EDTA) and 10 mM mercaptoethanol, pH 7.4, and centrifuged at 100,000 x g for 20 min. The supernatant was kept at - 2 0 C until assayed. [Carboxyl-14C]ornithine (New England Nuclear) was prepared for use as a substrate by acidifying it with O.lN HC1 and heating it under vacuum at 50 C for 30 minutes to remove volatile radio-labelled contaminants. It was then diluted tenfold with water and frozen until used. ODC activity was measured by adding 100 fx\ of the tissue supernatant to 100 /x.1 of a reaction mixture containing 25 mM Tris-HCl (pH 7.8), 0.5 mM EDTA, 0.1 mM pyridoxal-5-phosphate, 13 jiimol mercaptoethanol and 600 /xM ornithine containing 1.5 x 106 dpm (15). The reaction was carried out at 37 C in gas-tight tubes and was terminated after 90 min by injecting 400 ix\ of 2M citric acid into the reaction mixture. In each sealed tube a small polyethylene well had previously been suspended, containing 200 fi\ of Protosol to adsorb the 14CO2 generated. After incubating for an additional hour after the enzymic reaction had been terminated, these wells were removed, placed in Aquasol and counted. Activity was linear over the time and enzyme concentration used. Activity was expressed as pmol 14CO2 generated per mg protein in the tissue supernatant per hour of incubation. RNA polymerase assay The assay was performed in a final volume of 0.2 ml containing 100 mM Tris HC1 (pH 7.9 at 37 C), 10 mM NaF, 0.5 mM mercaptoethanol and
Endo • 1977 Vol 100 • No 4
0.6 mM ATP, GTP and CTP, with final ammonium sulfate concentrations as specified (13). Final template concentration was 160 /xg/ml native highly polymerized calf thymus DNA unless otherwise specified. Denatured DNA was prepared by heating native DNA (1 mg/ml 3 mM NaCl) in boiling water for 3 min then plunging it into ice water. Final UTP concentration was 0.1 mM containing 20-30 x 103 cpm [3H]UTP/ nmol. Enzyme preparations were added in a volume of 50 fil. The reaction, generally started by the addition of the UTP, was run for 10 min at 37 C and terminated by the addition of 2 ml of iced 10% trichloroacetic acid containing 8 mM sodium pyrophosphate. One hundred mg RNA and 300 mg bovine serum albumin were added as carriers and the precipitate was washed twice with 5% trichloroacetic acid (TCA) containing 5 mM sodium pyrophosphate, given an ether/ ethanol (1:3) wash, and counted. Unless otherwise noted, assays were run with substrate and template excess and were linear with respect to time and enzyme concentration. Activity was generally expressed as pmoles of UTP incorporated per mg protein per 10 min incubation. Preparation of RNA polymerase The following buffers were used: "TGED 30" —50 mM TRIS-HC1 (pH 7.9) at 20 C, 30% vol/vol glycerol, 0.1 mM Na2EDTA, 2.0 mM dithiothreitol (DTT) and 0.5 mMphenylmethylsulfonyl fluoride (PMSF). "TGED 50"—as above, but with 50% glycerol; "TGED 25"—as above but with 25% glycerol; "TGED 30/30"—TGED 30 with 30 mM (NH4)SO4; "TGMED 30"—TGED 30 with 5 mM MgCl2. DTT and PMSF were added to the buffers just prior to use. The method is a modification of that described by Janne et al. (13). Thyroids were obtained from calves within 15 min of slaughter, placed in iced KRB, cleaned and their volume determined by displacement. They were then minced and homogenized in 2M sucrose, 10 mM MgCl2, 2 mM DTT with 0.5 mM PMSF. All procedures were performed in a well-ventilated area. The homogenate was strained through cheese-cloth and centrifuged at 50,000 x g for 1 h. Electron microscopic examination of such pellets revealed mostly well-preserved nuclei (about 80% intact), occasionally contaminated with cytoplasmic tags. The nuclear pellet was resuspended in TGED 30 using one-half the original volume of thyroid. Iced 3M (NH4)2SO4, pH 9, was added dropwise
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 23:43 For personal use only. No other uses without permission. . All rights reserved.
1041
POLYAMINES AND THYROIDAL RNA POLYMERASE 12
FlG. 1. Effect of TSH on ornithine decarboxylase in thyroid slices. Calf thyroid slices were incubated in triplicate in Krebs Ringer bicarbonate buffer with or without TSH (50 mU/ml). After various incubations times, slices were removed and homogenized, and the supernatant was assayed for ornithine decarboxylase activity. Bars represent ± 1 SEM.
• Control o with TSH
10
a.
I6 CM
o
O 4
3 4 TIME (hr)
to give a final concentration of 0.3M. The gelatinous sample was sonicated with 8 ten-sec bursts at setting "6" on a Branson W-140 D ultrasonicator. Each burst was followed by a 30 sec cooling period in ice water. The liquified sample was then centrifuged at 50,000 x g for 30 min and the activity precipitated from the supernatant by adding 0.35 g/ml of (NH4)2SO4 gradually over 30 min. After stirring on ice for an additional 30 min, the precipitate was collected by centrifugation at 100,000 x g for an hour, then redissolved in 0.2 times the initial tissue volume of TGED 50, and frozen in liquid N2. After careful thawing, the solution was diluted with TGMED 30 until, the salt concentration was below 0. 1M, as determined by conductivity measurements. Protamine sulfate (1%), pH 5.5, was then added dropwise to the sample until a final concentration of 0.05% was obtained. After stirring on ice for 45 min, the precipitate was removed by centrifugation at 100,000 x g for 2 h.
The supernatant was then re-precipitated with (NH4)2SO4 as before and the pellet redissolved in TGED 50 (0.1 times the original tissue volume), and stored in liquid N2. DEAE Sephadex chromatography The enzyme preparation was diluted, then stirred with DEAE-Sephadex A-25 and poured into a column. The column was washed first with 30 mM, then 140 mM, and finally 400 mM (NH4)2SO4 (Fig. 2). Tubes containing RNA polymerase activity were pooled and dialyzed. Phosphocellulose chromatography The dialyzed peaks from DEAE-Sephadex chromatography were added to Whatman PII phosphocellulose columns. These columns were eluted with a linear gradient of (NH4)2SO4 (Fig. 3). Samples were assayed, and the tubes
TABLE 1. Purification of RNA polymerase activity
Whole homogenate Sonicated nuclei lst(NH 4 ) 2 SO 4 Ppt 2nd (NH4)2SO4 Ppt
Volume (ml)
Protein (mg/ml)
Total activity recovered (pmol UTP incorp)
Specific activity (pmol UTP incorp/mg protein)
950
28 4.4
102,000 44,200 51,200 18,500
3.8 72 133 203
140
107 26
3.6 3.5
250 cc of minced calf thyroid were homogenized with five strokes of a Teflon-glass homogenizer in five volumes of iced buffer (see text). The samples were all assayed with a final concentration of 30 mM (NH4)2SO4, 3 mM MnCl2, 6 mM MgCl2 and 160 /ig/ml native calf thymus DNA.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 23:43 For personal use only. No other uses without permission. . All rights reserved.
1042
SPAULDING
containing the greatest activity were pooled and stored in liquid nitrogen. Studies on salt, divalent cation requirements etc. were performed on these pools. Protein concentrations were determined on samples precipitated with 5% TCA, centrifuged at 3000 rpm x 20 min and then dissolved in 0.2 ml NaOH. Bovine serum albumin (BSA) standards were treated similarly. The samples were then assayed according to the method of Lowry et al. (16). PMSF and highly polymerized native calf thymus DNA were obtained from Sigma Chemicals, poly (dA-dT) from Miles Laboratories, and alpha amanitin from Calbiochem.
Endo • 1977 Vol 100 • No 4
90
(•) without
K
NOWLEDGE concerning the control of RNA synthesis is important in understanding the regulation of protein synthesis. In the thyroid, TSH reportedly causes a rapid increase in RNA synthesis both in vivo and in vitro (1,2) and longer TSH stimulation increases the RNA content per cell (3). Some evidence suggests that new RNA synthesis is necessary for the expression of several important metabolic responses to TSH (4-6). Clearly, one effect of TSH is simply the stimulation of nucleotide biosynthesis (7,8) but there are data suggesting that as in other tissues, the rate-limiting process in RNA synthesis involves the polymerization of the nucleotides. TSH has been reported to increase nuclear RNA synthesis (9) and to increase the amount of heterogeneous rapidly-labelled high molecular weight RNA in thyroid slices (10). RNA synthesis occurs by the transcription of DNA template by the enzyme RNA polymerase. RNA polymerase is a large Received August 25, 1976. Supported in part by the Margaret Duffy and Robert Cameron Troup Fund, Buffalo General Hospital, West Haven Conn. V.A. Hospital and NIH Grants AM 16878 and 17060.
from slices that had been incubated with 50 mU/ml TSH for various lengths of time. ODC activity was 380% of control in calf thyroid slices that had been incubated with TSH for 5 h. The addition of exogenous polyamine to the RNA polymerase II preparation enhanced the transcription of highly polymerized DNA: 3 mM spermine increased activity by 800%, while spermidine and putrescine were somewhat less stimulatory. Low concentrations of spermine slightly enhanced the activity of RNA polymerase I preparations. Polyamine synthesis could thus play a role in the longterm effects of TSH on RNA synthesis. (Endocrinology 100: 1039, 1977)
labile molecule made of multiple sub-units, and is predominantly associated with chromatin in eukaryotic cells (11). Three classes of RNA polymerase have been described in eukaryotic cells: Type I is apparently responsible for ribosomal RNA synthesis, Type II for heterogeneous nuclear RNA, mRNA precursors, and Type III for cytoplasmic tRNA precursors. The three forms display differential sensitivities to the inhibitory octapeptide alpha-amanitin (11). In purifying RNA polymerase, both stimulatory and inhibitory factors are removed, thus affecting the apparent activity, the degree of purification and the recovery obtained (11). One widespread class of compounds that can affect RNA polymerase activity is the polyamines (12,13). If physiological levels of polyamines alter thyroidal RNA polymerases, the control of polyamines could be important in the regulation of RNA synthesis in the thyroid. The enzyme ornithine decarboxylase (ODC), is thought to be rate-limiting in the synthesis of polyamines. It has one of the shortest half-lives of mammalian enzymes and is induced by hormones in many tissues, including TSH in the thyroid in vivo (14,15). It therefore
1039
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 23:43 For personal use only. No other uses without permission. . All rights reserved.
SPAULDING
1040
seemed of interest to determine whether ODC activity could be shown to increase in thyroid slices exposed to TSH and to see whether polyamines affect the activity of RNA polymerases in the thyroid. Materials and Methods Ornithine decarboxylase Bovine thyroid slices were incubated in triplicate in Krebs-Ringer bicarbonate buffer (KRB) with glucose (2 mg/ml) at 0 C for 30 min, then transferred to 10 ml fresh KRB with glucose containing 25 /xg/ml cephalexin (for bacteriostasis) with or without 50 mU/ml TSH (Armour Thytropar). After incubation at 37 C for various periods, the slices were removed, homogenized in 2 ml of iced 0.25M sucrose containing 0.5 mM ethylenediamine tetraacetic acid (EDTA) and 10 mM mercaptoethanol, pH 7.4, and centrifuged at 100,000 x g for 20 min. The supernatant was kept at - 2 0 C until assayed. [Carboxyl-14C]ornithine (New England Nuclear) was prepared for use as a substrate by acidifying it with O.lN HC1 and heating it under vacuum at 50 C for 30 minutes to remove volatile radio-labelled contaminants. It was then diluted tenfold with water and frozen until used. ODC activity was measured by adding 100 fx\ of the tissue supernatant to 100 /x.1 of a reaction mixture containing 25 mM Tris-HCl (pH 7.8), 0.5 mM EDTA, 0.1 mM pyridoxal-5-phosphate, 13 jiimol mercaptoethanol and 600 /xM ornithine containing 1.5 x 106 dpm (15). The reaction was carried out at 37 C in gas-tight tubes and was terminated after 90 min by injecting 400 ix\ of 2M citric acid into the reaction mixture. In each sealed tube a small polyethylene well had previously been suspended, containing 200 fi\ of Protosol to adsorb the 14CO2 generated. After incubating for an additional hour after the enzymic reaction had been terminated, these wells were removed, placed in Aquasol and counted. Activity was linear over the time and enzyme concentration used. Activity was expressed as pmol 14CO2 generated per mg protein in the tissue supernatant per hour of incubation. RNA polymerase assay The assay was performed in a final volume of 0.2 ml containing 100 mM Tris HC1 (pH 7.9 at 37 C), 10 mM NaF, 0.5 mM mercaptoethanol and
Endo • 1977 Vol 100 • No 4
0.6 mM ATP, GTP and CTP, with final ammonium sulfate concentrations as specified (13). Final template concentration was 160 /xg/ml native highly polymerized calf thymus DNA unless otherwise specified. Denatured DNA was prepared by heating native DNA (1 mg/ml 3 mM NaCl) in boiling water for 3 min then plunging it into ice water. Final UTP concentration was 0.1 mM containing 20-30 x 103 cpm [3H]UTP/ nmol. Enzyme preparations were added in a volume of 50 fil. The reaction, generally started by the addition of the UTP, was run for 10 min at 37 C and terminated by the addition of 2 ml of iced 10% trichloroacetic acid containing 8 mM sodium pyrophosphate. One hundred mg RNA and 300 mg bovine serum albumin were added as carriers and the precipitate was washed twice with 5% trichloroacetic acid (TCA) containing 5 mM sodium pyrophosphate, given an ether/ ethanol (1:3) wash, and counted. Unless otherwise noted, assays were run with substrate and template excess and were linear with respect to time and enzyme concentration. Activity was generally expressed as pmoles of UTP incorporated per mg protein per 10 min incubation. Preparation of RNA polymerase The following buffers were used: "TGED 30" —50 mM TRIS-HC1 (pH 7.9) at 20 C, 30% vol/vol glycerol, 0.1 mM Na2EDTA, 2.0 mM dithiothreitol (DTT) and 0.5 mMphenylmethylsulfonyl fluoride (PMSF). "TGED 50"—as above, but with 50% glycerol; "TGED 25"—as above but with 25% glycerol; "TGED 30/30"—TGED 30 with 30 mM (NH4)SO4; "TGMED 30"—TGED 30 with 5 mM MgCl2. DTT and PMSF were added to the buffers just prior to use. The method is a modification of that described by Janne et al. (13). Thyroids were obtained from calves within 15 min of slaughter, placed in iced KRB, cleaned and their volume determined by displacement. They were then minced and homogenized in 2M sucrose, 10 mM MgCl2, 2 mM DTT with 0.5 mM PMSF. All procedures were performed in a well-ventilated area. The homogenate was strained through cheese-cloth and centrifuged at 50,000 x g for 1 h. Electron microscopic examination of such pellets revealed mostly well-preserved nuclei (about 80% intact), occasionally contaminated with cytoplasmic tags. The nuclear pellet was resuspended in TGED 30 using one-half the original volume of thyroid. Iced 3M (NH4)2SO4, pH 9, was added dropwise
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 23:43 For personal use only. No other uses without permission. . All rights reserved.
1041
POLYAMINES AND THYROIDAL RNA POLYMERASE 12
FlG. 1. Effect of TSH on ornithine decarboxylase in thyroid slices. Calf thyroid slices were incubated in triplicate in Krebs Ringer bicarbonate buffer with or without TSH (50 mU/ml). After various incubations times, slices were removed and homogenized, and the supernatant was assayed for ornithine decarboxylase activity. Bars represent ± 1 SEM.
• Control o with TSH
10
a.
I6 CM
o
O 4
3 4 TIME (hr)
to give a final concentration of 0.3M. The gelatinous sample was sonicated with 8 ten-sec bursts at setting "6" on a Branson W-140 D ultrasonicator. Each burst was followed by a 30 sec cooling period in ice water. The liquified sample was then centrifuged at 50,000 x g for 30 min and the activity precipitated from the supernatant by adding 0.35 g/ml of (NH4)2SO4 gradually over 30 min. After stirring on ice for an additional 30 min, the precipitate was collected by centrifugation at 100,000 x g for an hour, then redissolved in 0.2 times the initial tissue volume of TGED 50, and frozen in liquid N2. After careful thawing, the solution was diluted with TGMED 30 until, the salt concentration was below 0. 1M, as determined by conductivity measurements. Protamine sulfate (1%), pH 5.5, was then added dropwise to the sample until a final concentration of 0.05% was obtained. After stirring on ice for 45 min, the precipitate was removed by centrifugation at 100,000 x g for 2 h.
The supernatant was then re-precipitated with (NH4)2SO4 as before and the pellet redissolved in TGED 50 (0.1 times the original tissue volume), and stored in liquid N2. DEAE Sephadex chromatography The enzyme preparation was diluted, then stirred with DEAE-Sephadex A-25 and poured into a column. The column was washed first with 30 mM, then 140 mM, and finally 400 mM (NH4)2SO4 (Fig. 2). Tubes containing RNA polymerase activity were pooled and dialyzed. Phosphocellulose chromatography The dialyzed peaks from DEAE-Sephadex chromatography were added to Whatman PII phosphocellulose columns. These columns were eluted with a linear gradient of (NH4)2SO4 (Fig. 3). Samples were assayed, and the tubes
TABLE 1. Purification of RNA polymerase activity
Whole homogenate Sonicated nuclei lst(NH 4 ) 2 SO 4 Ppt 2nd (NH4)2SO4 Ppt
Volume (ml)
Protein (mg/ml)
Total activity recovered (pmol UTP incorp)
Specific activity (pmol UTP incorp/mg protein)
950
28 4.4
102,000 44,200 51,200 18,500
3.8 72 133 203
140
107 26
3.6 3.5
250 cc of minced calf thyroid were homogenized with five strokes of a Teflon-glass homogenizer in five volumes of iced buffer (see text). The samples were all assayed with a final concentration of 30 mM (NH4)2SO4, 3 mM MnCl2, 6 mM MgCl2 and 160 /ig/ml native calf thymus DNA.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 23:43 For personal use only. No other uses without permission. . All rights reserved.
1042
SPAULDING
containing the greatest activity were pooled and stored in liquid nitrogen. Studies on salt, divalent cation requirements etc. were performed on these pools. Protein concentrations were determined on samples precipitated with 5% TCA, centrifuged at 3000 rpm x 20 min and then dissolved in 0.2 ml NaOH. Bovine serum albumin (BSA) standards were treated similarly. The samples were then assayed according to the method of Lowry et al. (16). PMSF and highly polymerized native calf thymus DNA were obtained from Sigma Chemicals, poly (dA-dT) from Miles Laboratories, and alpha amanitin from Calbiochem.
Endo • 1977 Vol 100 • No 4
90
(•) without
