Alain TIRARD | Aix-Marseille University (original) (raw)
Papers by Alain TIRARD
Le Centre pour la Communication Scientifique Directe - HAL - Université Francois Rabelais - Tours, Oct 27, 2009
The present invention relates to novel polypeptides constituting receptors for pheromones in rats... more The present invention relates to novel polypeptides constituting receptors for pheromones in rats, their cloning and sequencing of the genes encoding these polypeptides and their use. The present invention relates to novel polypeptides constituting receptors for pheromones in rats, as well as the genes encoding these polypeptides. The invention also relates to the use of these polypeptides for aroma detection, quality control, sample analysis, perfume analysis or comparison, detection of toxic substances, or trapping. odor.
Summary The Argentine ant, native to the area around Buenos Aires, has been spreading in the Medi... more Summary The Argentine ant, native to the area around Buenos Aires, has been spreading in the Mediterranean region from colonization foci. This invasive behavior is based upon a particuliar demographic strategy and social organization : the formation of super-colonies composed of several million individuals. So, a question follows: is the functional structure of the colony also concerned by the homogeneisation process? We show with a principal component analysis of the proportion of the different cuticular compounds, that the structuration remains at least at the level of the chemical cue of males, queens and workers.
Plant Molecular Biology Reporter, 2007
European journal of cell biology, 1987
When HT 29 cells grown as a monolayer were incubated in a synthetic medium in presence of 0.1 mic... more When HT 29 cells grown as a monolayer were incubated in a synthetic medium in presence of 0.1 microM [gamma 32P]-ATP, the radioactivity was incorporated predominantly into three major endogenous polypeptides of 123 kDa, 50 kDa and 46 kDa. The radioactive proteins could be detected as soon as 30 s after the addition of the labelled ATP. When exogenous substrates such as casein or phosvitin were added in the synthetic medium, these proteins became phosphorylated. The phosvitin-kinase activity was released in the culture medium following an incubation of the cells with phosvitin. Depletion of the enzymatic activity from the cell surface as well as competition between phosvitin and endogenous substrates led specifically to the inhibition of the 123 kDa polypeptide phosphorylation. At low density, endogenous phosphorylation increased with the cell number, whereas on the contrary it decreased at high cell density. We concluded that the surface of HT 29 cells expressed several protein kina...
Cellular and molecular biology (Noisy-le-Grand, France), 2000
Alpine Marmots (Marmota marmota) are a good model to study intraspecific chemical communication a... more Alpine Marmots (Marmota marmota) are a good model to study intraspecific chemical communication among mammals. This species has been subjected to several behavioural and biochemical studies regarding both their scent-marking behaviour by cheek-rubbing, and the chemical composition of their glandular secretions. However, no molecular study has been undertaken until today on proteins from the olfactory epithelium possibly implicated in chemical perception. In this study, we identified, to our knowledge for the first time, some olfatory receptors from this wild rodent. Starting with olfactory epithelium of an Alpine Marmot, and by mean of reverse transcriptase polymerase chain reaction technique (RT-PCR), we isolated fourteen partial sequences that exhibited a high degree of homology (45-92%) with olfactory receptors from other vertebrates. Conserved identities and structural features clearly defined these Alpine Marmot sequences as members of the seven transmembrane domain olfactory r...
European journal of cell biology, 1988
HT29-D4, a clone of the human colonic adenocarcinoma cell line (HT29), possesses at its cell surf... more HT29-D4, a clone of the human colonic adenocarcinoma cell line (HT29), possesses at its cell surface specific binding sites for the vasoactive intestinal peptide (VIP) (KD = 0.5 nM). Their molecular weight was previously estimated to 117 kDa and 64 kDa. This clone underwent functional and structural differentiation when grown in a glucose-free galactose-containing medium. The [125I]VIP binding capacity of cells grown in this medium gradually declined while the cell density increased and reached a value close to zero when cell monolayer was able to form hemicysts. At this time, cells presented numerous tight junctions and desmosomes and a well organized brush border. Binding capacity could be recovered when the post-confluent monolayers were previously disaggregated with EDTA. Neither the affinity for VIP nor the molecular weight of the [125I]VIP cross-linked polypeptides were modified in these cells compared to cells grown in glucose-containing medium. However, surface receptor numb...
The initial event of VIP action is its interaction with a specific receptor at the surface of a t... more The initial event of VIP action is its interaction with a specific receptor at the surface of a target cell. The understanding of the fine mechanism of action of VIP requires the characterization and of course the purification of the receptor. The understanding of the mechanism which regulates the number of receptor sites at the cell surface, if it occurs, is also a way to characterize the properties of VIP receptor. In this paper we report a number of data which represent several attempts to characterize VIP receptor in a human colonic adenocarcinoma cell (HT 29 cells). We have characterized a monoclonal antibody which partially inhibits 125I-VIP binding to HT 29 cells. We have specifically cross-linked, on intact HT 29 cells, a major polypeptide of Mr-64,000 with 125I-VIP using DTSP or DSS as cross-linking reagents. This polypeptide behaves like a high affinity binding site for VIP. We have demonstrated that VIP is rapidly internalized in HT 29 cells (in less than 10 minutes) and that simultaneously VIP receptors were no more detectable on the cell surface by cross-linking experiments. This suggests that VIP is internalized together with its receptor.
Journal of Molecular Biology, 1985
In order to understand how the phosphorylation of histones affects the chromatin structure, we us... more In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.
Journal of Endocrinological Investigation, 1980
Phosphoprotamine phosphatase activity in the rat thyroid was examined by studying the release of ... more Phosphoprotamine phosphatase activity in the rat thyroid was examined by studying the release of inorganic phosphate from [32P]-phosphorylated protamine. Rats were given a normal diet supplemented or not with thyroxine (T4) (3 mg/l in 0.05% bovine serum albumin solution) or propylthiouracil (1 g/l in 1% sucrose solution) in drinking water for various periods. TSH was injected ip, 100 mU per animal. A significant increase of phosphatase specific activity-units per mg protein (3-fold) as well as units per gland- was observed following goitrogen treatment, whereas a decrease (50%) was seen in response to T4. Both the soluble and particle activities were similarly affected. As total activity decreased after T4 treatment, it appeared that only the Mn2+-stimulating enzyme was concerned, the Mn2+-unstimulated part remaining unaltered. In vitro cyclic AMP or cyclic GMP at concentrations from 10 micro M to 200 micro M had no effect on these phosphatase activities. TSH injected in chronically T4-treated rats (32 days treatment) failed to produce any significant enhancement in phosphatase activity whatever the time of injection before sacrifice (from 2 to 6 h). On the contrary a single injection of TSH in animals subjected to short treatment with T4 (2 days) induced an elevation of the enzyme activity, restoring partially the initial level. These findings suggest that TSH controls protein dephosphorylation activities in the thyroid. Furthermore, they offer additional evidence that prolonged T4 administration leads to decreased responsiveness to TSH.
Annals of the New York Academy of Sciences, 1988
... MICHAL SVOBODA, PATRICK ROBBERECHT, FRANCOISE GOMEZ, JACQUES WINAND, AND JEAN CHRISTOPHE ... ... more ... MICHAL SVOBODA, PATRICK ROBBERECHT, FRANCOISE GOMEZ, JACQUES WINAND, AND JEAN CHRISTOPHE ... SDS-PAGE was performed on 5-20% T gradient, 2.7% polyacrylamide gels (80 x 80 x 1.5 mm) using the discontinuous system of Laemmli.' Dried gels were ...
imep-cnrs.com
Since the middle of the 20th century, improvements in analytical technologies have permitted the ... more Since the middle of the 20th century, improvements in analytical technologies have permitted the identification of cuticular hydrocarbons present on the cuticle of almost all insects.
Biochimica Et Biophysica Acta (bba) - Protein Structure, 1978
Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by... more Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.
European Journal of Endocrinology, 1976
Triiodothyronine (T3) may bind directly to receptors present in liver cell nuclei, or may be tran... more Triiodothyronine (T3) may bind directly to receptors present in liver cell nuclei, or may be transported into nuclei by receptor protein(s) present in the cytosol. To evaluate these possibilities, T3 binding was studied in vitro using liver cell nuclei isolated from rats exposed in vivo to very low (H), normal (N),or high levels of T3 (H + T3), and using nuclei incubated in vitro with added cytosol proteins. Ka for T3 was 0.075 +/- 0.05 x 10(10) M-1 in N, 0.1 + 0.04 in H, and 0.094 + 0.04 in H + T3, and pg T3 bound/100 mug DNA were 47 +/- 17, 31 +/- 14, and 29 +/- 8 in the three groups. The data indicate no difference in binding capacity between the groups related to prior in vivo exposure to T3, and that T3 may bind directly to empty nuclear receptor sites. Rat liver cytosol proteins added to the in vitro incubation medium always depressed T3 uptake by nuclei. Bovine serum albumin had a similar effect. Large amounts of rat serum proteins depressed uptake, but low levels augmented T3 binding through an unknown mechanism. It is probable that free T3 in serum is in equilibrium with free T3 in the cytosol and nucleus, and binds directly to nuclear receptor proteins without mediation by a cytosol receptor protein.
![Research paper thumbnail of Phosphorylation of purified thyroid plasma membranes incubated with [32P]ATP](https://attachments.academia-assets.com/49957027/thumbnails/1.jpg)
](Molecular and Cellular Endocrinology, 1975
have been prepared from porcine thyroid glands using sucrose gradients.
Biochimie, 1974
The distribution of cyclic AMP-dependent protein kinase activity in porcine thyroid glands has be... more The distribution of cyclic AMP-dependent protein kinase activity in porcine thyroid glands has been studied. Enzyme activity catalyzing phosphorylation of exogenous substrate (protamine) from ATP, and cyclic AMP binding were determined in parallel in subcellular fractions purified by differential centrifugation and flotation on sucrose density layers. Both activities were found in all the studied fractions ; they were quantitatively the highest in the cytosol but particles showed the highest specific activities.
Endocrinology, 1977
Protein kinase activities were determined in liver from normal, thyroidectomized and triiodothyro... more Protein kinase activities were determined in liver from normal, thyroidectomized and triiodothyronine (T3)-treated rats. Changes related to thyroid hormone were observed in cytosol and nuclear protein kinase activities. When protamine was used as substrate for phosphorylation, thyroidectomy induced a decrease of protein kinase activity associated with nuclei but an increase of activity was found in the cytosol. Fifteen hours after injection of T3 the levels in nuclei and cytosol were restored to normal. When casein was used as substrate, hypothyroidism led to a lowering of protein kinase activity in both fractions and T3 treatment augmented the activity in both. These studies suggest that thyroid hormones modify hepatic protein kinase activity. Results differ depending upon the substrate used. The hormones also appear to alter the subcellular distribution of some protein kinase activities.
Le Centre pour la Communication Scientifique Directe - HAL - Université Francois Rabelais - Tours, Oct 27, 2009
The present invention relates to novel polypeptides constituting receptors for pheromones in rats... more The present invention relates to novel polypeptides constituting receptors for pheromones in rats, their cloning and sequencing of the genes encoding these polypeptides and their use. The present invention relates to novel polypeptides constituting receptors for pheromones in rats, as well as the genes encoding these polypeptides. The invention also relates to the use of these polypeptides for aroma detection, quality control, sample analysis, perfume analysis or comparison, detection of toxic substances, or trapping. odor.
Summary The Argentine ant, native to the area around Buenos Aires, has been spreading in the Medi... more Summary The Argentine ant, native to the area around Buenos Aires, has been spreading in the Mediterranean region from colonization foci. This invasive behavior is based upon a particuliar demographic strategy and social organization : the formation of super-colonies composed of several million individuals. So, a question follows: is the functional structure of the colony also concerned by the homogeneisation process? We show with a principal component analysis of the proportion of the different cuticular compounds, that the structuration remains at least at the level of the chemical cue of males, queens and workers.
Plant Molecular Biology Reporter, 2007
European journal of cell biology, 1987
When HT 29 cells grown as a monolayer were incubated in a synthetic medium in presence of 0.1 mic... more When HT 29 cells grown as a monolayer were incubated in a synthetic medium in presence of 0.1 microM [gamma 32P]-ATP, the radioactivity was incorporated predominantly into three major endogenous polypeptides of 123 kDa, 50 kDa and 46 kDa. The radioactive proteins could be detected as soon as 30 s after the addition of the labelled ATP. When exogenous substrates such as casein or phosvitin were added in the synthetic medium, these proteins became phosphorylated. The phosvitin-kinase activity was released in the culture medium following an incubation of the cells with phosvitin. Depletion of the enzymatic activity from the cell surface as well as competition between phosvitin and endogenous substrates led specifically to the inhibition of the 123 kDa polypeptide phosphorylation. At low density, endogenous phosphorylation increased with the cell number, whereas on the contrary it decreased at high cell density. We concluded that the surface of HT 29 cells expressed several protein kina...
Cellular and molecular biology (Noisy-le-Grand, France), 2000
Alpine Marmots (Marmota marmota) are a good model to study intraspecific chemical communication a... more Alpine Marmots (Marmota marmota) are a good model to study intraspecific chemical communication among mammals. This species has been subjected to several behavioural and biochemical studies regarding both their scent-marking behaviour by cheek-rubbing, and the chemical composition of their glandular secretions. However, no molecular study has been undertaken until today on proteins from the olfactory epithelium possibly implicated in chemical perception. In this study, we identified, to our knowledge for the first time, some olfatory receptors from this wild rodent. Starting with olfactory epithelium of an Alpine Marmot, and by mean of reverse transcriptase polymerase chain reaction technique (RT-PCR), we isolated fourteen partial sequences that exhibited a high degree of homology (45-92%) with olfactory receptors from other vertebrates. Conserved identities and structural features clearly defined these Alpine Marmot sequences as members of the seven transmembrane domain olfactory r...
European journal of cell biology, 1988
HT29-D4, a clone of the human colonic adenocarcinoma cell line (HT29), possesses at its cell surf... more HT29-D4, a clone of the human colonic adenocarcinoma cell line (HT29), possesses at its cell surface specific binding sites for the vasoactive intestinal peptide (VIP) (KD = 0.5 nM). Their molecular weight was previously estimated to 117 kDa and 64 kDa. This clone underwent functional and structural differentiation when grown in a glucose-free galactose-containing medium. The [125I]VIP binding capacity of cells grown in this medium gradually declined while the cell density increased and reached a value close to zero when cell monolayer was able to form hemicysts. At this time, cells presented numerous tight junctions and desmosomes and a well organized brush border. Binding capacity could be recovered when the post-confluent monolayers were previously disaggregated with EDTA. Neither the affinity for VIP nor the molecular weight of the [125I]VIP cross-linked polypeptides were modified in these cells compared to cells grown in glucose-containing medium. However, surface receptor numb...
The initial event of VIP action is its interaction with a specific receptor at the surface of a t... more The initial event of VIP action is its interaction with a specific receptor at the surface of a target cell. The understanding of the fine mechanism of action of VIP requires the characterization and of course the purification of the receptor. The understanding of the mechanism which regulates the number of receptor sites at the cell surface, if it occurs, is also a way to characterize the properties of VIP receptor. In this paper we report a number of data which represent several attempts to characterize VIP receptor in a human colonic adenocarcinoma cell (HT 29 cells). We have characterized a monoclonal antibody which partially inhibits 125I-VIP binding to HT 29 cells. We have specifically cross-linked, on intact HT 29 cells, a major polypeptide of Mr-64,000 with 125I-VIP using DTSP or DSS as cross-linking reagents. This polypeptide behaves like a high affinity binding site for VIP. We have demonstrated that VIP is rapidly internalized in HT 29 cells (in less than 10 minutes) and that simultaneously VIP receptors were no more detectable on the cell surface by cross-linking experiments. This suggests that VIP is internalized together with its receptor.
Journal of Molecular Biology, 1985
In order to understand how the phosphorylation of histones affects the chromatin structure, we us... more In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.
Journal of Endocrinological Investigation, 1980
Phosphoprotamine phosphatase activity in the rat thyroid was examined by studying the release of ... more Phosphoprotamine phosphatase activity in the rat thyroid was examined by studying the release of inorganic phosphate from [32P]-phosphorylated protamine. Rats were given a normal diet supplemented or not with thyroxine (T4) (3 mg/l in 0.05% bovine serum albumin solution) or propylthiouracil (1 g/l in 1% sucrose solution) in drinking water for various periods. TSH was injected ip, 100 mU per animal. A significant increase of phosphatase specific activity-units per mg protein (3-fold) as well as units per gland- was observed following goitrogen treatment, whereas a decrease (50%) was seen in response to T4. Both the soluble and particle activities were similarly affected. As total activity decreased after T4 treatment, it appeared that only the Mn2+-stimulating enzyme was concerned, the Mn2+-unstimulated part remaining unaltered. In vitro cyclic AMP or cyclic GMP at concentrations from 10 micro M to 200 micro M had no effect on these phosphatase activities. TSH injected in chronically T4-treated rats (32 days treatment) failed to produce any significant enhancement in phosphatase activity whatever the time of injection before sacrifice (from 2 to 6 h). On the contrary a single injection of TSH in animals subjected to short treatment with T4 (2 days) induced an elevation of the enzyme activity, restoring partially the initial level. These findings suggest that TSH controls protein dephosphorylation activities in the thyroid. Furthermore, they offer additional evidence that prolonged T4 administration leads to decreased responsiveness to TSH.
Annals of the New York Academy of Sciences, 1988
... MICHAL SVOBODA, PATRICK ROBBERECHT, FRANCOISE GOMEZ, JACQUES WINAND, AND JEAN CHRISTOPHE ... ... more ... MICHAL SVOBODA, PATRICK ROBBERECHT, FRANCOISE GOMEZ, JACQUES WINAND, AND JEAN CHRISTOPHE ... SDS-PAGE was performed on 5-20% T gradient, 2.7% polyacrylamide gels (80 x 80 x 1.5 mm) using the discontinuous system of Laemmli.' Dried gels were ...
imep-cnrs.com
Since the middle of the 20th century, improvements in analytical technologies have permitted the ... more Since the middle of the 20th century, improvements in analytical technologies have permitted the identification of cuticular hydrocarbons present on the cuticle of almost all insects.
Biochimica Et Biophysica Acta (bba) - Protein Structure, 1978
Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by... more Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.
European Journal of Endocrinology, 1976
Triiodothyronine (T3) may bind directly to receptors present in liver cell nuclei, or may be tran... more Triiodothyronine (T3) may bind directly to receptors present in liver cell nuclei, or may be transported into nuclei by receptor protein(s) present in the cytosol. To evaluate these possibilities, T3 binding was studied in vitro using liver cell nuclei isolated from rats exposed in vivo to very low (H), normal (N),or high levels of T3 (H + T3), and using nuclei incubated in vitro with added cytosol proteins. Ka for T3 was 0.075 +/- 0.05 x 10(10) M-1 in N, 0.1 + 0.04 in H, and 0.094 + 0.04 in H + T3, and pg T3 bound/100 mug DNA were 47 +/- 17, 31 +/- 14, and 29 +/- 8 in the three groups. The data indicate no difference in binding capacity between the groups related to prior in vivo exposure to T3, and that T3 may bind directly to empty nuclear receptor sites. Rat liver cytosol proteins added to the in vitro incubation medium always depressed T3 uptake by nuclei. Bovine serum albumin had a similar effect. Large amounts of rat serum proteins depressed uptake, but low levels augmented T3 binding through an unknown mechanism. It is probable that free T3 in serum is in equilibrium with free T3 in the cytosol and nucleus, and binds directly to nuclear receptor proteins without mediation by a cytosol receptor protein.
Molecular and Cellular Endocrinology, 1975
have been prepared from porcine thyroid glands using sucrose gradients.
Biochimie, 1974
The distribution of cyclic AMP-dependent protein kinase activity in porcine thyroid glands has be... more The distribution of cyclic AMP-dependent protein kinase activity in porcine thyroid glands has been studied. Enzyme activity catalyzing phosphorylation of exogenous substrate (protamine) from ATP, and cyclic AMP binding were determined in parallel in subcellular fractions purified by differential centrifugation and flotation on sucrose density layers. Both activities were found in all the studied fractions ; they were quantitatively the highest in the cytosol but particles showed the highest specific activities.
Endocrinology, 1977
Protein kinase activities were determined in liver from normal, thyroidectomized and triiodothyro... more Protein kinase activities were determined in liver from normal, thyroidectomized and triiodothyronine (T3)-treated rats. Changes related to thyroid hormone were observed in cytosol and nuclear protein kinase activities. When protamine was used as substrate for phosphorylation, thyroidectomy induced a decrease of protein kinase activity associated with nuclei but an increase of activity was found in the cytosol. Fifteen hours after injection of T3 the levels in nuclei and cytosol were restored to normal. When casein was used as substrate, hypothyroidism led to a lowering of protein kinase activity in both fractions and T3 treatment augmented the activity in both. These studies suggest that thyroid hormones modify hepatic protein kinase activity. Results differ depending upon the substrate used. The hormones also appear to alter the subcellular distribution of some protein kinase activities.