3H]-Thyroliberin (TRH) binding to nuclei isolated from a pituitary clonal cell line (GH3).

[3H]Thyroliberin (TRH) has been previously shown to enter its target GH3 cells. Intracellular [3H]-TRH was found chemically unmodified and associated to organites, cytosol and nucleus. We studied the [3H]-TRH binding capacity of a highly purified nuclear fraction isolated by an original procedure from GH3 cells. The nuclei still presented their double nuclear envelope. They are able to bind [H]-TRH to the same extent as nuclei isolated from GH3 cells previously exposed to [3H]-TRH. The equilibrium of binding was reached after 2--5 min incubation at 25 degrees or 35 degrees C. The binding is stable at 4 degrees C and partially (50%) dissociated within 15 min at 25 degrees C. 50% of the binding was inhibited by large excess of unlabelled TRH. Nuclei obtained from a variant GH3 cell which has lost its responsiveness to TRH presented only the noncompetitive binding compartment. The binding was found dose dependent and not saturable. Two apparent dissociation constants were evaluated: 1.5--2.5 x 10--8M and 2.10--6M, respectively, for high and low doses of [3H]-TRH. The first one was identical to that previously found for intact GH3 cells. The present data show the existence of specific nuclear binding sites for TRH, establish their characteristics and suggest a possible nuclear site of action for that peptide hormone.     

RBC-mediated microinjection of chromatin components into cultured mammalian cells

Radiolabeled DNA fragments or nuclear proteins were encapsulated within human erythrocytes, and the erythrocytes were then fused with cultured mammalian cells using Sendai virus. Autoradiography revealed that 125I-labeled DNA fragments remained dispersed in the cytoplasm and disappeared with a half-life of 24 hours. In contrast, the nuclear proteins, HMG1, HMG2, HMG17 and histone H1, rapidly localized within HeLa nuclei and exhibited half lives greater than 80 hours. Several biochemical criteria indicate that the association of the injected nuclear proteins with chromatin faithfully mimics the behavior of their endogenous counterparts.     

Oestradiol binding to nuclei of anterior pituitary cells of the ram

The methodology to fully characterise nuclear receptor for oestradiol-17 beta (E2) in the ram pituitary has been investigated. Purified nuclei, clean under the electron microscope, were obtained from 2.4 M sucrose ultracentrifugation and were extracted for 2 h at 0 degrees C with 0.6 M NaCl. After centrifugation, the supernatant was incubated with [3H]E2 with or without a 100-fold excess of unlabelled E2. The main results were: the specific binding was maximum at 20 degrees C in 2-3 h and remained constant up to 19 h without significant metabolism; an incubation temperature of 25 degrees C reduces the binding, while at 0 degrees C maximum binding was attained at a much slower rate; the binding was linearly related to the dose of nuclear proteins; the binding was not affected by DNase and RNase but was suppressed by trypsin, pronase or a temperature of 56 degrees C; binding was specific for oestrogens; preincubation of cytosol with [3H]E2 and then coincubation with nuclei showed an uptake of the [3H]E2 receptor complex by nuclei; such a transfer was inhibited if cytosol was previously heated; after a prelabelled cytosol-nuclei coincubation, a specific binding peak was found in the nuclear extract submitted to sucrose gradient sedimentation (4.1S); in vivo injection of 100 micrograms E2 resulted in a sharp increase in nuclear receptor numbers 30 and 60 min later, with a concomitant drop in cytosolic receptor numbers. These results indicate that E2 can bind to pituitary nuclei in the ram.     

Skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin.

The proteasome (the multicatalytic endoproteinase complex) in mammalian tissues hydrolyzes proteins and several types of peptides. When this structure was isolated rapidly from rabbit skeletal muscle in the presence of glycerol, its various peptidase and protease activities showed a large reversible activation by physiological concentrations of ATP (Ka = 0.3-0.5 mM). Hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) was stimulated up to 12-fold by ATP, whereas degradation of casein and bovine serum albumin increased 4- to 7-fold. Neither ADP nor AMP had any effect. CTP, GTP, UTP, and the nonhydrolyzable analogs adenosine 5'-[beta,gamma-imino]triphosphate (AMPP[NH]P) and adenosine 5'-[alpha,beta-methylene]triphosphate (AMP[CH2]PP) increased peptide hydrolysis as well as ATP did. However, only ATP stimulated casein breakdown and only in the presence of Mg2+. Thus, nucleotide binding allows activation of the peptidase functions, but ATP hydrolysis seems necessary for enhanced degradation of proteins. The ATP effect on proteolysis was reversible and did not require ubiquitin. Sensitivity to ATP was labile, and with storage at 4 degrees C the enzyme became fully active in the absence of ATP or Mg2+. The ATP-activated form closely resembles the proteasome complex described previously, which did not show ATP dependence: both have molecular masses of 650 kDa, contain the same 8-10 subunits, and are precipitated by the same antibodies. A similar ATP-activated form was found in rabbit liver but not in rabbit reticulocytes. The proteasome seems to represent a ubiquitin-independent, ATP-stimulated proteolytic activity within nucleated mammalian cells.     

Role of nucleotides in tubulin polymerization: effect of guanylyl 5''-methylenediphosphonate.

Incubation of 48,000 X g rat brain supernatants for 30 min at 37 degrees with 1-2 mM guanylyl 5'-methylenediphosphonate [Gmp(CH2)pp] results in polymerization of 95-98% of the tubulin present. This is considerably more than the 50% polymerization that can be achieved with the natural nucleotide, GTP, under optimal conditions. Gmp(CH2)pp is also much more effective than GTP in inducing polymerization of purified tubulin. Assembly of microtubules with Gmp(CH2)pp occurs at tubulin concentrations one-third of those possible with GTP. Furthermore, with Gmp(CH2)pp, microtubule assembly does not require the high molecular weight basic proteins needed with GTP. Polymerization of tubulin by Gmp(CH2)pp is neither prevented nor reversed by concentrations of calcium (2 mM) that can either prevent microtubule assembly or disrupt already formed microtubules if the nucleotide used is GTP or guanylyl imidodiphosphate. When Ca2+ is added before or after microtubule assembly, electron microscopy of the Gmp(CH2)pp preparations reveals normal microtubules turning into twisted ribbons. Low temperature (4 degrees) can both prevent and disrupt the tubulin assembled Gmp(CH2)pp although disruption proceeds much more slowly when GTP is used.     

Resistance to phosphatase of thiophosphorylated epidermal growth factor receptor in A431 membranes.

Epidermal growth factor (EGF) increases the phosphorylation of its receptor and other membrane proteins, and these proteins can be rapidly dephosphorylated by membrane-bound protein phosphatase [Carpenter, G., King L., Jr., & Cohen, S. (1979) J. Biol. Chem. 254, 4884]. We report that [35S]-adenosine 5'-[gamma-thio]triphosphate is as effective as [gamma-32P]ATP as substrate for the EGF receptor-associated protein kinase in A431 membranes. Both the kinetics and the extent of the EGF-dependent thiophosphorylation at 0 degrees C are similar to those obtained with [gamma-32P]ATP, provided that ATP hydrolysis by the membrane preparation is inhibited by addition of adenosine 5'-[beta, gamma-imino]-triphosphate. The thiophosphorylation reaction requires Mn2+ but differs from the phosphorylation reaction in the inability of Mg2+ to serve as cofactor. Both EGF-dependent phosphorylated and thiophosphorylated membrane proteins yield the same two major bands of Mr 145,000-160,000 in autoradiograms of NaDodSO4/polyacrylamide gel electrophorograms. The rate of dephosphorylation of membrane proteins that have been thiophosphorylated in the presence of EGF is dramatically slower (factors of 1/20 to 1/40) than that of the phosphorylated proteins at both 0 degrees C and 32 degrees C. This increased metabolic stability of the thiophosphorylated proteins will be useful for investigation of the role of phosphorylation in the biological effects of EGF.     

Mechanism of elongation of primed DNA by DNA polymerase delta, proliferating cell nuclear antigen, and activator 1.

In the presence of a single-stranded-DNA-binding protein (SSB), the elongation of primed DNA templates by DNA polymerase delta (pol delta) is dependent on ATP and two protein factors, activator 1 (A1) and proliferating cell nuclear antigen (PCNA). We have examined the interaction of these proteins with (dA)4500.(dT)12-18 by measuring their ability to form stable complexes with this DNA. In the presence of ATP, A1, PCNA, and pol delta formed a stable complex with DNA that could be isolated by gel filtration. Incubation of the isolated complex with dTTP resulted in the synthesis of poly(dT). While ATP was required for the formation of this complex, it was not required for the subsequent elongation of DNA. The temporal requirements for complex formation were determined. A1 was found to bind first, followed by the ATP-dependent addition of PCNA to the A1.DNA complex, while pol delta was added last. Each of these complexes could be isolated by gel filtration, indicating that they possessed a high degree of stability. The binding of PCNA to the A1-SSB-coated primed DNA occurred with adenosine 5'-[gamma-thio]triphosphate as well as ATP. However, the binding of pol delta to the PCNA.A1-DNA complex was observed only when the latter complex was formed in the presence of ATP. The complete complex was formed after incubation at 37 degrees C for 2 min, whereas no complex was detected after incubation at 0 degree C. These results indicate that these proteins act in a manner analogous to the accessory proteins that play critical roles in the elongation reaction catalyzed by T4 phage DNA polymerase and Escherichia coli DNA polymerase III.     

Androgen binding in nuclear matrix of human genital skin fibroblasts from patients with androgen insensitivity syndrome

Specific sex steroid-binding sites are associated with the salt-insoluble nuclear matrix from which lipids, histones, and chromatin have been extracted. In intact cultured normal human genital skin fibroblasts incubated for 1 h at 37 C with a saturating concentration (2 nM) of [3H]dihydrotestosterone [( 3H]DHT), approximately 50% of the total intracellular androgen receptor-steroid complexes were found in the nucleus. Within isolated nuclei from such cells, 28-49% of the specific androgen receptor binding was associated with the nuclear matrix. The antiandrogen cyproterone acetate inhibited DHT binding within the nuclear matrix. Cultured genital skin fibroblasts from two unrelated patients with receptor-positive complete androgen insensitivity (CAIS, AR+), had normal (approximately 50%) nuclear binding of DHT, and 35% and 45% of it was localized to the nuclear matrix. Genital skin fibroblasts from a patient with receptor-negative complete androgen insensitivity (CAIS, AR-) had no specific DHT binding in isolated nuclei or nuclear matrix. Scatchard analysis of specific DHT binding in the nuclear matrix isolated from cells of normal subjects after an in vitro exchange assay (0 C; 24 h) revealed the presence of saturable (maximum binding, approximately equal to 200 fmol/mg nuclear DNA), high affinity (Kd approximately equal to 1.0 nM) binding sites. By contrast, in the nuclear matrix isolated from cells of a patient with CAIS, AR+, the binding affinity for DHT was 3-fold lower (Kd approximately equal to 3.0 nM). When cytosolic androgen receptor-DHT complexes prepared from cells preincubated at 37 C for 1 h with [3H]DHT were incubated at 0 C for 1 h with isolated nuclei and nuclear matrix in the presence of 0.15 M KCl, 40-60% of specific nuclear binding was associated with the nuclear matrix. In these cell-free in vitro experiments, radiolabeled DHT-receptor complexes prepared from normal or mutant cells were mixed with isolated nuclei and nuclear matrix prepared from cells of normal subjects or patients with CAIS, AR+ or CAIS, AR-. Under these conditions, specific DHT binding in nuclei and nuclear matrix was quantitatively similar in the presence of a mutant (CAIS, AR+) receptor-steroid complex or in the presence of nuclei or nuclear matrix from the mutant cells (CAIS, AR- or AR+) when compared simultaneously with the same subcellular fractions prepared from the cells of normal subjects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Existence of nuclear thyroid hormone receptors in the porcine thyroid gland and the rat thyroid cell line FRTL-5

We have investigated whether nuclear T3 receptors exist in the thyroid cell. Nuclear proteins extracted from porcine thyroid nuclei with 0.4 mol/l KCl were incubated with [125I]T3. The mixture was then analysed by sucrose density gradient ultracentrifugation which revealed that the T3-binding proteins migrated at the same position of 3.6 S as rat liver nuclear T3 receptors. Fractionation by high performance liquid chromatography using a size exclusion column and an ion exchanger column also demonstrated elution patterns of T3-binding similar to those of the rat liver receptor. Scatchard plots of crude nuclear extracts from porcine thyroid represented a curvilinear pattern. However, when the nuclear proteins partially purified by a DEAE column chromatography were analysed, a single binding component was found; the association constant was 4.1 x 10(10) l/mol and the maximal binding capacity was 602 fmolT3/mg protein. Displacement study with several T3 analogues showed a highly selective affinity for L-T3. Cultured rat thyroid cells of the FRTL-5 line also contained a single class of saturable, high affinity T3-binding site. Subconfluent cells in 100-mm dishes were incubated with increasing amounts of [125I]T3 at 37 degrees C for 3 h and radioactive T3 in isolated nuclei was counted. Scatchard analysis of data showed that the association constant and the maximal binding capacity were 3.44 +/- 0.63 x 10(10) l/mol and 63.7 +/- 17.8 fmolT3/mg protein, respectively. These results strongly suggest that there are nuclear T3 receptors, indistinguishable from the hepatic T3 receptors, in the porcine thyroid and rat FRTL-5 cells.     

Protein kinase activity associated with the D2 hybrid protein related to simian virus 40 T antigen: Some characteristics of the reaction products

Protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) has been found associated with the D2 hybrid protein, a highly purified protein of 107,000 daltons specified by the adenovirus-simian virus 40 (SV40) hybrid Ad2(+)D2, which has many properties associated with authentic SV40 T antigen [Tjian, R. & Robbins, A. (1979) Proc. Natl. Acad. Sci. USA 76, 610-614]. We have now examined some of the biochemical characteristics of the reaction products. Acceptors for the terminal phosphoryl group of [gamma-(32)P]ATP are the purified protein itself and at least four proteins extracted from nuclei of uninfected cells. Purified histones do not serve as substrate for the enzyme. Phosphorylation is markedly reduced by heating the D2 hybrid protein to 50 degrees C for 30 min. The products of phosphorylation are stable to treatment with ethanol/ether, DNase, and RNase, but completely degraded by digestion with Pronase, demonstrating their protein nature. The phosphate bonds are liable to hot alkali and sensitive to digestion with alkaline phosphatase but stable to treatment with hot acid or hydroxylamine. These results provide evidence that (32)P is incorporated into O-phosphoserine or O-phosphothreonine residues of acceptor proteins, indicating that the enzymatic activity is characteristic for protein kinase, and that cell-specified nuclear proteins other than histones may serve as substrates for the enzyme.     

Intracellular transport of 3,5,3'-triiodo-L-thyronine in rat skeletal myoblasts

Since in skeletal muscle circulating L-T3 is the only source for the hormone bound to nuclei, we investigated the intracellular and intranuclear transport of L-T3 in L6E9 rat skeletal muscle cells. The characteristics of this process were assessed by analyzing the nuclear bound L-T3 as a marker of the internalized hormone and by determining the initial rate of L-T3 uptake. [125I]L-T3 cellular uptake at 37 C reached a plateau at 2 h when the nuclear uptake, after an initial lag phase, was still increasing and represented 4.7% of the cellular uptake. Incubation at 4 C caused [125I]L-T3 cellular uptake to decrease by 77% and nuclear uptake to be abolished. A similar effect on [125I]L-T3 nuclear uptake was obtained after myoblasts were incubated at 37 C with a 1000-fold excess of unlabeled L-T3. The addition of various inhibitors of ATP production, cytoskeleton integrity, endocytosis, and Na+, K+-ATPase that did not interfere with [125I]L-T3 binding to the cell surface or to isolated nuclei caused a dose-dependent reduction of both extranuclear and nuclear uptake, ranging from 34-85%. Scatchard analysis revealed the presence on the myoblast surface of L-T3 high affinity (Ka = 1.6 X 10(9) M-1) and low affinity (Ka = 5.4 X 10(6) M-1) binding sites; other iodothyronines exhibited lower affinity for both sites. Kinetic analysis of L-T3 transport after 60-sec incubation at 23 C defined a process with a Km of 17 +/- 5.6 nM and a maximum velocity of 83 +/- 35 pmol/mg DNA. These results indicate the existence in rat myoblasts of a temperature-dependent, energy-requiring, saturable, and stereospecific L-T3 uptake mechanism, probably mediated through an endocytotic pathway, located on the myoblast plasma membrane, that may regulate L-T3 action in skeletal muscle.     

Regulation of DNA replication in S phase nuclei by ATP and ADP pools

Synchronized 3T6 (mouse fibroblast) ghost monolayers (isolated nuclei) were utilized to study the effects of ATP and ADP levels on DNA replication in vitro. A system yielding discontinuous semiconservative DNA replication (without any detectable repair) in synchronized S phase nuclei has been developed. Lack of initiation of new sites has been observed in isolated S phase 3T6 nuclei without the presence of cytoplasmic material; DNA synthesis is comprised only of elongation at sites where initiation had previously taken place. DNA synthesis in S phase nuclei proceeded optimally at an ATP concentration of 4-5 mM. High ATP levels as well as high ATP/ADP ratios (produced by an ATP-regenerating system at a variety of ATP concentrations) yielded marked inhibition of [(3)H]dTTP incorporation. The cellular and nuclear pools of ATP and ADP in intact synchronized 3T6 cells were accurately determined by high-pressure liquid chromatography. A good correlation with the studies on isolated nuclei has been observed. Whereas total cellular ATP pools increase during the progression of 3T6 cells from G(1) to S phase of the cell cycle, nuclear ATP pools do not increase, and the nuclear ATP/ADP ratios decrease once the cells enter the S phase of their cycle. These experiments suggest that nuclear ATP pools and ATP/ADP ratios act as S phase controls, regulating DNA elongation at sites where its synthesis has previously been initiated by cytoplasmic factors.     

Thyroid hormone receptors in a human hepatoma cell line: multiple receptor forms on isoelectric focusing

The nuclear thyroid hormone receptors isolated from cultured human hepatoma cells (Hep G2) were characterized and compared with those from cultured human fibroblasts and rat liver. The Hep G2 nuclear thyroid hormone receptors had an affinity constant (Ka) of 2.1 X 10(10) M-1 and maximal binding capacity (MBC) of 21.0 fmol/100 micrograms DNA for T3 in assays performed on isolated nuclei. 16% of nuclear receptors were released into the media during incubation and had the same Ka. Salt-extracted receptors had a Ka of 1.8 X 10(10) M-1 and MBC of 0.1 pmol/mg protein for T3. Density gradient sedimentation and gel filtration chromatography revealed a sedimentation coefficient of 3.4 S and Stokes radius of 34 A. From these values, a molecular weight of 49,000 and total frictional ratio (f/f0) of 1.4 were calculated, suggesting an asymmetrical shape of the receptor molecule. Heat inactivation occurred with t1/2 of 28.1, 18.0, and 7.9 min at 38, 43, 45 degrees C, respectively. Isoelectric focusing (IEF) of Hep G2 nuclear receptors demonstrated T3 binding proteins at pH 5.3-5.5, 5.7, and 5.9. Evidence that these are nuclear thyroid hormone receptors includes the following: Triiodothyroacetic acid was the most potent competitor of [125I]T3 binding to these proteins followed by L-T3, and L-T4. Cytosolic protein, human serum, and fetal calf serum failed to show the same T3 binding proteins. Ka of these proteins measured by T3 displacement was 1.1-3.2 X 10(9) M-1. Human fibroblast nuclear extract showed similar T3 binding pattern in IEF, except for a slight difference in pI of an acidic band.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triple-helix formation of DNA oligomers with methylthiourea-linked nucleosides (DNmt): a kinetic and thermodynamic analysis

Complementary short-strand DNA homooligomers and methylthiourea-linked homonucleosides associate and form triplexes in solution. The melting temperatures, Tm, the association and dissociation kinetic and thermodynamic parameters, and activation energies were determined by UV thermal analysis for the triplexes of short-strand DNA homooligomers [d(pA)10-d(pA)23] and poly(dA) with the methylthiourea-linked nucleoside [5'-NH3+-d(Tmt)4-T-OH [DNmt5]]. Circular dichroism studies show evidence of triple-helical association dependent on the length of the target homooligomer. The melting and cooling curves exhibit hysteresis behavior in the temperature range of 10-95 degrees C at 0.13 deg/min thermal rate. From these curves, the rate constants and the energies of activation for association (kon, Eon) and dissociation (koff, Eoff) were obtained. Tm decreases with the ionic strength and increases with increase in length of the monomers. The rate constants kon and koff at a given temperature (288 K-310 K) are dependent on the DNA strand length and also decrease and increase respectively with the ionic strength. The energies of activation for the association and dissociation processes are in the range of -18 to -38 kcal/mol and 3 to 18 kcal/mol, respectively. The equilibrium constant for the formation of the triplexes [5'-NH3+-d(Tmt)4-T-OH)2.d(pA)x, x = 10-23] is several orders of magnitude greater when compared with the triplexes of DNA. The number of base triplets in the nucleus of the DNmt2.DNA triple-helix (nucleation-zipping model) increases with decreased DNA oligomer length and with increased ionic strength. The values of DeltaH degrees calculated from the activation parameters are between -30 and -50 kcal/(mol base) and the values of DeltaG degrees are between -6 and -11 kcal/(mol base) for short-strand DNA.     

Cells injected with guanosine 5''-[alpha, beta-methylene]triphosphate, an alpha, beta-nonhydrolyzable analog of GTP, show anomalous patterns of tubulin polymerization affecting cell translocation, intracellular movement, and the organization of Golgi elements.

Injection of the alpha, beta-nonhydrolyzable GTP analog, guanosine 5'-[alpha, beta-methylene]triphosphate (pp[CH2]pG) into PtK2, A549, and Swiss 3T3 cells produced dramatic changes in the normal pattern of long radiating microtubules displayed by the cells before injection. Injection of pp[CH2]pG into cells growing in normal medium resulted in the formation of microtubule bundles resistant to depolymerization by Colcemid and calcium. Cells injected with pp[CH2]pG after incubation with Colcemid for 2 hr showed polymerization of tubulin into long wavy ribbons within 2 hr after injection. Removal of Colcemid 1 hr after the injection of pp[CH2]pG resulted in assembly of tubulin into short single randomly oriented microtubules. All cells injected with pp[CH2]pG showed impeded translocation and restriction or absence of intracellular movement. pp[CH2]pG also prevented the fragmentation of Golgi elements in A549 cells treated with Colcemid. Cells first treated with Colcemid and then injected with pp[CH2]pG failed to reassemble the Golgi elements after the removal of Colcemid. Cells in intimate membrane contact with cells injected with pp[CH2]pG showed similar changes in microtubule polymerization, cell movement, and organization of Golgi elements.     

Nuclear retinoic acid binding protein in human prostate adenomas

A retinoic acid binding protein has been detected in salt extracts of nuclei obtained from human prostate adenoma. The binding was characterized by competition experiments, temperature/time studies and saturation analysis. Substantial binding was only observed after sonication of nuclei and charcoal-pretreatment of a salt extract. The binding of radiolabelled all-trans-retinoic acid was displaced by all-trans-retinoic acid, retinol and to a lesser extent retinal and two synthetic retinoids, RO 10-1670 and RO 13-7410. Testosterone and dihydrotestosterone, at a 100-fold excess, had little effect on the binding. The association between retinoic acid and nuclear protein was both temperature and time dependent. At 37 degrees C, equilibrium was rapidly reached (30 min) whereas at 4 and 25 degrees C, ligand binding occurred at a slower rate. Saturation analysis performed under steady-state conditions yielded a dissociation constant of 15 +/- 2 nmol/l. Metabolism studies failed to show conversion of either radiolabelled all-trans-retinol or [3H]retinoic acid in vitro; these data suggest that both acid and alcohol forms of vitamin A are recognized by the extracted nuclear protein. The effect of three enzyme inhibitors on [3H]retinoic acid binding was studied. Binding was unaltered in the presence of aprotinin and phenylmethylsulphonyl fluoride but sodium molybdate (10 mmol/l) increased binding by 18%. The presence of a specific retinoid binding protein in prostate nuclei suggests that retinoids may play some role in the function of the gland.     

Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

The direct addition of insulin to highly purified nuclear envelopes prepared from the livers of diabetic rats resulted in a decrease in the incorporation of 32P into trichloroacetic acid-precipitable proteins. Autoradiography of 32P-labeled envelopes, solubilized in sodium dodecyl sulfate and subjected to electrophoresis, revealed that insulin decreased the phosphorylation of all major protein bands. Insulin produced detectable effects at concentrations between 0.1 and 1 pM, maximal effects at 10 pM, and progressively diminished effects at higher concentrations. Two insulin analogs, desdipeptide proinsulin and desoctapeptide insulin, had approximately 10% and 1%, respectively, the activity of native insulin. When nuclear envelopes were first phosphorylated with [gamma-32P]ATP and insulin was then added with an excess of unlabeled ATP, dephosphorylation was enhanced, suggesting that insulin was regulating nuclear envelope phosphatase activity. The direct addition of insulin to isolated rat liver nuclei in the presence of ATP stimulated the release of previously 14C-labeled trichloroacetic acid-precipitable mRNA-like material, and the direct addition of insulin to nuclear envelopes stimulated the activity of nucleoside triphosphatase, the enzyme that participates in mRNA nucleocytoplasmic transport. Moreover, the dose-response curves for these functions mirrored insulin's inhibition of nuclear envelope phosphorylation. These data suggest, therefore, a mechanism whereby insulin directly inhibits the phosphorylation of the nuclear envelope, leading in turn to the regulation of mRNA metabolism.     

Saturable triiodothyronine-binding sites in the pituitary nuclei of salmonid teleost fish

High-affinity, limited-capacity, 3,5,3'-triiodo-L-thyronine (T3)-binding sites were established by in vitro saturation analysis in cell nuclei of the pituitary gland of arctic charr. The sites were extracted from the purified nuclei using 0.4 M NaCl and incubated with [125I]T3 in the presence of 0.2 M NaCl. T3 saturable binding attained equilibrium after 18-24 hr of incubation at 4 degrees. The association constant ranged from 6.7 to 20.1 liters.mol-1 x 10(9), indicating a T3 affinity greater than that for T3-binding sites in rainbow trout liver. The maximal binding capacity ranged from 0.93 to 2.05 10(-13) mol.mg DNA-1, representing a mean site abundance corresponding to 60% of that for nuclei from trout liver. Thyroxine (T4) completely displaced [125I]T3 in the pituitary nuclei of arctic charr and T3 completely displaced [125I]T4 in the pituitary nuclei of rainbow trout, suggesting that in salmonids both T4 and T3 bind to the same single class of sites. However, the site affinity for T4 was approximately 20-50x less than that for T3. The possible roles of these sites in pituitary function as well as their relationship to other nuclear T3-binding sites in salmonid fish are discussed.     

Histone and high mobility group protein phosphorylation in the thyroid: regulation by cyclic nucleotides

A variety of cyclic nucleotide analogs and other agents that affect thyroid cyclic nucleotide metabolism were used to investigate the role of cAMP and cGMP in regulating nuclear protein phosphorylation in calf thyroid slices labeled in vitro with [32P]orthophosphate. Two major groups of acid-soluble proteins were studied. Group I consisted of proteins whose phosphorylation is stimulated by TSH [histones H1 and H3, high mobility group (HMG) protein 14, and the HMG 14/17-like protein PS.3]; group II included representatives of a spectrum of proteins whose phosphorylation is unaffected by TSH (histones H2A, H2B, and H4, HMG 17, the HMG 14/17-like protein PS.2, and the nonhistone protein AS.1). The effects of TSH (50 mU/ml) on the 32P labeling of group I proteins were partially reproduced by (Bu)2cAMP (1 mM), 8-bromo-cAMP (1 mM), and butyrate (2 mM), and closely mimicked by 8-(4-chlorophenylthio)cAMP (1 mM), forskolin (25 microM), and butyrate (10 mM). (Bu)2cGMP (1 mM), 8-bromo-cGMP (1 mM), and carbachol (50 microM) had no effect on protein phosphorylation. NaNO2 (20 mM), which markedly increases cGMP concentration in calf thyroid slices, decreased the 32P labeling of group I proteins and also affected, to varying extents, the phosphorylation of the group II proteins. The phosphodiesterase inhibitor methylisobutylxanthine (0.5 mM) had generally minor effects on 32P labeling; however, it did counteract the effects of NaNO2 on group I protein phosphorylation. Our results provide strong support for the hypothesis that TSH-dependent phosphorylation of group I proteins is mediated by cAMP, but they provide little evidence of cGMP regulation of histone or HMG protein phosphorylation.     

Triiodothyronine binding to liver nuclear solubilized proteins in vitro.

Nuclear proteins extracted from purified nuclei with 0.4M KCl at pH 7.4 OR 8.5 are able to bind L-triiodothyronine (T3) giving rise to nuclear thyroid hormone binding protein-T3 (NTBP-T3) complexes. Binding is maximum in 3 h at 20 C. It is thermolabile even at 36 C, inhibited by p-hydroxymercuribenzoate and markedly enhanced by dithiothreitol. Optimum pH is between 7.8 and 8.5. Divalent cations are not necessary. The NTBP-T3 complex exhibits similar anodal electrophoretic migration in polyacrylamide gel at pH 8.5, whether formed in vivo or in vitro. Scatchard plots obtained with various amounts of T3 from 0.15 nM TO 0.15 MUM and either unlabeled nuclear proteins or in vivo formed NTBP-[125I]-T3 complexes, give apparent association constants K-a of 0.2 X 10-10 M minus at pH 7.4 and 0.8 X 10-10 M minus 1 at pH 8.5. Capacity is about 0.5 pmol T3 per mg protein or 800 pg/g liver. The presence of dithiothreitol markedly enhances the Ka. The nuclear binding sites are not highly specific for L-T3 since they are able to bind D-T3 with almost equal affinity and triiodothyroacetic acid with a higher affinity. L-thyroxine (T4) can also displace L-T3 but with about 10-fold lesser effectiveness. Nuclear binding proteins of low capacity and high affinity have been demonstrated in vitro. The NTBP-T3 complexes formed in vivo, with whole nuclei, or in vitro are indistinguishable.