Formation and characterisitcs of hepatic dexamethasone-receptor complexes of different molecular weight

The dexamethasone-binding receptor protein in rat liver cytosol has a Stokes radius of 61 Å and a sedimentation coefficient of 4.0 S. In contrast, cell nuclei labelled with [³H]dexamethasone in vivo or in vitro (reconstitution experiments with [³H]dexamethasone-labelled cytosol and isolated unlabelled nuclei) contain a high-salt-extractable dexamethasone-receptor complex with a Stokes radius of 30–36 Å and a sedimentation coefficient of 3.2 S. Exposure of liver homogenate or 1000 × g homogenate supernatant to low ionic strenght during preparation of cytosol resulted in conversion of the 61 Å to a 36 Å complex very similar to the intranuclear form of dexamethasone receptor. 61 → 36 Å complex-verting activity was present in both the 100 × g ?10 000 × g sediment of liver homogenate, from which it could be extracted by hypotonic media, and in the liver cell nuclei, from which it could be extracted by hypertonic media. Mild digestion of the 61 Å dexamethasone-receptor complex with trypsin also gave rise to a complex with a Stokes radius of 36 Å. Reconstitution experiments with isolated liver cell nuclei indicated that both the 61 Å and 36 Å dexamethasone-receptor complexes were taken up by the nuclei; reextraction of the nuclei incubated with the 61 Å complex revealed that this form had been converted to the 30–36 Å complex.Further digestion of teh 61 and 36 Å [³H]dexamethasone-receptor complexes with hypotonic extract of the 1000 × g ?10 000 × g sediment of liver homogenate or with trypsin resulted in formation of a third complex with a Stokes radius of 19 Å and a sedimentation coefficient of 2.5 S. The approximate molecular weights of the 61, 36 and 19 Å dexamethasone-receptor complexes were calculated as 102 000, 46 00 and 19 000, respectively, and the frictional ratios of the molecules as 1. 84, 1. 38 amd 1.00, respectively.It is concluded that the nuclear 30–36 Å dexamethasone-receptor complex is formed from the cytosol 61 Å complex by proteolytic digestion and that this latter protein contains at least two sites with a relatively high sensitivity to protelytic cleavage.     

Interaction of ATP with a macromolecular translocation inhibitor of the nuclear binding of “activated” receptor-glucocorticoid complex

Incubation of 1–5 mM ATP with nuclei and partially purified “activated” receptor-[³H]triamcinolone acetonide complex from rat liver cytosol had no significant effect on association of the activated complex with the nuclei. However, when the nuclear uptake was reduced by the macromolecular translocation inhibitor in the rat liver cytosol, addition of 5 mM ATP restored the uptake to the level without inhibitor. ADP and AMP as well as other nucleotides tested could not overcome the inhibitory effect of macromolecular inhibitor.     

Effects of theophylline on the activation and nuclear translocation of the hepatic glucocorticoid receptor at low temperature.

$\text{In vitro}$ nuclear binding of the (³H) dexamethasone-receptor complex from rat liver cytosol takes place at a slow rate when nuclei are incubated at low temperature. However, if theophylline is included during the incubation there is a threefold increase in the rate of nuclear binding. The activation by theophylline is independent of its known effect on cyclic AMP levels since the cyclic nucleotide has no effect on nuclear binding either in the absence or presence of theophylline. Activation ability is specific to methylxanthines and not to nucleoside derivatives. Theophylline may be acting directly on the (³H) dexamethasone-receptor complex converting it to an active form.     

Interaction of sodium thiocyanate with rat hepatic glucocorticoid-receptor complexes

0.1–0.3 M sodium thiocyanate greatly enhanced the rate of inactivation of unbound rat hepatic glucocorticoid receptors in vitro at 4°C. Prior treatment of the unbound glucocorticoid receptor with 10 nM molybdate (at 25°C for 30 min) protected the receptor from 0.3 M KCl, but not from 0.3 M NaSCN inactivation. When the [³H]dexamethasone-receptor complex was examined on sucrose density gradients containing 0.1 M NaSCN, the receptor sedimented as a 4 S complex rather than the 7 S form observed in 0.1 M KCl gradients. NaSCN was found to be more effective in the extraction of both in vivo and in vitro nuclear-bound [³H]dexamethasone-receptor complexes than KCl. At a concentration of 0.3 M, NaSCN extracted most of the specific nuclear-bound receptor. 50 mM NaSCN significantly blocked the thermal activation of preformed [³H]dexamethasone-receptor complexes. The chaotropic salt, NaSCN, appears therefore to have significant effects on glucocorticoid receptors in vitro. In addition, NaSCN appears to be a useful agent in quantitative extraction of steroid from nuclear-bound steroid-receptor complexes.     

Effects of sodium tungstate on the nuclear uptake of glucocorticoid-receptor complex from rat liver

Effects of sodium tungstate on the nuclear uptake of rat liver cytosolic glucocorticoidreceptor complex were examined at pH 7. The nuclear uptake of heat-activated [³H]triamcinolone acetonide-receptor complex was blocked completely in the presence of 1 mm tungstate. A preincubation of nuclear preparation with tungstate (>0.1 mm) blocked the subsequent uptake of [³H]triamcinolone acetonide-receptor complex. When the tungstate-treated nuclear preparation was washed with 0.3 M KCl, its [³H]triamcinolone acetonide-receptor complex binding capacity recovered to 50% of that of control samples with no tungstate treatment. A preincubation of chromatin with tungstate yielded similar results. The nuclear-bound [³H]triamcinolone acetonide-receptor complex, formed either by an in vivo administration of [³H]triamcinolone acetonide or by an in vitro incubation of glucocorticoid-receptor complex with isolated nuclei, was extracted by tungstate in a concentration-dependent manner. The majority of nuclear-bound [³H]triamcinolone acetonide could be extracted with 0.1 and 1 mm tungstate from in vitro- and in vivo-labeled nuclei, respectively. The tungstate-extracted steroid-receptor complexes sedimented in 4–5 S and 3.3–3.5 S region in 10 mm KCl- and 0.3 mm KCl-containing sucrose gradients, respectively. Tungstate treatment caused an irreversible loss of the nuclear binding capacity of [³H]triamcinolone acetonide-receptor complex which could not be recovered after dialysis. These studies indicate that tungstate affects both glucocorticoidreceptor complex and certain nuclear or chromatin proteins.     

Studies of kidney plasma membrane adenosine-3′,5′-monophosphate-dependent protein kinase

Porcine kidney cortex was utilized for the preparation of plasma-membrane-enriched and soluble cytoplasmic (cytosol) fractions for the purpose of examining the relative properties of cyclic [³H]AMP receptor and cyclic AMP-dependent protein kinase activities of these preparations. The affinity, specificity and reversibility of cyclic [³H]AMP interaction with renal membrane and cytosol binding sites were indicative of physiological receptors.Binding sites of cytosol and deoxycholate-solubilized membranes were half-saturated at approx. 50nM and 100 nM cyclic [³H]AMP. Native plasma membranes exhibited multiple binding sites which were not saturated up to 1 mM cyclic [³H]AMP. Modification of the cyclic phosphate configuration or 2′-hydroxyl of the ribose moiety of cyclic AMP produced a marked reduction in the effectiveness of the cyclic AMP analogue as a competitor with cyclic [³H]AMP for renal receptors. The cyclic [³H]AMP interaction with membrane and cytosol fractions was reversible and the rate and extent of dissociation of bound cyclic [³H]AMP was temperature dependent. With the plasma-membrane preparation, dissociation of cyclic [³H]AMP was enhanced by ATP or AMP.Assay of both kidney subcellular fractions for protein kinase activity revealed that cyclic AMP enhanced the phosphorylation of protamine, lysine-rich and arginine-rich histones but not casein. The potency and efficacy of activation of renal membrane and cytosol protein kinase by cyclic AMP analogues such as N⁶-butyryl-adenosine cyclic 3′,5′-monophosphate or N⁶,O²-dibutyryl-adenosine cyclic 3′,5′-monophosphate supported the observations on the effectiveness of cyclic AMP analogues as competitors with cyclic [³H]AMP in competitive binding assays.This study suggested that the membrane cyclic [³H]AMP receptors may be closely associated with the membrane-bound catalytic moiety of the cyclic AMP-dependent protein kinase system of porcine kidney.     

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.     

Protamine increases the affinity of 3′-phosphoadenosine 5′-phosphosulfate toward a sulfotransferase from chicken embryo epiphyseal cartilage

A 3′-phosphoadenosine 5′-phosphosulfate (PAPS):chondroitin sulfate sulfotransferase from chicken embryo epiphyseal cartilage, which was partially purified, exhibited a molecular mass of 150 kDa. The enzymatic sulfation of totally desulfated chondroitin was activated up to 12-fold by protamine while the sulfation of partially sulfated chondroitin was activated only 3-fold. Protamine increased the affinity of the enzyme for PAPS about 4-fold when partially desulfated chondroitin was used as sulfate acceptor. The S_0.5 for the totally desulfated chondroitin was not affected by protamine, while high PAPS concentration slightly increased the affinity of the enzyme for the same sulfate acceptor. The possible role of these substances in the regulation of the sulfation of chondroitin sulfate is discussed.     

The steroid ligands of estrogen binding proteins in Xenopus laevis liver cytosol are primarily metabolites of 17β-estradiol

The interactions in vitro between [³H]estradiol and liver proteins from Xenopus laevis have been examined to determine if the binding reaction meets criteria of steroid-receptors which may function in the induction of vitellogenesis. Estrogenic hormones associated with proteins in serum and liver cytosol from Xenopus laevis. However, the interactions between soluble liver proteins and estrogens apparently do not result from serum contamination of liver as specific binding was distinguishable by ligand affinity and by differential mobility on polyacrylamide gels. Steroid ligands bound by liver proteins during incubation in vitro were examined by solubility and by thin-layer chromatography. Only a small percentage (13%) of the bound radioactive ligand was recovered as the original tritium-labeled steroid, 17β-estradiol. The major ligand was recovered as a water-soluble metabolite of estradiol which was identified tentatively as an estradiol-glucoside. To investigate whether the protein-bound estradiol metabolite(s) merely masks a small amount of authentic estradiol-receptor complexes or if the metabolite could be an intermediate in estrogen function, isolated liver nuclei were incubated with liver cytosol containing ³H-labeled steroid-protein complexes or with serum protein-bound [³H]estradiol. Nuclei preferentially accumulated ³H-labelea steroids from liver cytosol protein-steroid complexes relative to [³H]estradiol from serum proteins. However, analysis of the steroids recovered in the nuclei after incubation with liver cytosol revealed that both 17β-[³H]estradiol and the ³H-labeled water-soluble metabolite were retained in vitro by nuclei.     

Age-dependent activation of glucocorticoid receptors in the liver of male rats

The binding of hepatic [3H] dexamethasone-receptor complexes to DNA-cellulose and purified nuclei was studied in the immature (3-week) and mature (26-week) Long-Evans male rats to determine the age-associated changes, if any, in the physicochemical properties of glucocorticoid-receptors. Our data show that heat activation (for 45 min at 25 degrees C) significantly enhances the binding of [3H] dexamethasone-receptor complexes to DNA-cellulose and purified nuclei at both the ages, with a greater magnitude in mature rats. Cross-mixing experiments (i.e. binding of activated cytosol from mature rats to nuclei of immature and vice-versa) show receptor specificity. Ca2+ activation (20mM Ca2+ for 45 min at 0 degrees C) also enhances the nuclear and DNA-cellulose binding at both the ages but to a similar extent. These findings indicate that some of the physicochemical properties (e.g. heat activation) of glucocorticoid receptor change, while others (e.g. Ca2+ activation) remain unchanged at these phases of the life span. The observed changes may lead to functional alterations in the tissue response as a function of age.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

Difference in extractability of estradiol- and tamoxifen-receptor complex in the nuclei from MCF-7 cells with nonidet P-40

The extraction of [³H]estradiol- and [³H]tamoxifen-receptor complex in the nuclei from MCF-7 cells with the nonionic detergent Nonidet P-40 has been studied. We found that there is a striking difference in the extractability of estradiol- and tamoxifen-receptor complex from nuclei with 0.5% Nonidet P-40. The nuclear bound estradiol-receptor complex is scarcely extractable with Nonidet P-40. In contrast, almost all of the nuclear bound tamoxifen-receptor complex is extractable. The nuclear [³H]tamoxifen-receptor complex extracted in the presence of Nonidet P-40 sediments in two peaks at 7 S and 5 S. The latter sedimentation rate is the same with that of the nuclear [³H]tamoxifen-receptor complex extracted with 0.4 M KC1. The nuclear [³H]estradiol-receptor complex extracted with 0.4 M KC1 sediments at 4 S.The results suggest that interaction of tamoxifen-receptor complex with chromatin is different from that of estradiol-receptor complex.     

In situ protamine release: A versatile sample preparation method for the electrophoretic analysis of nuclear proteins on acid/urea-based gels

Protamine sulfate is used to release histones and other basic proteins from the DNA of chromatin. This phenomenon becomes the basis of a versatile method for analysis of the nucleic acid and protein composition of nucleoprotein samples, which is termed here in situ protamine release. When protamine is added to a nucleoprotein sample in 5% acetic acid and 8 m urea, at a concentration of 1.0%, ≥94% of the histones are released from the DNA of chromatin, comparable to the release of histones using sodium dodecyl sulfate. This makes in situ protamine release the method of choice for the analysis of acid-soluble proteins on acid/urea-based gels, where the DNA must be removed from the protein prior to electrophoresis. Compared to DNase I release or acid extraction, protamine release is found to be the simplest, most reliable, and most effective method for removing the acid-soluble proteins from DNA. Protamine is either added to the sample (very much like the detergent, sodium dodecyl sulfate), or electrophoresed through a gel containing nucleoprotein, thus displacing proteins in its path. A serendipitous advantage of protamine is that it can also serve as a carrier for the precipitation of dilute nucleoprotein samples with ethanol, and 3 mm Mg²⁺, to concentrate the nucleoprotein in preparation for analysis. A unique feature of the in situ protamine-release method is that the DNA is not lost or destroyed and can therefore be used for subsequent analysis.     

A low molecular weight inhibitor of steroid receptor activation.

Dilution at 0 degrees of rat liver cytosol incubated with [3H]triamcinolone acetonide provoked an enhanced binding of steroid-receptor complexes to nuclei. The explanation of this phenomenon was found to be an "activation" of the complexes. Dilution acted by decreasing the concentration of a cytosol inhibitor. This reaction was irreversible at 0 degrees: once activated the complexes could not be reversed to the nonactivated state by the addition of inhibitor. The presence of hormone was necessary, since hormone-free receptor molecules could not be activated by dilution. Removal of the inhibitor did not lead to activation of all complexes: after 24 h a "plateau" was attained where 55 to 70% of the complexes were activated. The inhibitor was shown to be a low molecular weight molecule by dialysis, Sephadex G-25 chromatography, ammonium sulfate precipitation, and ultrafiltration. Thus [3H]triamcinolone acetonide-receptor complexes present in a cytosol from which the inhibitor had been removed by Sephadex G-25 chromatography became spontaneously activated at low ionic strength and at 0 degrees. The inhibitor is not a steroid (at least of usual polarity) since it cannot be extracted by methylene chloride or adsorbed by activated charcoal. It is thermostable (resists to 30 min at 100 degrees). Its removal by incubation with a cation exchange resin suggests that it may be positively charged, however it is not complexed by EDTA. This inhibitor must be distinguished from a previously described inhibitor of steroid-receptor complexes binding to nuclei. The latter compound has been shown in various systems to be responsible for an artifactual saturation of nuclear acceptor by steroid-receptor complexes. It inhibits the binding to nuclear acceptors of already activated complexes and is probably a macromolecule. It is thus different from the low molecular weight activation inhibitor described in the present paper.     

Cytosolic and nuclear binding proteins for 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat thymus

The existence of a high-affinity, low-capacity 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-binding species was demonstrated in cytosol from rat thymus. It was sensitive to heat and to pronase, trypsin or chymotrypsin but not to DNAase or RNAase, indicating that it was a protein. An excess of unlabelled 2,3,7,8-tetrachlorodibenzofuran or β-naphthoflavone displaced [³H]TCDD from the binder whereas phenobarbital, pregnenolone-16-α-carbonitrile or dexamethasone did not compete. Using a dextra-coated charcoal assay, the apparent dissociation constant (K_d) of the [³H]TCDD-binder complex was determined to 0.36 nM and the apparent maximum amount of binding sites (B_max) to 68 fmol/mg of cytosolic protein. When analyzed by sucrose density-gradient centrifugation at high ionic strength, the [³H]TCDD-binder complex sedimented at 4?5 S; at low ionic strength the complex sedimented more rapidly, probably due to aggregation. All these data support the interpretation that the demonstrated cytosolic TCDD-binder represents the receptor protein for TCDD, as previously described for rat and mouse liver. Following intravenous administration of [^3H]TCDD, a low-capacity [³H]TCDD-macromolecule complex was extractable from thymic cell neuclei; this complex behaved identically to the cytosolic [³H]TCDD-receptor complex when exposed to heat or to hydrolytic enzymes and was therefore alos identified as a protein. The nuclear [³H]TCDD-protein complex sedimented at 4–5 S at high ionic strength. Furthermore, a maximum uptake of [³H]TCDD in thymic nuclei was observed simultaneously with a decline in cytosolic radioactivity (at 3 h post-injection). These findings suggest that the nuclear [³H]TCDD-protein complex represented [³H]TCDD-receptor complex translocated from the cytoplasm. In conclusion, the rat thymus contains a cytosolic TCDD receptor at a concentration similar to that of the rat hepatic receptor. However, in vivo experiments showed that the nuclear uptake of [³H]TCDD (expressed as dpm/mg GNA) in the thymus was only about 6% of that in liver. Further studies are needed for an understanding of the mechanism behind this discrepancy.     

Degradation without apparent change in size of molybdate-stabilized nonactivated glucocorticoid-receptor complexes in rat thymus cytosol

We have investigated the stability of the [3H]dexamethasone 21-mesylate-labeled nonactivated glucocorticoid-receptor complex in rat thymus cytosol containing 20 mM sodium molybdate. Cytosol complexes were analyzed under nondenaturing conditions by gel filtration chromatography in the presence of molybdate and under denaturing conditions by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. When analyzed under nondenaturing conditions, complexes from fresh cytosol and from cytosol left for 2 h at 3 degrees C eluted from gel filtration as a single peak of radioactivity with a Stokes radius of approximately 7.7 nm, suggesting that no proteolysis of the complexes had occurred in either cytosol. When analyzed under denaturing conditions, however, whereas the fresh cytosol gave a receptor band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr approximately 90,000 (corresponding to the intact complex), the cytosol that had been left for 2 h at 3 degrees C gave only a fragment (Mr approximately 50,000). This fragment, just as the intact complex, could be thermally activated to a DNA-binding form. Proteolysis of the receptor could be blocked by preparing the cytosol in the presence of EGTA, leupeptin, or a heat-stable factor present in the cytosol of rat liver and WEHI-7 mouse thymoma cells. From these results we conclude: (i) 20 mM molybdate does not protect the nonactivated glucocorticoid-receptor complex present in rat thymus cytosol against proteolysis under conditions which are commonly used for cell-free labeling of the receptor, and (ii) the demonstration of a Stokes radius of approximately 8 nm for the nonactivated glucocorticoid-receptor complex is not sufficient to indicate that the receptor complex is present in its intact form.     

In Vitro Gibberellin A(1) Binding in Zea mays L

The first and second leaf sheaths of Zea mays L. cv Golden Jubilee were extracted and the extract centrifuged at 100,000g to yield a supernatant or cytosol fraction. Binding of [³H]gibberellin A₁ (GA₁) to a soluble macromolecular component present in the cytosol was demonstrated at 4°C by Sephadex G-200 chromatography. The binding component was of high molecular weight (HMW) and greater than 500 kilodaltons. The HMW component was shown to be a protein and the ³H-activity bound to this protein was largely [³H]GA₁ and not a metabolite. Binding was pH sensitive but only a small percentage (20%) appeared to be exchangeable on addition of unlabeled GA₁. Both biologically active and inactive GAs and non-GAs were able to inhibit GA₁ binding. [³H]GA₁ binding to an intermediate molecular weight (IMW) fraction (40-100 kilodaltons) was also detected, provided cytosol was first desalted using Sephadex G-200 chromatography. Gel filtration studies suggest that the HMW binding component is an aggregate derived from the IMW fraction. The HMW binding fraction can be separated into two components using anion exchange chromatography.     

The use of a tritium release assay to measure 6-N-trimethyl-L-lysine hydroxylase activity: synthesis of 6-N-[3-3H]trimethyl-DL-lysine

6-N-[3-³H]Trimethyl-dl-lysine was synthesized from 6-N-acetyl-l-lysine by the following chemical scheme: 6-N-acetyl-l-lysine → 2-keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid oxime → 6-N-[3-³H]acetyl-dl-lysine → dl-[3-³H]lysine → 2-N-[3-³H]formyl-dl-lysine → 2-[3-³H]formyl-6-N-trimethyl-dl-lysine → 6-N-[3-³H]trimethyl-dl-lysine. Using a 70% ammonium sulfate fraction obtained from a high-speed rat kidney supernatant, the cosubstrate and cofactor requirements for 6-N-trimethyl-l-lysine hydroxylase activity as measured by tritium release from 6-N-[3-³H]trimethyl-dl-lysine were: α-ketoglutarate, ferrous ions, l-ascorbate, and oxygen, with added catalase showing a slight but distinct stimulatory effect. On incubation with the crude rat kidney preparation, the release of tritium from 6-N-[3-³H]trimethyl-dl-lysine was linear with both time of incubation and protein concentration. Hydroxylation of 6-N-trimethyl-l-lysine, as measured by tritium release from the labeled substrate, was examined in rat kidney, heart, liver, and skeletal muscle tissues, and found to be most active in the kidney.     

Activated steroid-receptor complex comparison of assays using DNA-cellulose or homologous nuclei

When soluble steroid-receptor complexes are exposed to DNA-cellulose only activated complexes bind. The specificity of the binding was shown by its dependence on the presence of hormone during activation. However, prolonged incubation of non-activated steroid-receptor complexes with DNA-cellulose led to a progressive activation of these complexes. When the same hepatic cytosol containing heat-activated [³H]triamcinolone acetonide-receptor complexes was titrated by high concentrations of nuclei or DNA-cellulose the former bound 75% of the complexes, the latter only 40%. This decreased binding was due on the one hand to a lower initial interaction between DNA-cellulose and activated complexes than between nuclei and these complexes and on the other hand to increased losses during washes when DNA-cellulose was used. For these reasons nuclei and not DNA-cellulose should be used when accurate measurements of the concentration of activated complexes are required. When only comparative data are needed DNA-cellulose may, however, be employed.