Reconstitution of ciliary membranes containing tubulin

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.     

Primary biliary cirrhosis sera recognize not only gp210 but also proteins of the p62 complex bearing N-acetylglucosamine residues from rat liver nuclear envelope. Anti-p62 complex antibody in PBC

We have recently observed reactivity of primary biliary cirrhosis (PBC) sera with several proteins bearing N-acetylglucosamine residues from rat liver nuclear envelopes. The aim of this study was to characterize the reactive antigens. Sera from 31 patients with PBC, 30 with rheumatoid arthritis (RA) and 30 with Sj?gren's syndrome (SS) were examined. Rim-like immunofluorescence staining was observed in 15 of 31 (48%) sera from patients with PBC, in 1 of 30 with RA and in 1 of 30 with SS. Upon immunoblotting using preparations of whole rat liver nuclear envelopes and their Triton X 100-KCl extract as antigen sources, a 200 kDa protein band was observed in 9 of sera with PBC. Furthermore, upon immunoblotting using the wheat germ aggulutinin-bound fraction of rat liver envelope as antigen, 62, 60 and 54 kDa protein bands corresponding to components of the p62 complex in the nuclear pore complex (Kita et al. Biochem. 113, 377-382) were observed in 7, 5 and 6 samples respectively, of the 31 PBC sera. Our data suggest that PBC sera recognize not only the 210 kDa protein but also the p62 complex proteins.     

Dimerization of 8-OH-DPAT increases activity at serotonin 5-HT1A receptors

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCl (polymerase M1) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg2+ in preference to Mn2+ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn2+ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata beta-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCl DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCl, used both Mg2+ and Mn2+ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is approximately 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase M1 shows similarities to the Crithidia fasciculata beta-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.     

Phosphatidyl inositol-phospholipase C in squid photoreceptor membrane is activated by stable metarhodopsin via GTP-binding protein, Gq

Phosphatidyl inositol-phospholipase C (PI-PLC) in squid retina was studied by immunoblotting and its activities were determined using [3H]phosphatidyl inositol bisphosphate ([3H]PIP2) as substrate. PI-PLC activity was found mostly in soluble fraction when the retina homogenate was treated with 400 mM KCl, but was associated with rhabdomal membranes under low salt conditions (20 mM Hepes). A protein with apparent molecular mass of 130kD was recognized by an antibody against PLC beta 4/norp A in both 400 mM KCl soluble and rhabdomal membrane fractions. A 42 kD protein recognized by antibody against the C-terminus of Gq alpha was also present in these two fractions. GTP gamma S stimulated only the PI-PLC activity associated with membrane and was magnesium dependent. PI-PLC activity was found to be (i) highly dependent upon calcium concentrations, (ii) enhanced by GTP but not by other nucleotides, and (iii) significantly stimulated by light at lower concentrations of GTP gamma S. The stimulation by light was still observed when irradiated membrane was incubated at 10 degrees C for 10 min and then mixed with GTP gamma S. These results suggest that stable metarhodopsin stimulates a PLC beta 4/norp A-like enzyme via a G-protein, Gq.     

In memoriam Frank Morrell: a man ahead of his time. June 4, 1926-October 22, 1996

Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed by electron microscopy and by the content of beta-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes, this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl.     

Structure, subunit topology, and actin-binding activity of the Arp2/3 complex from Acanthamoeba

The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actin-related proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of approximately 13 x 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 microM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actin-rich regions of Acanthamoeba.     

On the possible role of brain protein synthesis in functional barbiturate tolerance

Pentobarbital pellet implantation increased more than 200% the ED50 dose of pentobarbital required to induce loss of the righting reflex within 2 min of i.p. injection and increased the onset of barbital-induced sleep. Both tests of functional barbiturate tolerance were blocked by the intraventricular injection of cycloheximide. The effects of acute (45 mg/kg i.p.) and chronic (pellet implantation) pentobarbital treatment on the incorporation of 3/-lysine into the protein of various subcellular fractions of the cortex and subcortex were studied. In the subcortex, chronic pentobarbital treatment significantly stimulated protein synthesis 40-50% in the microsomal, soluble and mitochondrial fractions. Both acute and chronic pentobarbital treatments significantly increased (3H-lys)-protein accumulation in a fraction of synaptic plasma membranes derived from a population of nerve ending particles (NEP) enriched in gamma-aminobutyric acid (GABA). The possible significance of these data to pentobarbital tolerance and dependence development is discussed.     

Variation in cytoskeletal assembly during spreading of progressively subcultivated human embryo fibroblasts (IMR-90)

The cytoskeletons of early and late passage IMR-90 human diploid fibroblasts have been directly imaged in replicas of Triton X-100 extracted cells during spreading following reseeding. All cells from both young and sensescent cultures exhibit a cytoskeletal network of actin microfilaments, intermediate (10 nm) filaments, microtubules, and interconnecting thin filaments (6-8 nm in diameter) which do not interact with heavy meromyosin. Early passage cells assemble linear aggregates of actin filaments within 1 h of spreading. By 4 h of incubation, these bundles establish a structural bond with the cell membrane which results in resistance by the plasmalemma to detergent extraction at these sites. Furthermore, these membrane regions are associated with developing stress fibers of well-spread cells. In contrast, late passage cells exhibit slower spreading which correlates with a retarded assembly of actin bundles. In addition, by 8 h of spreading, cells of older cultures do not exhibit the regions of membrane-actin interaction which impart detergent resistance to the plasmalemma. We conclude that the ability to reassemble actin-actin and actin-membrane association during cell spreading is reduced with increased serial subcultivation of cells.     

The germinal vesicle nucleus of Xenopus laevis oocytes as a selective storage receptacle for proteins

The amorphous nucleoplasm of the germinal vesicle nucleus of Xenopus laevis oocytes has been selectively extracted under conditions which leave the nuclear formed elements morphologically intact. The nucleoplasm contains about 97% of the total nuclear proteins and on SDS-polyacrylamide gels some 68 polypeptides can be distinguished. On the basis of solubility differences, the nucleoplasmic proteins can be classified into two categories. The first consists of soluble or easily solubilized proteins which comprise about 34 polypeptides making up 87% of the nucleoplasm. A few of these proteins show electrophoretic mobilities similar to those of soluble proteins of the cytoplasm, but most are unique to the nucleus. The residual 13% of the nucleoplasmic proteins are tightly bound to a nucleoplasmic gel and can be extracted only by solubilizing the gel. The solubility characteristics of the proteinaceous gel suggest a complex held together by salt, nonpolar, hydrogen, and possibly disulfide bonding. Some 34 polypeptides can be distinguished in this gel fraction, including prominent and highly enriched polypeptides of about 115,000 and 46,000 daltons. The relatively soluble fraction of the nucleoplasm does not contain informofers and contains little or no nucleic acid. Evidence is presented that if histones are present in the germinal vesicle, they can comprise no more than about 8% of the total protein. The possibility is discussed that the unique polypeptides of the nucleoplasm may be sequestered there by selective adsorption to or in the nuclear gel.     

Ubiquitin is conjugated to the cytoskeletal protein alpha-spectrin in mature erythrocytes

Ubiquitination of red blood cell (RBC) proteins was investigated by encapsulation of 125I-ubiquitin into human erythrocytes using a procedure of hypotonic dialysis, isotonic resealing, and reannealing. Incubation (37 degrees C, up to 2 h) of 125I-ubiquitin-loaded cells resulted in the recovery of 125I-ubiquitin with the cytosolic proteins (9.22 +/- 0.4 micrograms/ml RBC) and conjugated to membrane proteins (2.18 +/- 0.05 micrograms/ml RBC). This conjugation was time-dependent, and the predominant membrane protein band that became labeled showed an apparent molecular mass of 240 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Western blotting experiments with three different anti-ubiquitin antibodies revealed that this protein is also ubiquitinated in vivo. Cell-free experiments have shown that fraction II (a DEAE-bound protein fraction eluted by 0.5 M KCl) prepared from both mature erythrocytes and reticulocytes is able to conjugate ubiquitin to this protein. Ubiquitin conjugation was ATP-dependent (Km 0.09 mM), time-dependent, and fraction II-dependent (8 +/- 0.5 pmol of 125I-ubiquitin/h/mg of fraction II). Isolation of the major RBC membrane protein that is ubiquitinated was obtained by using biotinylated ubiquitin. Membrane proteins, once ubiquitinated with this derivative, were extracted and purified by affinity chromatography on immobilized avidin. The major components retained by the column were two peptides of molecular masses 220 and 240 kDa. Both proteins are recognized by a monoclonal anti-spectrin antibody, but only the 240-kDa component is detected by streptavidin peroxidase conjugate. That indeed the ubiquitinated membrane protein of 240-kDa is alpha-spectrin was confirmed by immunoaffinity chromatography using 125I-ubiquitin and a monoclonal anti-spectrin antibody. Antigen-antibody complexes were purified by protein A chromatography and analyzed by SDS-PAGE and autoradiography. Again two bands of 240 and 220 kDa were eluted (alpha- and beta-spectrin), but only one band corresponding to the electrophoretic mobility of alpha-spectrin was detected by autoradiography. Thus, alpha-spectrin is a substrate for the ATP-dependent ubiquitination system, suggesting that the cytoskeleton is covalently modified by ubiquitination both in reticulocytes and mature RBC.     

Molecular cloning and characterization of the noncatalytic subunit of the Rab3 subfamily-specific GTPase-activating protein

We recently purified and characterized from rat brain a GTPase-activating protein (GAP) specific for the Rab3 small G protein subfamily implicated in Ca2+-dependent exocytosis. Rab3 GAP showed two bands with Mr of about 130,000 (p130) and 150,000 (p150) on SDS-polyacrylamide gel electrophoresis. p130, but not p150, showed the catalytic activity. Because p150 was likely the subunit of Rab3 GAP, here we cloned the cDNA of p150, determined its primary structure, and characterized it. The tissue and subcellular distribution patterns of p150 and p130 were similar, and both the proteins were enriched in the synaptic soluble fraction. p150 was co-immunoprecipitated with p130 from this fraction. Recombinant p150 formed a heterodimer with recombinant p130 as estimated by sucrose density gradient ultracentrifugation. Recombinant p150 neither showed the Rab3A GAP activity nor affected the activity of recombinant p130. When p150 and p130 were co-expressed in the cells, the subcellular localization of each protein did not change. These results indicate that p150 is the noncatalytic subunit of Rab3 GAP.     

A G-protein-coupled 130 kDa phospholipase C isozyme, PLC-beta 4, from the particulate fraction of bovine cerebellum

A 130 kDa PLC isozyme was purified from the particulate fraction of bovine cerebellum. This PLC was recognized by a polyclonal antiserum generated against the purified 97 kDa PLC-beta 4. Reconstitution of the purified 130 kDa PLC with the membranes of C6 Bu-1 cells in the presence of GTP gamma S or AlF4- resulted in PLC activation as well as the association of PLC with the membranes. Both the association and activation were revoked when the membrane was washed with 2 M KCl. The 97 kDa PLC-beta 4 did not associate with membranes. These data suggest that the 130 kDa PLC is the intact form of PLC-beta 4 the activity of which is likely to be regulated by a G-protein on the membrane.     

Cytoskeletal-associated protein kinase and phosphatase activities from cerebral cortex of young rats

We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 microM cyclic AMP (cAMP), 1 mM calcium and 1 microM calmodulin (Ca2+/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 microM phorbol 12,13-dibutyrate (Ca2+/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca2+/calmodulin, respectively, but was unaffected in the presence of Ca2+/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 microM okadaic acid and 0.01 microM microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.     

Association of influenza virus NP and M1 proteins with cellular cytoskeletal elements in influenza virus-infected cells

We have investigated the association of the influenza virus matrix (M1) and nucleoprotein (NP) with the host cell cytoskeletal elements in influenza virus-infected MDCK and MDBK cells. At 6.5 h postinfection, the newly synthesized M1 was Triton X-100 (TX-100) extractable but became resistant to TX-100 extraction during the chase with a t1/2 of 20 min. NP, on the other hand, acquired TX-100 resistance immediately after synthesis. Significant fractions of both M1 and NP remained resistant to differential detergent (Triton X-114, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS], octylglucoside) extraction, suggesting that M1 and NP were interacting with the cytoskeletal elements. However, the high-molecular-weight form of the viral transmembrane protein hemagglutinin (HA), which had undergone complex glycosylation, also became resistant to TX-100 extraction but was sensitive to octylglucoside detergent extraction, indicating that HA, unlike M1 or NP, was interacting with TX-100-insoluble lipids and not with cytoskeletal elements. Morphological analysis with cytoskeletal disrupting agents demonstrated that M1 and NP were associated with microfilaments in virus-infected cells. However, M1, expressed alone in MDCK or HeLa cells from cloned cDNA or coexpressed with NP, did not become resistant to TX-100 extraction even after a long chase. NP, on the other hand, became TX-100 insoluble as in the virus-infected cells. M1 also did not acquire TX-100 insolubility in ts 56 (a temperature-sensitive mutant with a defect in NP protein)-infected cells at the nonpermissive temperature. Furthermore, early in the infectious cycle in WSN-infected cells, M1 acquired TX-100 resistance very slowly after a long chase and did not acquire TX-100 resistance at all when chased in the presence of cycloheximide. On the other hand, late in the infectious cycle, M1 acquired TX-100 resistance when chased in either the presence or absence of cycloheximide. Taken together, these results demonstrate that M1 and NP interact with host microfilaments in virus-infected cells and that M1 requires other viral proteins or subviral components (possibly viral ribonucleoprotein) for interaction with host cytoskeletal components. The implication of these results for viral morphogenesis is discussed.     

Brefeldin A inhibits the constitutive-like secretion of a sulfated protein in pancreatic acinar cells

Sulfation is a common posttranslational modification of secretory proteins and serves as a valuable marker of constitutive and regulated secretory pathways. We investigated the cellular localization and the secretory behavior of sulfated macromolecules in the mouse pancreatic acinar cell. The major sulfated proteins of the cell were present in isolated zymogen granules, as determined by metabolic labeling with [35S]sulfate and subcellular fractionation. The sulfated proteins fell into three groups: gp300 is not secreted and is a component of the zymogen granule membrane; pancreatic lipase (56 kDa) and a 40 kDa protein are soluble and exhibit regulated secretion kinetics; and p82 is initially granule membrane associated, but is released from the cell with constitutive-like kinetics as a 75 kDa protein (p75). Secretion of p75 could be stimulated for up to 4 h after pulse labeling, presumably from immature secretory granules, but not after 6 h of chase. Treatment of cells with brefeldin A (BFA) at the start of the [35S]sulfate pulse resulted in almost total inhibition of sulfation. Addition of BFA during the chase (0-2 h) allowed normal basal and stimulated secretion of regulated secretory proteins, but reversibly inhibited the constitutive-like secretion of p75. In this case, the behavior of p75 was maintained as that of a regulated secretory protein for up to 6 h of chase. In untreated cells, immunofluorescence of p82/p75 was along the acinar lumen, and in small punctate structures in the apical cytoplasm. In BFA-treated cells, immunolabeling of p82/p75 was lost from the acinar lumen, and cytoplasmic labeling was finer and appeared to be associated with the secretory granule membranes. These data suggest a role for brefeldin A-sensitive coat formation in maturation of secretory granules after they bud from the TGN.     

Cytochalasin B inhibits actin-related gelation of HeLa cell extracts

When the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 is incubated at 25 degrees C, it gels, and actin and a HMWP are progressively enriched in the extract and in gel isolated from extract. CB (greater than or equal to 0.25 muM) inhibits gelation and specifically lowers the concentrations of actin and the HMWP in the fraction which sediments at 100,000 g after incubation. These results indicate that actin and HMWP are partly disaggregated by cytochalasin treatment, and thus that their aggregation is related gelation. Inasmuch as previous results showed that actin is present and HMWP is enriched in the plasma membrane fraction of HeLa cells, the results also point to a possible relation between plasma membrane-associated gel and in vivo effects of CB.     

Protein-nucleic acid recognition: simulation of base and "model" amino acids complexes in DMSO by the Monte Carlo method

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in discrete steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate. Water-soluble cIP was used as the substrate to study the cyclic phosphodiesterase activity and interfacial behavior of PI-PLC. Different detergent micelles and phospholipid vesicles were used to examine if "interfacial activation" of the enzyme could occur toward a soluble substrate. Almost all detergents examined activated the enzyme at least 2-fold, with PC species yielding the largest increases in PI-PLC specific activity. Kinetic parameters were measured in the absence and presence of several representative detergents (e.g., Triton X-100 and diheptanoylphosphatidylcholine (diC7PC)). Gel filtration experiments showed that, under these conditions, the cIP did not partition to any measurable extent with these detergent micelles. The concentration at which half the maximum activation was observed occurred near the detergent CMC. Both Km and Vmax were altered by the presence of a surface: Km decreased to different degrees depending on the detergent, while Vmax increased substantially. The Km for cIP was 90 mM without detergent and decreased to 29 mM with diC7PC micelles added; Vmax increased almost 7-fold in the presence of diC7PC micelles. The enzyme efficiency (Vmax/Km) in the presence of diC7PC increased more than 21-fold, but it was still 20-fold lower than initial phosphotransferase activity for monomeric dihexanoylphosphatidylinositol. The poor efficiency of the cyclic phosphodiesterase activity is largely due to substrate binding affinity. The dependence of rate on substrate concentration exhibits cooperative behavior, especially without detergent. This cooperativity is discussed in terms of protein aggregation and ligand binding sites on the enzyme.     

pICln predominantly localizes at luminal surface membranes of distal tubules and Henle''s ascending limbs

We produced a highly specific antibody to the C-terminal peptide sequence of pICln. It recognized pICln with a 38-kDa molecular mass on SDS-polyacrylamide gel electrophoresis, coinciding with that previously reported. During native polyacrylamide gel electrophoresis, three immunoreactive bands (38, 70, and 130 kDa) were detected. The isoelectric point of pICln was calculated to be 4.0. Subcellular localization study showed the presence of pICln in the soluble and microsomal fraction. pICln can be easily solubilized from the membrane fraction with Triton X-100. From immunohistochemical observations, we found pICln to be obviously located on the luminal surface membranes of the distal tubules and Henle's loop ascending limbs, and it can also be found inside proximal tubular cells. The present results suggested that pICln functions as a "cytosolic anchor = membrane insertion" model, and it plays important roles in the "urine dilution segment" cells of nephrons.     

Antiestrogen action: differential nuclear retention and extractability of the estrogen receptor

Since there is a much longer uterine nuclear retention of the U-11, 100A (antiestrogen) receptor complex (UARC) than of the estradiol receptor complex (ERC) at 4-12 hrs after injection, experiments were designed to determine if there is a difference between the relative nuclear affinities for the two RCs as determined by extraction with various ionic strength mediums. Although the UARC was retained longer in the nuclear fraction in vivo, the UARC was completely extractable with 0.3M KCl or 50mM spermine, whereas the ERC demonstrates a salt-resistant form. This suggests that the ERC is more tightly bound to nuclear components through this salt-resistant form of the receptor. In addition, various intercalating agents were used to distinguish the different nuclear chromatin DNA sites where the UARC and ERC may be binding. With actinomycin D (50 uM) more ERC than UARC was retained in the nuclear fraction. However, with ethidium bromide (100uM) less ERC than UARC was retained. Also, the ERC selectively released by ethidium bromide is precisely that fraction not released by salt. These results indicate that the UARC and ERC bind to different chromatin loci.