Cytosolic 25-hydroxycholesterol binding activity of Chinese hamster ovary cells

DEAE-Sephadex chromatography of cytosols of Chinese hamster ovary cells incubated with tritium-labeled 25-hydroxycholesterol shows a peak of specific binding activity. This binding activity can be assayed by determining the amount of labeled 25-hydroxycholesterol in cytosol which is refractory to adsorption to activated charcoal at high specific activity but can be made to adsorb to charcoal in the presence of a 50-fold excess of unlabeled 25-hydroxycholesterol. The binding activity shows positive cooperatively (Hill coefficient = 2.3 ± 0.3) and high affinity (dissociation constant = 1.4 × 10^?7m). Inactivation of binding by trypsin or boiling suggests that the binding activity is a protein. The sedimentation coefficient of the binding activity is 5 S. Binding of 25-hydroxycholesterol is competitive with several other sterols and correlates well with the concentrations of these compounds that inhibit cholesterol biosynthesis.     

24,25-Epoxysterol metabolism in cultured mammalian cells and repression of 3-hydroxy-3-methylglutaryl-CoA reductase

In view of the potential importance of 24,25-epoxysterols as intracellular regulators of 3-hydroxy-3-methylglutaryl-CoA reductase, the C-24 epimers of 24,25-oxidolanosterol and 24,25-epoxycholesterol were tested for their biological activity and metabolism in cell cultures. All four compounds produced repression of the reductase in cultured mouse fibroblasts (L cells), and both 24(S)- and 24(R),25-epoxycholesterol exhibited high affinity binding to the cytosolic oxysterol-binding protein. However, binding of the epimeric 24,25-oxidolanosterols was not detected. 24(S),25-Epoxycholesterol was not rapidly metabolized in either L cells or Chinese hamster lung (Dede) cells. 24(S),25-Oxidolanosterol was rapidly converted to 24(S),25-epoxycholesterol in both cell lines. 24(R),25-Oxidolanosterol was converted to 24(R)-hydroxycholesterol in Dede cells, but was converted instead to 24(R),25-epoxycholesterol in L cells, which lack sterol delta 24-reductase activity. Although 24(S),25-oxidolanosterol does not appear to accumulate in these cell cultures, it was found in human liver in about one-fifth the amount of 24(S),25-epoxycholesterol. 24(R),25-Epoxycholesterol was also converted to 24(R)-hydroxycholesterol in Dede cells, but not in L cells. Triparanol inhibited the reduction of the 24(R),25-epoxides in Dede cells, consistent with the idea that this reaction is catalyzed by the delta 24-reductase. 24(R)-Hydroxycholesterol and its 24(S) epimer exhibited affinity for the binding protein and repressed 3-hydroxy-3-methylglutaryl-CoA reductase.     

Different forms of the oxysterol-binding protein. Binding kinetics and stability

Based upon measurements of the sedimentation coefficient and the Stokes radii, three forms of the oxysterol-binding protein were identified. The unliganded binding protein was the largest (7.7 S, Stokes radius = 71.6 A, Mr = 236,000) was relatively asymmetric (f/f0 = 1.7), and was composed of at least three subunits. Binding of 25-hydroxycholesterol was associated with a reduction in the size of the protein (7.5 S, Stokes radius = 50 A, Mr approximately 169,000) and an increase in symmetry (f/f0 = 1.4), due to the loss of a subunit of Mr approximately 67,000. At pH 6 or lower, the Mr = 169,000 sterol-protein complex was altered so that reversible dissociation to give a smaller (4.2 S, Stokes radius = 53 A, Mr = 97,000) more asymmetric (f/f0 = 1.8) sterol-protein complex occurred when it was sedimented in a sucrose gradient buffered at pH 7.4 containing 0.3 M KCl and 2.5 M urea. Irreversible dissociation of the 7.5 S, Mr = 169,000 form to a 4.2 S form occurred spontaneously when the complex in whole cytosol buffered at pH 7.8 was allowed to stand overnight at 0 degree C, or when the partially purified complex was incubated at pH 5.5 at 0 degree C for several days. The partially purified, unliganded binding protein was unstable at 0 degree C (approximately 75% loss of binding activity in 24 h) whereas the liganded protein was stable for 7 days at 0 degree C although irreversible conversion to a 4.2 S form occurred under some conditions. Rates of sterol binding and dissociation were increased in the presence of 2.5 M urea at pH 7.4 or when the pH was lowered to 5.5 Kd values were not greatly altered under the various incubation conditions.     

cDNA cloning and expression of oxysterol-binding protein, an oligomer with a potential leucine zipper

Feedback repression of the genes encoding the low density lipoprotein receptor and several enzymes of the cholesterol biosynthetic pathway is mediated by 25-hydroxycholesterol and other oxysterols. In this study, we have cloned a rabbit cDNA encoding an oxysterol-binding protein that may play a role in this regulation. The predicted amino acid sequence revealed a protein of 809 amino acids with two distinctive features: 1) a glycine- and alanine-rich region (63% of 80 residues) at the NH2 terminus, and 2) a 35-residue leucine zipper motif that may mediate the previously observed oligomerization of the protein. When transfected into simian COS cells, the rabbit cDNA produced a protein that exhibited the same affinity and specificity for sterols as the previously purified hamster liver protein. Immunoblotting analysis showed that the rabbit cDNA encodes both the 96- and 101-kilodalton forms of the oxysterol-binding protein that were previously observed. The availability of an expressible cDNA for the oxysterol-binding protein should help elucidate its role in sterol metabolism.     

Specific binding of oestradiol to guinea-pig prostate cytosol and nuclear fractions

A high affinity (Kd approximately 0.15 nM), saturable oestradiol binding site, which is specific for natural and synthetic oestrogens has been identified in guinea-pig prostate cytosol fractions. The binding site is protein in nature (heat- and protease-sensitive) and has a sedimentation coefficient of approx. 8S on glycerol gradients. A high affinity (Kd approximately 0.16 nM), saturable oestradiol binding site was also identified in salt-extracted (0.5 M KC1) nuclear fractions. The optimum incubation conditions for measuring the cytosolic and nuclear oestradiol binding sites were determined to be 20 h at 4 degrees C. Saturation analysis studies revealed that following oestrogen treatment of intact animals, approx. 80% of the specific oestradiol binding sites in prostatic cytosol fractions were transferred into the nucleus. The presence of a specific oestradiol binding protein with characteristics of an oestrogen receptor in the guinea-pig prostate, is consistent with oestrogen having biological activity in this tissue. In view of the abundance of stroma in the prostate of this species, and the consistent finding that the stroma of male accessory sex tissues is oestrogen sensitive, the guinea-pig may be an appropriate experimental animal for further investigating the role of oestrogen in the growth and development of the prostate.     

Covalent stabilization of the nontransformed chick oviduct cytosol progesterone receptor by chemical cross-linking

The nontransformed forms of the chick oviduct cytosol progesterone receptor of sedimentation coefficient approximately 8 S (8S-PR) are heterooligomers including one hormone binding molecule, either B, approximately 110,000, or A, approximately 79,000, and two non-hormone binding subunits recently identified as heat-shock protein Mr approximately 90,000 (hsp 90) [Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., & Baulieu, E. E. (1984) Biochemistry 23, 6016-6023]. In the crude cytosol, bisimidates reacted under mild conditions and gave rise to complexes, binding progesterone and reacting with BF4, an anti-hsp 90 monoclonal antibody. These complexes have a sedimentation coefficient of 8.4 S and Rs of 8.1 nm in the presence of 0.4 M KCl and in the absence of molybdate ions, i.e., in conditions that would transform non-cross-linked 8S-PR to Rs approximately 5 nm forms of approximately 4-S sedimentation coefficient. All bisimidates tested, of an effective reagent length between 0.73 and 1.09 nm, gave comparable results in the cytosol prepared with or without molybdate ions, confirming that the latter were not responsible for the formation of the cross-linked 8S complexes. It was found that the dimethyl pimelimidate cross-linked 8S-PR was more resistant to inactivating conditions, urea, or heat treatment than the non-cross-linked 8S-PR. The 8S-PR cross-linked in the cytosol was purified by affinity chromatography in the absence of molybdate ions. After purification, it also reacted with the monoclonal antibody BF4 and had the same Rs (8.0 nm), sedimentation coefficient (approximately 8.5 S), and thus Mr (approximately 290,000) as the original cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [3H]estradiol-17beta are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [3H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4--5 S and the other had a sedimentation coefficient of 8--9 S. The two components differed from each other regarding steroid specificity and various physiocochemical parameters. [3H]estradiol binding to the 4--5 S component was not inhibited by estrogens, 5alpha-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appear to be saturable and label was rapidly stripped from it by charcoal. Estradiol binding to the 8--9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4--5 S moiety. The specific binding protein has a Kd of 3.05 . 10(-10) M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incubation of [3H]estradiol with mature male liver cytosol at 0--5 degrees C polar metabolites of estradiol are produced.     

Correlation between oxysterol binding to a cytosolic binding protein and potency in the repression of hydroxymethylglutaryl coenzyme A reductase

Support for the role of a cytosolic oxysterol-binding protein in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was obtained by correlating the relative binding affinities of a wide range of oxysterols to their potency in suppressing HMG-CoA reductase activity in mouse fibroblast cell cultures. Forty-seven oxysterols encompassing a 100-fold range of activity in both assays were tested and the two parameters were closely correlated for 35 of the sterols. Twelve sterols showed poor binding when compared to their ability to suppress HMG-CoA reductase activity in cell cultures. Among these were seven sterols with a ketone function at C-3. For this group, the discrepancy could be explained by their rapid conversion within cells to the 3 beta-hydroxy derivatives which have a much higher affinity for the binding protein. One sterol with 3-keto-4-ene grouping was not reduced to its 3 beta-hydroxy derivative in cells and thereby showed no discrepancy in the two assays. The remaining five sterols exhibiting discordant activities in the two tests contained 4,4-dimethyl moieties and were relatively weak suppressors of HMG-CoA reductase activity. Cellular metabolism of these sterols was not detected. Possible reasons for their apparent inactivity in the binding assay are discussed.     

A specific glucocorticoid binding macromolecule of rabbit uterine cytosol

A high affinity (K_d=2.7 × 10^?10M at 0°) dexamethasone binding macro-molecule has been identified in the cytosol fraction of rabbit uteri. Competition studies show high specificity for glucocorticoids since binding of labeled dexamethasone is inhibited by cortisol and corticosterone but not by progesterone, testosterone, or estradiol 17β. The binding component has a sedimentation coefficient of 8S and its concentration in uterine cytosol is about 0.2 pmoles per mg protein. Uptake of labeled dexamethasone by isolated uterine nuclei requires the presence of cytosol and is temperature dependent. The KCl-extractable nuclear complex sediments at 4S. Thus the dexamethasone binding components of the rabbit uterus have properties similar to those described for steroid hormone receptors present in target tissues. Specific dexamethasone binding could not be demonstrated in rat uterine cytosol.     

Identification of a novel binding partner of phospholipase cβ1: translin-associated factor X

Mammalian phospholipase Cβ1 (PLCβ1) is activated by the ubiquitous Gα(q) family of G proteins on the surface of the inner leaflet of plasma membrane where it catalyzes the hydrolysis of phosphatidylinositol 4,5 bisphosphate. In general, PLCβ1 is mainly localized on the cytosolic plasma membrane surface, although a substantial fraction is also found in the cytosol and, under some conditions, in the nucleus. The factors that localize PLCβ1in these other compartments are unknown. Here, we identified a novel binding partner, translin-associated factor X (TRAX). TRAX is a cytosolic protein that can transit into the nucleus. In purified form, PLCβ1 binds strongly to TRAX with an affinity that is only ten-fold weaker than its affinity for its functional partner, Gα(q). In solution, TRAX has little effect on the membrane association or the catalytic activity of PLCβ1. However, TRAX directly competes with Gα(q) for PLCβ1 binding, and excess TRAX reverses Gα(q) activation of PLCβ1. In C6 glia cells, endogenous PLCβ1 and TRAX colocalize in the cytosol and the nucleus, but not on the plasma membrane where TRAX is absent. In Neuro2A cells expressing enhanced yellow and cyano fluorescent proteins (i.e., eYFP- PLCβ1 and eCFP-TRAX), F?rster resonance energy transfer (FRET) is observed mostly in the cytosol and a small amount is seen in the nucleus. FRET does not occur at the plasma membrane where TRAX is not found. Our studies show that TRAX, localized in the cytosol and nucleus, competes with plasma-membrane bound Gα(q) for PLCβ1 binding thus stabilizing PLCβ1 in other cellular compartments.     

Physico-chemical properties of the hydroxysterol binding protein of human lymphocyte cytosol. Effects of high salt concentrations and molybdate

Side chain-hydroxylated derivatives of cholesterol (OH sterol) inhibiting lymphoblastic transformation bind with high affinity and specificity to a hydroxysterol binding protein (OHSBP) in the cytosol of human lymphocytes. These binding properties of OHSBP suggested some analogies with that of steroid hormone receptors. The observation of a nuclear binding of 25-OH[3H]cholesterol prompted us to apply to the cytosolic OH sterol-OHSBP complex the physico-chemical treatments known to 'activate' the steroid hormone receptors. A change of sedimentation coefficient from 8.3 to 4.3 S was observed in hypertonic buffer (0.4 M KCl) but the resulting 4.3 S complex dissociates easily whereas the 'native' 8.3 S form does not. Moreover, molybdate did not prevent the 8.3----4.3 S transformation induced by KCl and neither ammonium sulfate precipitation nor increasing temperature had any effect on the sedimentation coefficient of the 8.3 S complex. Thus, several physico-chemical features differentiate the OH sterol-OHSBP complex from steroid hormone receptors.     

Oestrogen receptors in chick oviduct. Characterization and subcellular distribution.

Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2--4 weeks, untreated by hormone) chicken oviduct. 7n the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA = 4 - 109 M-1 at 4 degrees C. The binding of [3H] oestradiol is abolished by 1 muM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low-ionic-strength medium. The estimated number of binding sites per nucleus is about 5000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two-step mechanism. The half-life time for the faster dissociation step is 18 h at 0 degrees C, 25 min at 20 degrees C and 10 min at 30 degrees C, and for the slower one is 180 h, 115 min and 60 min, respectively. In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor. After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA = 7 - 10(8) M-1 at 37 degrees C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken. In the high-speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (less than or equal to 100 sites/cell) and at the limits of sensitivity of the methods used. In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell.     

OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle.     

Effects of 25-hydroxycholesterol, cholesterol, and isoprenoid derivatives on the G1 progression in Swiss 3T3 cells

The effect of inhibition of 3-Hydroxy-3-methylglutaryl Coenzyme A reductase (HMG CoA reductase) on cell cycle progression in proliferating 3T3 cells was studied. It was found that short transient exposures to the HMG CoA reductase inhibitor 25-hydroxycholesterol temporarily blocked the cell cycle traverse in the postmitotic half of G1 (G1pm), whereas cells in the subsequent cell cycle phases were unaffected. The kinetics of the cell cycle delay, induced by 25-hydroxycholesterol, resembled the kinetics of the delay induced by serum depletion, which also inhibited the activity of HMG CoA reductase. In contrast to the case of serum depletion, platelet derived growth factor (PDGF), which efficiently prevented the decrease of HMG CoA reductase in serum-free medium, was not capable of preventing the growth inhibitory effect following treatment by 25-hydroxycholesterol. However, cholesterol and two isoprenoids, dolichol and coenzyme Q, were effective in this respect. In addition, dolichol counteracted the cell cycle delay following short periods of serum starvation.     

Modifications in the aminoether side chain of clomiphene influence affinity for a specific antiestrogen binding site in MCF 7 cell cytosol

Studies on the binding of a series of clomiphene derivatives to a specific high affinity antiestrogen binding site in MCF 7 cell cytosol revealed that substitutions in the aminoether side chain influenced affinity for this site. Removal of the aminoether side chain completely eliminated binding while changes in length of the C chain, and changes in the number and size of the substituents on the terminal amino group modified binding affinity. Conversion of the ether linkage to an amine also markedly reduced affinity. It is concluded that the aminoether side chain which is essential for antiestrogenic activity is a major structural determinant of the binding of synthetic triphenylethylene antiestrogens to the specific antiestrogen binding site.     

Characterization of an antiestrogen-binding protein in high salt extracts of human breast cancer tissue

An antiestrogen binding protein which binds [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)-phenyl]1,2-diphenylbut-1(Z)-ene) with high affinity (Kd = 1.1 X 10(-9) M) is present in high salt (0.6 M KCl) extracts of washed breast cancer tissue pellets. Its concentration in high salt extract is higher than its concentration in cytosol. The characteristics of the antiestrogen binding protein from cytosol and salt extract of breast cancer tissue are indistinguishable. It specifically binds triphenylethylene and other nonsteroidal antiestrogens and displays little or no binding affinity for estrogens, progesterone, dihydrotestosterone and cortisol. The antiestrogen binding protein is of unusually large size as judged by gel filtration on agarose 0.5 m and sedimentation analysis on 5-20% sucrose density gradients. Differential centrifugation studies indicate that it is not principally microsomal in origin. This protein is more thermostable than the estrogen receptor from which it can also be distinguished by ion exchange chromatography. The antiestrogen binding protein was eluted from DEAE-Sephacel by 0.05 M KCl indicating that it is less negatively charged than the estrogen receptor which was eluted by 0.1 M KCl. Lipoprotein fractionation of breast cancer cytosol using potassium bromide density gradients did not reveal specific antiestrogen binding activity associated with any recognized class of lipoprotein. Specific [3H]tamoxifen binding sites were pelleted in potassium bromide gradients consistent with the apparent large size of this protein. The physical characteristics of the antiestrogen binding protein in normal human tissue (myometrium) and neoplastic tissue (breast cancer) are remarkably similar, possibly reflecting a highly conserved structure.     

The N terminus controls sterol binding while the C terminus regulates the scaffolding function of OSBP

Previously we reported that when cell cholesterol is acutely lowered with beta-methyl-cyclodextrin the amount of activated ERK1/2 in caveolae dramatically increases. We traced the origin of this novel method of pERK1/2 accumulation to a macromolecular complex with dual specific phosphatase activity that contains the serine/threonine phosphatase PP2A, the tyrosine phosphatase HePTP, the oxysterol-binding protein OSBP and cholesterol. When cell cholesterol is lowered, or oxysterols is introduced, the complex disassembles and pERK1/2 increases. In an effort to better understand how OSBP functions as a cholesterol-regulated scaffolding protein, we have mapped the functional parts of the molecule. The command center of the molecule is a centrally located, 51 amino acids (408-459) long sterol-binding domain that can bind both cholesterol and 25-hydroxycholesterol. This domain is functional whether attached to the N- or the C-terminal half of OSBP. Introduction of a Y458S mutation impairs binding. Even though 25-hydroxycholesterol will compete for cholesterol binding to OSBP(408-809), it will not compete for cholesterol binding in full-length OSBP. Upon further analysis we found that a glycine-alaninerich region at the N-terminal end of OSBP works with the PH domain to control cholesterol binding without affecting 25-hydroxycholesterol binding. Finally, we found that HePTP and PP2A bind the C-terminal half of OSBP, HePTP binds a coiled-coil domain (amino acids 732-761), and PP2A binds neither the coiled-coil nor HePTP. On the basis of this information we propose a new model for how OSBP is able to sense both membrane cholesterol and oxidized sterols and link this information to the ERK1/2 signaling pathway.     

Protein transduction assisted by polyethylenimine-cationized carrier proteins

Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99, 95-103]. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled anti-S100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.     

Receptor for 1,25-dihydroxyvitamin D in a vascular smooth muscle cell line derived from rat aorta

The presence of a specific receptor for 1,25-dihydroxyvitamin D (1,25(OH)2D) was investigated in a cell line A7r5 derived from fetal aorta. 1,25(OH)2[3H]D3 binding to cytosol was saturated at 0.6-1 nM, and Scatchard analysis yielded dissociation constant and binding sites, (3.02 +/- 0.4) X 10(-11) M and 33.9 +/- 5.8 fmol/mg protein, respectively. Sucrose density gradient analysis revealed the sedimentation constant 3.2 S. Furthermore, the receptor protein had affinity for DNA-cellulose column and eluted with 0.2 M KCl. These data suggest that vascular smooth muscle cell may be a target tissue of vitamin D.