Multiple aflatoxin B1 binding proteins exist in rat liver cytosol

The in vitro binding of aflatoxin B1 to rat liver cytosolic proteins was investigated. Aflatoxin B1 binding activity was assayed with protein purified by gel permeation chromatography, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Twenty-five percent of the total binding activity was associated with proteins eluted by 0 and 0.1 M NaCl. Over 50% of the total binding activity was associated with protein present in the 0.2 M NaCl fraction. Glutathione S-transferase activity was also monitored and found only in the low salt (less than 0.2 M NaCl) fractions. The proteins eluted by 0.2 M NaCl were further purified by hydroxylapatite column chromatography and binding was found predominantly in a single fraction. The protein purification steps resulted in a 20-fold increase in the specific binding activity over that initially observed in the cytosol. These results indicate that multiple proteins are capable of binding aflatoxin B1 in rat liver cytosol.     

Putative inositol 1,4,5-trisphosphate binding proteins in rat brain cytosol.

In previous works, we synthesized a series of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) analogs, with a substituent on the second carbon of the inositol ring. Using these analogs, the Ins(1,4,5)P3 affinity media were also synthesized (Hirata, M., Watanabe, Y., Ishimatsu, T., Yanaga, F., Koga, T., and Ozaki, S. (1990) Biochem. Biophys. Res. Commun. 168, 379-386). When the cytosol fraction from the rat brain was applied to an Ins(1,4,5)P3 affinity column, an eluate with a 2 M NaCl solution was found to have remarkable Ins(1,4,5)P3-binding activity. The active fraction was further fractionated with gel filtration chromatography, and two proteins with an apparent molecular mass of 130 or 85 kDa were found to be Ins(1,4,5)P3-binding proteins but with no Ins(1,4,5)P3 metabolizing activities. Partial amino acid sequences determined after proteolysis and reversed-phase chromatography revealed that the protein with an apparent molecular mass of 85 kDa is the delta-isozyme of phospholipase C and that of 130 kDa has no sequence the same as the Ins(1,4,5)P3-recognizing proteins hitherto examined. Ins(1,4,5)P3 at concentrations greater than 1 microM strongly inhibited 85-kDa phospholipase C delta activity, without changing its dependence on the concentrations of free Ca2+ and H+. Among inositol phosphates examined, Ins(3,4,5,6)P4 inhibited the binding of [3H]Ins(1,4,5)P3 to the 130-kDa protein at much the same concentrations as seen with Ins(1,4,5)P3. This report seems to be the first evidence for the presence of soluble Ins(1,4,5)P3-binding proteins in the rat brain, one of which is the delta isozyme of phospholipase C.     

Presence of three different estradiol binding proteins in rat prostate cytosol

Depending upon the experimental conditions, three distinct [³H]-17β-estradiol binding entities can be detected in rat prostate cytosol. A protein with characteristics similar to other estrogen receptors is found in prostate cytosol from intact rats. This protein has a sedimentation coefficient of 8S on sucrose gradients. It has a high affinity only for natural and synthetic estrogens. It decreases rapidly after castration (less than 20% of binding activity after 24 hr) and reappears progressively after 8–10 days.A second binding entity with a sedimentation coefficient of 4–5S on sucrose gradients is also observed. It is found both in intact and castrated rats. At low temperatures, the binding of estradiol increases progressively and reaches a maximum after 24–48 hr of incubation as determined by charcoal assay. This binding species is highly specific for natural estrogens but not for diethylstilbestrol and is probably identical with the 4–5S binder.Finally, a third binding entity is found in 24 hr castrated rats with short incubations at 0°C This binding is labile even at low temperatures. Natural and synthetic androgens compete more strongly than estradiol for this binding. This behaviour suggests that the third estradiol binding entity is identical with the androgen receptor.     

Studies on the fatty acid binding proteins in cytosol of rat liver.

Gel filtration on Sephadex G-75 of crude rat liver supernatant preincubated with [1-14C]oleic acid yields three peaks of radioactivity which are attributed to the presence in these fractions of fatty acid binding proteins. We have confirmed these observations with binding assays by phase partition, polyacrylamide gel electrophoresis, and thin layer electrofocusing. Peak I (mol. wt. 60,000 pI 5.01 was shown to be albumin, which mainly arises from a contamination of the liver preparation by blood. Peak II (mol. wt. 10,000, pI 5.9) is a fatty acid binding protein. Finally peak III (mol. wt. 1500, pI 5.7) is a fatty acid binding component, the chemical nature of which was not elucidated. These fatty acid binding fractions have no effect on the reaction of acyl-CoA synthetase whereas the crude liver supernatant does stimulate the activation of fatty acid as shown earlier. In consequence, the physiological role of these fatty acid binding fractions is not yet elucidated.     

Haem-binding proteins of the rat liver cytosol

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino [³h] laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [³H] haem-labelled mitochondria, [³H] haem-labelled microsomes or with [³H] haemin.These results are discussed with particular reference to ligandin.     

Corticosterone binding by rat brain cytosol

Significant quantities of corticosterone were associated with macromolecules of the brain cytosol following intrathecal administration of [³H]corticosterone to adrenalectomized rats. Fifteen times more steroid was found associated with proteins from adrenalectomized rats than from control animals or adrenalectomized animals pretreated with corticosterone. Pretreatment of adrenalectomized rats with 11-dehydrocorticosterone, Cortisol and cortisone decreased the amount of [³H]corticosterone found associated with protein, whereas progesterone, oestradiol and testosterone did not interfere with the association of [³H]corticosterone with macromolecules of the cytosol. Further evidence for protein-steroid interaction was obtained by incubating [³H]corticosterone (B), [³H]cortisol (F), or 11-[³H]deoxycortisol (S) with brain cytosols. The degree of binding was in the order B > F > S.     

Further characterization of estrogen binding to rat testis cytosol

Binding of estradiol (E2), estriol (E3), RU16117, and moxestrol to testis cytosol from adult male rats was investigated. High-affinity binding sites were identified in the 8-9S region of sucrose density gradients; a second, high-capacity binding component in the 4S region was probably due to contamination with serum. Thermodynamic properties of the testicular estrogen binding site were quite similar to those of the uterine receptor. E2 had the highest affinity for testicular cytosol binding sites (Ka: E2 much greater than moxestrol greater than E3 greater than RU16117). Comparison of association rate (E2 greater than E3 greater than moxestrol = RU16117) and dissociation rate constants (E3 = RU16117 greater than E2 much greater than moxestrol) as well as studies in vivo revealed moxestrol as a long-acting and RU16117 as a short-acting compound. This difference may be useful for evaluation of the mediation of estrogen effects in the rat testis.     

Thyroid hormone and uncoupling proteins

p53 is a representative tumor suppressor whose dysfunction is a major cause of human cancer syndrome. Here we isolated flies lacking Dmp53, which encodes the single Drosophila orthologue of mammalian p53 family. Dmp53 null mutants well developed into adults, only displaying mild defects in longevity and fertility. However, genomic stability and viability of Dmp53 mutants dramatically decreased upon ionizing irradiation. Moreover, mutating Dmp53 abolished irradiation-induced apoptosis and reaper induction. These results indicate that Dmp53 is a central component of DNA damage-dependent apoptotic signaling.     

3H-estradiol binding to cytosol receptors of the rat olfactory epithelium

Two types of cytosol receptors of 3H-estradiol with high affinity and limited quantity of binding sites (KDI = 1-2 nM, BmaxI = 8 fmoles/mg protein; KDII = 10 nM, BmaxII = 8 fmoles/mg protein) were determined in the rat olfactory tissue. The amount of high affinity receptors of type I does not change with maturation of the rats, and has no sex difference. The role of estradiol receptors in the olfactory tissue of the rats is discussed.     

Two high affinity estrogen binding proteins of different specificity in the immature rat uterus cytosol

The presence of two high affinity estrogen binding proteins in the uterine cytosol of the immature rat has been observed.Besides the 8 S cytosol estrogen $\text{receptor}$, there is a 4–5 S fraction binding estradiol and estrone with a large capacity. In fact, the two binding systems have a different affinity for estradiol and estrone, the receptor binding more the former and the 4–5 S fraction more the latter. Exposure of the cytosol to specific anti-α₁-Fetoprotein antibodies suppresses a large part of the 4–5 S binder, if not the totality. Moreover the estrogen binding 4–5 S fraction decreases with increasing age until puberty, while the $\text{receptor}$ increases. These results suggest therefore that the estradiol-estrone binding 4–5 S peak of the uterine cytosol is mainly made up of Estradiol Binding Plasma Protein-α₁-Fetoprotein (EBP-AFP). Also they confirm that “cytosol” should be taken as an operational fraction which may include extracellular components.During the course of these experiments, it has been observed that the increase of the estradiol $\text{receptor}$ is more rapid than the other uterine cytosol proteins until the 8th day, and that there is a second period of growth when it follows the development of the uterus and of the animal, as if it had reached a constant number of binding sites per cell.     

Haem-binding proteins of the rat liver cytosol.

Gel filtration of soluble supernatant fraction obtained from livers of rats 10 min after an injection of the haem precursor 5-amino[3H]laevulinic acid shows the presence of a major radioactive fraction which upon gel filtration is similar in elution volume to ligandin. 20 min after administration of the precursor four previously minor components also come into prominence. This pattern is a characteristic of in vivo binding since a different elution pattern is obtained if soluble supernatant fraction from rat liver is labelled in vitro by incubation either with [3H]haem-labelled mitochondria, [3H]haem-labelled microsomes or with [3H]haemin. These results are discussed with particular reference to ligandin.     

Thyroid hormone modulation of agonist - beta-adrenergic receptor interactions in the rat heart

We have investigated alterations in beta-adrenergic receptors in rat myocardial membranes derived from hypothyroid and hyperthyroid animals. (-)Isoproterenol competition curves with (-)[³H]dihydroalprenolol revealed that isoproterenol binds to the beta-adrenergic receptor with two distinct affinity states having high (K_H) and low (K_L) dissociation constants. In the presence of guanine nucleotides the isoproterenol competition curve steepened and had a higher EC₅₀ (50% displacement). This was due to a transition of the high affinity state to a uniformly low affinity state. Using computer modeling of these competition curves, we have demonstrated that in hyperthyroidism, the isoproterenol curve in the absence of guanine nucleotides is shifted to the left with the EC₅₀ changing from 180 ± 40 to 80 ± 20 nM (p < .02). The fold shift (4 fold) in K_H (nM) 30 ± 9 to 7 ± 2 (p < .001) is greater than that (1.6 fold) in K_L (nM) 595 ± 56 to 376 ± 34 (p < .001) such that the K_L/K_H ratio shifted from 20 ± 3 to 54 ± 9 (p < .001). The ratio, K_L/K_H, for a particular agonist appears to be related to its efficacy in activating adenylate cyclase.There was no significant alteration in any of these parameters in hypothyroid animals. Receptor number was decreased in hypothyroidism, 16 ± 3 fmol/mg protein (p < .03) and increased in hyperthyroidism 44 ± 4 (p < .03) compared to control 26 ± 2.In the rat heart agonist affinity and receptor number are modulated in hyperthyroidism, but only receptor number in hypothryoidism. Thus thyroid hormone can modify not only receptor number but agonist affinity as well.     

From binding proteins to hormone receptors?

Phytohormones exert in responsive plant cells specific biochemical and physiological effects. It is a widely held view that phytohormones are first recognized by specific receptors which initiate the transduction of the hormonal signal. While hormone receptors are well studied in many eukaryotes ranging from yeast to man, we are lacking a detailed understanding of phytohormone receptors. Phytohormone binding proteins have been suspected to provide candidates for such receptors. In this review recent progress towards molecular analysis of such proteins and their genes will be summarized.