Purification and characterization of fatty-acid-binding proteins from rat heart and liver

Fatty-acid-binding proteins were purified from delipidated cytosols of rat heart and liver by gel filtration and anion-exchange chromatography at pH 8.0 and by repeated gel filtration, respectively. Homogeneity of both proteins was demonstrated by a single band on polyacrylamide gels; each had a molecular weight of about 14 000. Liver fatty-acid-binding protein is more basic (pI, 8.1) than that of heart (pI, 7.0) and contains more basic amino acids. Examination of fatty acid binding by the binding proteins from heart and liver revealed the presence of a single class of fatty-acid-binding sites in both cases with an apparent dissociation constant for palmitate of about 1 microM. Liver fatty- acid-binding protein shows similar binding characteristics for palmitate, oleate and arachidonate. Palmitate bound to heart fatty- acid-binding protein was a good substrate for oxidation by rat heart mitochondria. The results show that the fatty-acid-binding proteins from rat heart and liver are closely related, but that they are distinct proteins.     

Interactions of selenium and cadmium with metallothionein-like and other cytosolic proteins of rat kidney and liver

Following injection of rats with CdCl2 and [75Se]selenite using five different protocols, the metallothionein-like proteins (MTLPs) of kidney and liver cytosols were fractionated by Sephadex G-75 gel filtration and DEAE Sephacel ion-exchange chromatography. Cd and 75Se distribution in gel-filtration elution profiles was influenced mainly by the time that elapsed between administration of these elements and by the sequence of their administration. There was no Cd redistribution to high molecular weight proteins after long-term Cd injection when rats were killed 48 hr after 75Se injection. Cd was redistributed from MTLP to high molecular-weight proteins in the liver when Cd and 75Se were injected within 1-3 hr of each other. Incorporation of 75Se into MTLP of kidney and liver was independent of Cd injection. The strength of 75Se binding of MTLP was comparable to the covalent binding of 75Se to glutathione peroxidase. Cd and 75Se did not share binding sites on MTLP. In ligand-exchange studies, 1000 ppm Cd did not displace 75Se from MTLP, but 2% 2-mercaptoethanol displaced 10% of the presumably nonspecifically bound 75Se from kidney and liver MTLP. This study provides new information regarding the apparent covalent binding of Se to low molecular-weight, Cd-containing proteins in kidney and liver.     

Influence of zinc on copper binding in tissue proteins of steers

Two experiments were conducted with steers fed diets containing 270 ppm copper either with or without 2050 ppm zinc. Liver biopsies were taken from steers biweekly for 10 wk for analysis. The steers were then killed; tissues were removed, homogenized, and centrifuged, and the pellets were extracted with mercaptoethanol (BME), and selected cytosols and extracts were subjected to gel filtration (Sephadex G-75). Copper and zinc were determined on the BME extracts, pellets after extraction, cytosols, and gel-filtration fractions. Copper accumulated at about the same rate in BME extract and in the extracted pellet, with the smallest amount in the cytosol. In contrast, over 70% of the zinc was present in the hepatic cytosols. Gel filtration of BME extracts revealed the greatest amount of copper in a low-molwt (MW) peak in addition to three minor peaks of copper. Within the hepatic cytosols, the greatest amount of copper accumulated in proteins of MW>75,000, the next greatest amount in 30,000-MW proteins, and the least amount with metallothionein (MT) of steers fed the diet with only copper added. In contrast, the greatest amount of copper was present with MT in hepatic cytosols of the steer fed a diet that included copper plus zinc. Hence the zinc status of steers influences the deposition of copper in the cytosolic proteins (as demonstrated by liver, kidney, and pancreas), but not in the intracellular fractions.     

Liver cytosolic fatty acids binding proteins in rats and Psammomys obesus: modulation in diabetes

The effect of insulin on [3H]oleate binding to delipidated liver cytosolic proteins was studied in four groups of animals: untreated rats, streptozotocin induced diabetic rats, Psammomys obesus fed salt bush diet, and Psammomys obesus fed ordinary laboratory chow. The distribution of the protein bound [3H]oleate between low and high molecular weight cytosolic proteins in Psammomys differed from the distribution found in rats. Diet induced high insulin diabetes in Psammomys and streptozotocin induced low insulin diabetes in rats, modulated [3H]oleate binding in the same manner.     

The effect of feeding high corn oil on fatty-acid-binding-protein isolated from rat liver

Fatty-acid-binding-protein isolated from liver of rats receiving normal or high fat diet was studied by three different methods. The effect of high fat diet on the thermal stability of the protein was determined employing differential scanning calorimetry. Fatty acids have a stabilizing effect on the thermal stability of the protein. In order to determine the relative binding affinity of native and delipidated protein a Sephadex G-50 assay was employed using [1-14C] oleate as ligand. The delipidated protein exhibited greater binding of oleate than did the native material. Increases in the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes in vitro were also observed when protein obtained from both sources were delipidated. The results suggest that high corn oil diet would modify the properties of fatty-acid-binding-protein in the uptake and cytosolic transport of long-chain fatty acids.     

Fatty acid binding proteins from developing human fetal brain: Characterization and binding properties

Two fatty acid binding proteins (FABPs) of identicalM _r, 13 kDa, have been isolated from developing human fetal brain. A delipidated 105,000 g supernatant was incubated with [1 -¹⁴C]oleate and subjected to a Sephacryl S-200 column followed by gel filtration chromatography on a Sephadex G-75 column and ion-exchange chromatography using a DEAE-Sephacel column. Purity was checked by UV spectroscopy, SDS-PAGE, isoelectric focusing and immunological cross-reactivity. The two FABPs designated as DE-I (pI 5.4) and DE-II (pI 6.9) showed cross-reactivity with each other and no alteration at the antigenic site during intrauterine development. Anti-human fetal brain FABP does not cross-react with purified human fetal heart, gut, lung or liver FABPs. The molecular mass of DE-I and DE-II is lower than those of fetal lung and liver FABPs. Like liver FABP, these proteins bind organic anions, fatty acids and acyl CoAs but differ in their binding affinities. Both DE-I and DE-II have been found to exhibit higher affinity for oleate (K _d = 0.23 μM) than palmitate (K _d = 0.9μM) or palmitoyl-CoA (K _d = 0.96 μM), with DE-I binding less fatty acids than DE-II. DE-II is more efficient in transferring fatty acid from phospholipid vesjcles than DE-I indicating that human fetal brain FABPs may play a significant role in fatty acid transport in developing fetal brain.     

Binding of bile acids by 100 000g supernatants from rat liver.

1. The binding of glycocholic acid, chenodeoxycholic acid and lithocholic acid to rat liver 1000 000g supernatants was studied by equilibrium dialysis. 2. The binding characteristics of the bile acids suggest that the binding components are involved in bile acid transport. 3. When mixtures of [14C]lithocholic acid and liver supernatants were eluted from columns of Sephadex G-75, a prominent peak of [14C]lithocholic acid appeared with proteins of mol.wt. approx. 40000. A second, smaller, peak of [14C]lithocholic acid was eluted with proteins of mol.wt. approx. 100000. 4. The inclusion of cholic acid, glycocholic acid or chenodeoxycholic acid in the eluting buffer decreased the amount of [14C]lithocholic acid that was eluted with the higher-molecular-weight component.     

The red cell membrane and its cytoskeleton.

Gel-filtration (Sephadex G-75) analysis of hepatic cytosol reveals both qualitative and quantitative sex differences in oestrogen-binding proteins. The elution profile of [³H]oestradiol-labelled cytosol shows four species of oestrogen-binding proteins (peaks I, II, IV and V) common to both sexes. The amount of [³H]oestradiol binding in peak I is equivalent in both males and females and corresponds quantitatively to the specific oestrogen receptor. The amount of binding in the remaining three peaks is greater in males than females. In addition, an oestrogen-binding protein (peak III) is present that is unique to male cytosol. Proteinase-inhibition studies demonstrate that the observed multiplicity of oestrogen-binding proteins is not an artefact of proteolytic breakdown. Sex differences in oestrogen-binding proteins are absent in immature male and female animals; the oestrogen-binding protein profile in immature rats resembles that of an adult female. Gonadectomy of adult animals does not affect the oestrogen-binding-protein profile. In contrast, neonatal (day 1) castration results in partial feminization of the characteristic oestrogen-binding protein profile seen in the adult male; the appearance of Peak III is suppressed and marked decreases in the amount of oestradiol binding occurs in the remaining peaks. Hypophysectomy of adult animals results in near abolishment of the observed sex differences; the male oestrogen-binding protein profile is partially feminized and the female profile is partially masculinized, as characterized by the appearance of [³H]oestradiol binding in the region of peak III and increased amounts of binding in peaks IV and V. The present studies demonstrate a multiplicity of oestrogen-binding proteins in liver cytosol and raise the possibility that the presence of some of these proteins may be imprinted at birth through the hypothalamic–pituitary axis, by a mechanism requiring neonatal androgen exposure.     

Distribution of radioactive silver in the subcellular fractions of various tissues of the rat and its binding to low molecular weight proteins

In view of the electron microscopic evidence that silver does not penetrate cellular barriers, the distribution of radioactive silver in rat blood and subcellular fractions of liver, kidneys, spleen, and forebrain was studied. It was found that 24 h after a single intraperitoneal injection high levels of radioactivity were reached which decreased at different rates in the various tissues studied. In plasma, liver, and kidneys there was an initial rapid loss of radioactivity which was followed by a slower rate of loss. In the blood, forebrain, and spleen the loss of radioactivity was linear and somewhat slower than in the other three tissues. The cytosols of the liver and kidneys contained 60% while those of the forebrain and spleen contained 30% of the total radioactivity found in the tissue homogenates. Gel filtration on Sephadex G-75 showed that all cytosols contained two peaks of radioactivity; a high molecular weight peak which eluted just after the void volume and a low molecular weight peak. The amount of radioactivity in both peaks was, however, much lower in the chromatographic peaks of the forebrain and spleen than that found in those of the liver and kidneys. Furthermore, the spleen had a comparatively very small low molecular weight radioactive peak. In vitro experiments with liver cytosol showed similar results to those found in vivo in that the high molecular weight radioactive peak could be removed by heat. It is concluded that silver does enter cells and that silver thionein exists in the cytosols of forebrain, spleen, kidney, and liver.     

Effect of ethanol on hepatic phosphatidate phosphohydrolase: dose-dependent enzyme induction and its abolition by adrenalectomy and pyrazole treatment

Protein synthesis, measured as the incorporation of [¹⁴C]valine into cell proteins and into proteins secreted into the medium, and albumin production were studied in isolated rat liver hepatocytes. Protein synthesis was substantially higher in cells from fed rats than in cells from fasted rats. Addition of carbohydrates or amino acids increased protein synthesis in cells from fasted rats, whereas no effect was seen in cells from fed rats. Addition of oleate had no effect on protein synthesis. Ethanol inhibited protein synthesis in cells from fasted rats, whereas no or only small effect was seen in cells from fed rats. Simultaneous addition of carbohydrates diminished the inhibitory effect of ethanol, whereas addition of oleate increased the inhibitory effect of ethanol. It is suggested that the rate of protein synthesis in cells from fasted rats could be restricted by lack of precursors for synthesis of nonessential amino acids. The effect of ethanol is explained by an inhibition of gluconeogenesis.     

Interactions between cadmium,selenium and glutathione peroxidase in rat testis

In rats given a minimal damaging dose of ¹⁰⁹CdCl₂ (0.011 mmole/kg, s.c.), a visible hemorrhagic response was evident after 48 h when testicular Cd uptake exceeded a level of approx. 150 ng/g. Glutathione peroxidase (GSH-Px) activity was elevated in homogenates of these damaged testes. In rats whose testes were not damaged, the Cd levels were below 150 ng/g and the GSH-Px activity was similar to that of control animals injected with sodium acetate. Rat testis cytosol was found to contain two different GSH-Px activities when assayed with cumene hydroperoxide. These could be separated by gel filtration chromatography. The larger species (GSH-Px A) was eluted in the void volume on Sephadex G-150 and incorporated ⁷⁵Se from Na₂⁷⁵SeO₃ given 4 weeks earlier. The smaller species, of approx. 42 000 molecular weight (MW) (GSH-Px B), did not incorporate ⁷⁵Se and could be distinguished from GSH-Px A by its insensitivity to cyanide (10 mM). CdCl₂ (1 mM) did not inhibit GSH-Px activity when added in vitro to GSH-Px A or B from testicular cytosol, or to purified GSH-Px isolated from ovine erythrocytes. When ¹⁰⁹CdCl₂ was given in vivo to rats injected 4 weeks previously with a tracer dose of Na₂⁷⁵SeO₃ or added in vitro to cytosol prepared from similarly labeled rats, Sephadex G-150 chromatography of cytosol showed that most of the ¹⁰⁹Cd was eluted in a major peak of 34 000 MW. Little or no ¹⁰⁹Cd was found in association with ⁷⁵Se (major peak 140 000 MW) or GSH-Px activity. When ¹⁰⁹CdCl₂ was injected into rats given an equimolar dose of Na₂⁷⁵SeO₃ 30 min previously, ¹⁰⁹Cd uptake in cytosol was increased and both ¹⁰⁹Cd and ⁷⁵Se was shifted into a peak of 110 000 MW.The ¹⁰⁹Cd-binding peak of approx. 30 000–34 000 MW was the major Cd-binding fraction in cytosol of 7-week-old rats but was not detectable in 4-week-old rats. Susceptibility of the testes to Cd did not correlate with the presence of this peak, however, since 4-week-old rats were occassionally damaged by CdCl₂.     

Liberation and analysis of protein-bound arsenicals

Protein-bound arsenicals were liberated from binding sites on liver cytosolic proteins by exposure to 0.1M CuCl at pH 1. This method released greater than 90% of the arsenicals associated with biological matrices. Ultrafiltrates of CuCl-treated cytosols were subjected to thin-layer chromatography to speciate and quantify inorganic and methylated arsenicals. For rat liver cytosol in an in vitro methylation assay and for liver and kidney cytosols from arsenite-treated mice, most inorganic arsenic was protein bound. Appreciable fractions of the organoarsenical metabolites present in these cytosols were also protein bound. Therefore, CuCl treatment of cytosols releases protein-bound arsenicals, permitting more accurate estimates of the pattern and extent of arsenic methylation in vitro and in vivo.     

Generation of corticosteroid binder IB from binder II by a sulfhydryl dependent renal cytosolic factor

It has long been debated whether binder IB represents a unique form of the glucocorticoid receptor or is derived from the larger molecular weight form, binder II, by limited proteolysis. Transformed glucocorticoid receptors in kidney, liver and mixed kidney/liver cytosols were examined using anion exchange and gel filtration chromatography. The transformed receptor in liver cytosols chromatographs as binder II on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of approx 6.0 nm. The transformed receptor in kidney cytosols chromatographs as binder IB on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of 3.0-4.0 nm (3.2 nm on agarose; 3.0-4.0 nm on Sephadex G-100). Using cytosols prepared from mixed homogenates (2 g kidney plus 8 g liver tissue), our experiments show that binder II is converted to a lower molecular weight form (Rs = 3.2 nm on agarose; Rx = 3.9 nm on Sephadex G-100) that is identical to binder IB in its elution position from DEAE-Sephadex anion exchange resin. Identical results are obtained using kidney/liver/cytosols mixed in vitro in which only the hepatic receptor, binder II, is labelled with [3H]TA. These results support the hypothesis that the renal receptor, binder IB, is a proteolytic fragment of binder II and does not represent a polymorphic form of the glucocorticoid receptor. The renal converting activity is dependent on free-SH for full activity but is insensitive to the protease inhibitors leupeptin, antipain, and PMSF. The conversion of hepatic binder II to binder IB in in vitro mixing experiments can be prevented if kidney cytosol is gel filtered on Sephadex G-25 and the eluted macromolecular fraction is adjusted to 10 mM EGTA (or EDTA) prior to mixing with the [3H]TA labelled hepatic cytosol.     

Influence of free fatty acid anion on the binding of warfarin to cytoplasmic proteins from rat liver

In vitro binding studies have shown that warfarin binds strongly to both ligandins (Y) and Z protein obtained from rat liver cytosol with dissociation constants of 11.7 and 10.1 μM respectively. Increasing concentrations of oleate ion significantly increased the dissociation constant of warfarin with either protein, whereas laurate ion showed the same behavior only with Z protein. On the other hand, the binding of warfarin to liver cytoplasmic proteins in vivo was decreased in 72-h-pre-fasted rats, although such fasting failed to produce any increase in the in vivo levels of the cytoplasmic free fatty acids (FFA). However, based on the results of the in vitro binding study, it is suggested that changes in the composition of hepatic cytoplasmic free fatty acids as a result of fasting could reduce the in vivo binding of warfarin to Y and Z proteins and hence could lead to an increase of unbound warfarin in liver cytosol.     

Suppression of Receptors for Prolactin and Estrogen in Rat Liver Due to Treatment with the Growth Hormone Analogue Produced by the Tapeworm Spirometra Mansonoides

Abstract

Somatogenic hormones play an important role in regulation of receptors for prolactin (PRL) and estrogen. Plerocercoids of the tapeworm, S. mansonoides produce a factor which mimics some, but not all of the actions reported for GH. Intact female rats were subjected to a constant infusion of plerocercoid growth factor (PGF) via a subcutaneous infection for two weeks to determine if PGF influences receptors for PRL, GH or estradiol. The rate of weight gain in the PGF-treated rats was accelerated in spite of a marked reduction in serum GH. Partially-purified PGF specifically displaced [¹²⁵I]hGH from rat liver receptors but microsomes prepared from rats treated with PGF specifically bound significantly less [¹²⁵I]hGH than microsomes from control rats. The reduction in [¹²⁵I]hGH binding was not due to occupancy or to a change in affinity but to a suppression in receptor concentration. Scatchard analysis of [³H]estradiol binding in rat liver cytosols shows a 50% reduction in receptor concentration in the PGF-treated group. Specific binding of [³H]estradiol in anterior pituitary was also suppressed by PGF treatment.     

Copper binding components of blood plasma and organs,and their responses to influx of large doses of <Superscript>65</Superscript>Cu,in the mouse

To establish for the first time how mice might differ from rats and humans in terms of copper transport, excretion, and copper binding proteins, plasma and organ cytosols from adult female C57CL6 mice were fractionated and analyzed by directly coupled size exclusion HPLC-ICP-MS, before and after i.p. injection of large doses of ⁶⁵Cu. Plasma from untreated mice had different proportions of Cu associated with transcuprein/macroglobulin, ceruloplasmin and albumin than in humans and rats, and two previously undetected copper peaks (Mr 700 k and 15 k) were observed. Cytosols had Cu peaks seen previously in rat liver (Mr > 1000 k, 45 k and 11 k) plus one of 110 kDa. ⁶⁵Cu (141 μg) administered over 14 h, initially loaded plasma albumin and mainly entered liver and kidney (especially 28 kDa and 11 kDa components). Components of other organs were less (but still significantly) enriched. ⁶³Cu/⁶⁵Cu ratios returned almost to normal by 14 days, indicating a robust system for excreting excess copper. We conclude that there are significant differences but also strong similarities in copper metabolism between mice, rats and humans; that the liver is able to buffer enormous changes in copper status; and that a large number of mammalian copper proteins remain to be identified.     

Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver

Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.     

Hyperthermic stress affects the thermal modulation of glucocorticoid–receptor affinity

In vitro binding of a steroid hormone (triamcinolone acetonide) to hepatic glucocorticoid receptors was studied in liver cytosols prepared from untreated control rats and rats sacrificed after being exposed in vivo to whole body hyperthermia (41 or 42°C). Positive temperature modulation of glucocorticoid–receptor affinity (decrease of affinity with increasing temperature) was well expressed in all preparations. In preparations from hyperthermic rats, alterations of a possible functional (adaptive) significance have been recorded: the amplitude of positive thermal modulation was reduced, and its lower-temperature limit shifted towards higher temperatures.     

Hepatic estrogen-binding proteins in male and female vervet monkeys

Hepatic cytosols from male and female vervet monkeys (Cercopithecus pygerythrus) were found to contain the same levels of high affinity estrogen-binding proteins. Multipoint saturation analyses revealed that male liver cytosols contain two distinctly different binding components: a high affinity (HAEB) and a low affinity estrogen binder (LAEB). Female livers appeared to contain only the HAEB. Sucrose density gradient (SDG) analyses, however, clearly established the presence of a 3.8 S as well as an 8.1 S estrogen-binding component in the hepatic cytosols of both sexes. The 3.8 S binding component appeared to be more prominent in male SDG profiles. Cytosols, prepared in the presence of sodium molybdate (cyt +) exhibited significantly lower (50%) levels of specific estrogen-binding than cytosols prepared in the absence of the oxyanion (cyt-). SDG analyses, however, indicated that in cyt+ the 8.1 S binding component was stabilized at the cost of the 3.8 S binder. This phenomenon was observed in both sexes. Large excess levels of cortisol did not have any effect on specific estrogen binding by hepatic cytosols. The hepatic estrogen-binding proteins displayed a lower relative binding affinity for diethylstilbestrol than for its native ligand and higher affinities for estriol and estrone than expected.     

Studies on the relationship between glutathione S-transferase phenotype and bile acid binding by human liver cytosol

The possibility that the GST1 phenotype of human liver cytosol is a determinant of bile salt binding has been investigated by using equilibrium dialysis and gel-exclusion chromatography. Binding of bile salts was non-saturable and whereas the glutathione S-transferases did not appear to be major bile salt binders, other binding components with molecular weights of 35 000 and 11 000 were identified in both fetal and adult cytosols.