Chicken Liver Membranes Research Articles

Preparation and Characterization of Recombinant Chicken Growth Hormone (chGH) and Its Putative Antagonist chGH G119R Mutein

Synthetic cDNA of chicken GH (chGH) and its G119R mutein was synthesized after being optimized for expression in E. coli. The respective cDNAs were inserted into expression vector, expressed and found almost entirely in the insoluble inclusion bodies (IBs). The IBs were isolated, the proteins solubilized in 4.5 M urea, at pH 11.3 in presence of cysteine, refolded, and purified to homogeneity by anion-exchange chromatography on Q-Sepharose. The overall yields were 400 to 500 mg from 5 L of fermentation. Both proteins were > 98% pure, as evidenced by SDS-PAGE, and contained at least 95% monomers, as documented by gel-filtration chromatography under non-denaturing conditions. Circular dichroism analysis revealed that both proteins have identical secondary structure characteristic of cytokines, namely > 50% of alpha helix content. Chicken GH was capable of forming a 1:2 complex with recombinant oGH receptor extracellular domain, but its affinity, as determined by RRA, was 11-fold lower than that of ovine GH (oGH). Correspondingly, its bioactivity, assessed using FDC-P1 3B9 cells stably transfected with rabbit GHR, was 30-40-fold lower, whereas chGH G119R mutant did not bind to oGHR-ECD and was devoid of any biological activity in FDC-P1 3B9 cells. However, in binding experiments that were carried out using chicken liver membranes, both oGH and chGH showed similar IC(50) values in competition with (125)I-oGH, while the IC(50) of G119R mutein was 10-fold higher. These results emphasize the importance of species specificity and indicate the possibility of antagonistic activity of chGH G119R.

Modulation of regulatory factors involved in cholesterol metabolism in response to feeding of pravastatin- or cholesterol-supplemented diet in chickens

mRNA transcripts encoding multiple proteins from the cholesterol biosynthetic and uptake pathways in livers are controlled by sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound mammalian transcription factor. The aims of the present study were to investigate whether SREBP-2 responds to plasma cholesterol levels and modulates expression of factors involved in the cholesterol metabolism of chickens. Supplementing the diets of chickens with 0.1% pravastatin, a drug used to control hypercholesterolemia, decreased plasma LDL-cholesterol concentrations. It also increased levels of the nuclear forms of SREBP-2 and increased gene expression of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and low-density lipoprotein receptor (LDLr) in livers, relative to a control group. In contrast, feeding of 3% cholesterol-supplemented diet increased plasma total- and LDL-cholesterol concentrations but decreased levels of nuclear forms of SREBP-2 and reduced gene expression of HMGR and LDLr. However, the LDL-binding activities of chicken liver membranes were not affected by plasma cholesterol concentrations or by hepatic levels of the nuclear form of SREBP-2. LDL-binding proteins were detected as bands of 90 and 515 kDa on a ligand blot and the intensity of these bands was unaffected by pravastatin and cholesterol supplementation. These findings suggest that the cholesterol biosynthetic pathway is regulated by the nuclear form of SREBP-2 in chickens as well as in mammals, but that there may be species-specific differences in the regulatory mechanisms of hepatic cholesterol uptake.

Purification, characterization, cloning, and expression of the chicken liver ecto-ATP-diphosphohydrolase.

We previously demonstrated that the major ecto-nucleoside triphosphate phosphohydrolase in the chicken liver membranes is an ecto-ATP-diphosphohydrolase (ecto- ATPDase) [Caldwell, C., Davis, M.D. & Knowles, A.F. (1999) Arch. Biochem. Biophys. 362, 46-58]. Enzymatic properties of the liver membrane ecto-ATPDase are similar to those of the chicken oviduct ecto-ATPDase that we have previously purified and cloned. Using antibody developed against the latter, we have purified the chicken liver ecto-ATPDase to homogeneity. The purified enzyme is a glycoprotein with a molecular mass of 85 kDa and a specific activity of approximately 1000 U.mg protein-1. Although slightly larger than the 80-kDa oviduct enzyme, the two ecto-ATPDases are nearly identical with respect to their enzymatic properties and mass of the deglycosylated proteins. The primary sequence of the liver ecto-ATPDase deduced from its cDNA obtained by RT-PCR cloning also shows only minor differences from that of the oviduct ecto-ATPDase. Immunochemical staining demonstrates the distribution of the ecto-ATPDase in the bile canaliculi of the chicken liver. HeLa cells transfected with the chicken liver ecto-ATPDase cDNA express an ecto-nucleotidase activity with characteristics similar to the enzyme in its native membranes, most significant of these is stimulation of the ATPDase activity by detergents, which inhibits other members of the ecto- nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. The stimulation of the expressed liver ecto-ATPDase by detergents indicates that this property is intrinsic to the enzyme protein, and cannot be attributed to the lipid environment of the native membranes. The molecular identification and expression of a liver ecto-ATPDase, reported here for the first time, will facilitate future investigations into the differences between structure and function of the different E-NTPDases, existence of liver ecto-ATPDase isoforms in different species, its alteration in pathogenic conditions, and its physiological function.

The polypeptide PHI discriminates a GTP-insensitive form of vip receptor in livermembranes

In early reports on125I-VIP binding experiments in liver membranes, it has been proposed that, the VIP binding sites were partially sensitive to GTP. Here we confirm that the VIP binding sites of chicken liver membranes consisted mainly in bivalent VIP/PACAP receptors and that about 50% of the125I-VIP binding capacity was not affected by the GTP analogue GppNHp. Part of these bivalent receptors also appeared to represent PHI binding sites. In GppNHp-treated membranes, the GTP-insensitive VIP binding sites displayed a 17-fold higher relative affinity than in control membranes for the VIP analogue PHI. Such data suggested that GTP-insensitive VIP receptors may correspond to a subclass of high-affinity PHI receptors. Cross-linking of125I-VIP or125I-PHI to their receptors, revealed 2 components of 48 and 60kDa. The radiolabelling of the 60kDa component was strongly affected by increasing concentrations of the GTP analogue but was modestly abolished by an excess of PHI. Conversely, the radiolabelling of the 48kDa molecular form was not affected by the GTP analogue but was efficiently abolished by increasing concentrations of PHI. Taken together, the data suggest that the 48kDa component expressed in chicken liver membranes display the properties of a GTP-insensitive VIP/PHI receptor that can be pharmacologically discriminated from the GTP-sensitive 60kDa form, through its much higher affinity for PHI.

Characterization of a specific binding site for angiotensin II in chicken liver.

We have characterized a specific binding site for angiotensin II (AngII) in chicken liver membranes. Pseudo-equilibrium studies at 22 degrees C for 30 min have shown that this binding site recognizes AngII with a high affinity (pKD of 8.13 +/- 0.21). The binding sites are saturable and relatively abundant (maximal binding capacity varies from 0.318 to 0.88 pmol/mg of protein). Nonequilibrium kinetic analyses at 22 degrees C revealed a calculated kinetic pKD of 8.77 +/- 0.20. The binding site is pharmacologically distinct from the classic AngII receptors AT1 and AT2. Competitive binding studies with chicken liver membranes demonstrated the following rank order of effectiveness: AngII (human; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > AngI(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) > AngIII(Arg-Val-Tyr-Ile-His-Pro-Phe) > AngIV (Val-Tyr-Ile-His-Pro-Phe) > Ang(1-7) (Asp-Arg-Val-Tyr-Ile-His-Pro) > PD123319 (1-[4(dimethylamino)3-methylphenyl] methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo [4,5-c]pyridine-6-carboxylic acid) > DuP753 (2-n-butyl-4-chloro-5 hydroxymethyl-1-[(2'-1H-tetrazol-5-yl)biphenyl-4-yl)methyl] imidazole. This atypical AngII binding site (chicken AT) was sensitive to increasing concentrations of DTT and Mn2+. The structure-activity relationship on position 1 of AngII showed that the primary N-terminal amine was essential for binding affinity ([Asp1]AngII > [Suc1]AngII > or = [Sar1]AngII), but modifications of the side chain in position 1 had less influence on the affinity ([Gly1]AngII > [Cys1]AngII approximately [aminoisobutyryl1]AngII approximately [Ser1]AngII > > > [Sar1]AngII). The presence of substantial quantities of this binding site in chicken liver membranes suggests the possibility that the chicken AT may play an important, yet unrecognized, role in the renin-angiotensin system.

Characterization of the alpha 1B-adrenergic receptors of chicken hepatocytes. Signal transduction and actions.

1. In chicken hepatocytes, alpha 1-adrenoceptor activation increased: (a) phosphatidylinositol labeling; (b) production of inositol trisphosphate; (c) cytosol calcium; and (d) phosphorylase activity. 2. Prazosin (Ki approximately 0.2-0.4 nM) was more potent in inhibiting these actions than 5-methyl-urapidil (Ki approximately 30-60 nM); these actions were sensitive to chlorethylclonidine suggesting the involvement of alpha 1B-adrenoceptors. 3. The stimulation of phosphoinositide turnover was insensitive to pertussis toxin. 4. In chicken liver membranes, [3H]prazosin binding sites (Bmax 872 fmol/mg protein) with high affinity for prazosin (KD 0.3 nM; Ki 0.4 nM) and lower affinity for 5-methyl-urapidil (Ki 46 nM) were detected, consistent with the presence of alpha 1B-adrenoceptors.

Insulin receptor and insulin sensitivity in a chicken hepatoma cell line

Insulin receptors have been characterized in a cell line recently isolated from a chicken hepatoma (LMH). The binding of 125I-insulin to LMH cells or membranes displayed the expected criteria for insulin receptors: affinity, temperature dependency, curvilinearity of Scatchard plot, rank order of potency for insulin analogs and insulin induced down-regulation. The α-subunit of LMH cell insulin receptors exhibited a normal size of 135 kDa. Following autophosphorylation, LMH WGA-purified receptors revealed a 95 kDa β-subunit and a 72 kDa protein (pp72). Both proteins were phosphorylated in a time-, insulin- (and insulin-like growth factor 1; IGF-1) and manganese-dependent manner, and were precipitated by antiphosphotyrosine and two anti-insulin receptor antibodies. The 72 kDa protein was not present under non-reducing condition PAGE or in normal chicken liver. These results strongly suggest that pp72 is either a truncated form of the insulin receptor β-subunit specific to LMH cells or a degradation product. Lectin-purified insulin receptors from LMH cells or chicken liver membranes exhibited similar tyrosine kinase activity, using artificial substrate poly(Glu-Tyr) 4:1. Finally, amino acid uptake by LMH cells was insulin stimulatable.

Insulin sensitivity and liver insulin receptor structure in ducks from two genera

Insulin sensitivity and liver insulin receptor structure were studied in 5-wk-old ducks from two genera (Muscovy and Pekin). In the fasting state, both duck types were equally resistant to exogenous insulin compared with chicken. Despite the low potency of duck insulin, the number of insulin receptors was lower in Muscovy duck and similar in Pekin duck and chicken liver membranes. After 125I-insulin cross-linking, the size of the alpha-subunit of the receptors from the three species was 135,000. Wheat germ agglutinin-purified receptors from the three species were contaminated by an active and unusual adenosinetriphosphatase (ATPase) contaminant (highest activity in Muscovy duck). Sequential purification of solubilized receptor from both duck types on lentil and then wheat germ agglutinin lectins led to a fraction of receptors very poor in ATPase activity that exhibited a beta-subunit size (95,000) and tyrosine kinase activity similar to those of ATPase-free chicken insulin receptors. Therefore the ducks from the two genera exhibit an alpha-beta-structure for liver insulin receptors and a clear difference in the number of liver insulin receptors. Their sensitivity to insulin is, however, similarly decreased compared with chicken.

Insulin-like growth factor receptors in chicken liver membranes: binding properties, specificity, developmental pattern and evidence for a single receptor type

Two distinct receptors for the insulin-like growth factors (IGF-I and IGF-II) have been identified in mammalian tissues, but so far only a receptor structurally related to the type I receptor has been identified in chicken embryonic tissues. This study was designed to characterize binding sites for IGF peptides in chicken liver microsomal membranes prepared from hatch to 10 weeks of age which is the period of most rapid growth. Binding of both human (h) IGF-I and hIGF-II was displaceable by either peptide and exhibited similar pH, time and temperature dependency. Human IGF-II was more potent than hIGF-I in competing for the binding of the iodinated ligands with half-maximum effective concentrations of 3-5 micrograms/l and 7-13 micrograms/l respectively. Porcine insulin was also a potent competitor. Affinity cross-linking studies, followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions demonstrated that both IGF peptides were linked to a protein with a molecular weight of about 130,000 Da characteristic of the alpha-subunit of the type I receptor. There was no evidence for the presence of a type II receptor similar to that found in mammals. Specific binding of both peptides was low on the day of hatch, increased about threefold by day 3 of age and remained high for the first 3 weeks of life before returning to a lower steady state level up to 10 weeks of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Avian growth hormone receptor assay: use of chicken and turkey liver membranes

A sensitive avian growth hormone (GH) radioreceptor assay (RRA) was developed using recombinant chicken growth hormone (rcGH) and a membrane receptor preparation of chicken or turkey livers. The specific binding of 125I-labeled rcGH to a 47800 × g pellet was 33–36% in a 16–20 h incubation period at 4°C. Binding was time, temperature and pH dependent. Scatchard analysis indicated a single class of high affinity GH binding sites in chicken and turkey livers, with binding affinities of 1.03 × 10 10 liter/M and 1.11 × 10 10 liter/M, respectively, and corresponding binding capacities of 10.7 fmol and 21 fmol per mg protein. The sensitivity of the assay was 0.41 ng of rcGH. Intra- and inter-assay coefficients of variation were 5.3% and 9.7%, respectively. Bovine GH, ovine GH, and porcine GH competed effectively for the GH binding sites in chicken and turkey livers. Turkey prolactin (PRL) and porcine PRL showed little cross-reaction (< 0.07%), while cross-reaction of ovine and bovine PRL was greater (< 10%). Standard rcGH (0.5–30 ng) was added to sera from hypophysectomized chickens and turkeys (hypox sera) and to tissue culture medium and was measured quantitatively. Untreated medium (10–100 μ1) and hypox sera (5–40 μ1) did not inhibit rcGH binding. These studies report the existence of specific binding sites for avian GH in chicken and turkey liver and validate a sensitive RRA for measurement of bioactive GH in sera and tissue culture medium.

The discovery of a lipid-linked glucuronide and its synthesis by chicken liver.

Upon incubation with uridine diphosphate-[14C]glucuronic acid, membrane fractions from adult and phenobarbital-induced embryonic liver synthesize a single glucuronide, which is soluble in chloroform:methanol (2:1). The compound is completely hydrolyzed and glucuronic acid released by either mild acid or beta-glucuronidase, whereas mild base hydrolysis results in a mixture of glucuronic acid and glucuronic acid-1,2-cyclic phosphate. These data and the behavior of the lipid-linked glucuronide on DEAE-cellulose chromatography indicate that the compound contains a monophosphate diester of glucuronic acid, which is beta-linked to a lipid. The synthesis of the lipid-linked glucuronide in uninduced normal embryonic liver is very low (5-15 pmol product/mg/5 min) at all developmental ages up to hatching, but the introduction of phenobarbital into the air space of a 9-10-day-old embryo causes a premature increase of activity (75-150 pmol products/mg/5 min) within 7 days. The glucuronyltransferase in adult and induced embryonic liver has a Km for UDPGlcUA of 0.17 x 10(-3) M and a broad pH optimum between pH 6 and 7. Glucuronic acid is released from the lipid-linked glucuronide by a beta-glucuronidase in liver that is active at neutral pH and is not inhibited by saccharolactone. This glycosidase activity appears, therefore, to be distinct from the previously characterized lysosomal beta-glucuronidase. Fractionation of adult chicken liver membranes by differential centrifugation indicates that over 70% of the glucuronyltransferase is associated with the nuclear and mitochondrial fractions. The endogenous beta-glucuronidase capable of hydrolyzing the lipid-linked glucuronide was not separated from the glucuronyl-transferase activity during fractionation. The data available suggests that the lipid-linked glucuronide is involved directly in the generation of free glucuronic acid for further metabolism.

The insulin receptor of embryonic chicken cartilage.

Highly purified plasma membranes have been obtained from embryonic chicken cartilage by physical means rather than enzymatic digestion. Rapid and reversible binding of [125I]iodoinsulin to these membranes is demonstrated. Similar to the insulin-binding properties of rat liver and adipocytes and human mononuclear cells, optimal specific binding of insulin to chondrocyte plasma membranes has a sharp pH optimum at 8.0, and maximal binding occurs at 2--4 C. Analysis of equilibrium binding reveals a curvilinear Scatchard plot, whose high affinity segment generates a maximum affinity of 1.0 X 10(9) M-1, and a receptor concentration of 0.4 pmol/mg membrane protein. This affinity constant is similar to those generated for insulin binding to membranes prepared from embryonic chicken liver (2.5 X 10(9) M-1), rat liver (1.4 X 10(9) M-1), and mouse liver (0.6 X 10(9) M-1), whereas the receptor concentration is less than that of embryonic chicken liver membranes (1.1 pmol/mg), which in turn was less than those of rat liver membranes (2.8 pmol/mg) and mouse liver membranes (3.5 pmol/mg). Kinetic studies show augmentation of insulin-receptor dissociation by excess insulin when initial receptor occupancy, is low, suggesting that negative cooperativity is present. There is little or no interaction of other hormones with the chondrocyte insulin receptor, with the exception of proinsulin and the insulin-like growth factors. Porcine proinsulin, bovine proinsulin, somatomedin C, and nonsuppressible insulin-like protein prevent [125I]iodoinsulin binding to chondrocyte plasma membranes with dose-response curves which are parallel to that of unlabeled porcine insulin itself, but with molar potencies relative to porcine insulin of 15%, 9%, 2.5%, and 1.4%, respectively. Porcine insulin and proinsulin both prevent binding of [125I]iodosomatomedin C to chondrocyte plasma membranes but with molar potencies less than 1% that of unlabeled somatomedin C. These observations are consistent with the presence of a specific independent insulin receptor in embryonic chicken cartilage which is similar in its characteristics to the insulin receptor in previously described tissues. Insulin has a weak interaction with the chondrocyte receptor for somatomedin C. Interaction with the somatomedin receptor may be the mechanism by which insulin exerts anabolic effects on cartilage when used in pharmacological amounts.

Adhesive specificity of juvenile rat and chicken liver cells and membranes.

Liver cells, isolated from either juvenile rats or chickens by a collagenase perfusion technique, reaggregated when maintained in suspension. The cells exhibited marked adhesive specificity; when suspensions contained both cell types, the aggregates consisted primarily of either rat or chicken cells. Adhesive specificity was also observed with plasma membrane fractions isolated from rat liver homogenates, and with comparable fractions from chicken liver. These membranes stimulated aggregation of the homologous but not the heterologous cell type. Other membrane fractions had little or no effect on the aggregation of the homologous cell type. These and other properties of the liver cell and membrane preparations suggest that biochemical studies on cell-cell recognition and adhesion can most effectively be conducted with cells from juvenile and adult animals.