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1.
The rat vitamin D binding protein (Gc-globulin) gene is syntenic with the rat albumin and alpha-fetoprotein genes on chromosome 14 总被引:1,自引:0,他引:1
Using a panel of mouse X rat somatic cell hybrids and a characterized rat vitamin D binding protein (DBP) cDNA probe, we assigned the gene coding for rat DBP to chromosome 14. We conclude that the genes for DBP, albumin, and alpha-fetoprotein, all of which derive from a common precursor, are syntenic in the rat, as they are in man. 相似文献
2.
W C Okulicz L D Johnson 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1987,185(4):478-483
The relative binding affinity (RBA) of diethylstilbestrol (DES) was determined in nuclear fractions of the rat uterus. DES displayed a two- to threefold greater affinity (RBA = 245 +/- 36) than estradiol (RBA = 100) for nuclear E receptor. The RBA of DES to nuclear E receptor was lowered significantly in the presence of rat serum (43 +/- 1) or human serum (52 +/- 7). Dilution of human serum resulted in a progressive increase in the RBA of DES which approached that observed in the absence of serum. Addition of purified human serum albumin mimicked the decrease in RBA of DES that was observed with serum. The IC50 of estradiol was not changed in the presence of either rat serum or albumin. These data show that DES possesses a greater affinity for nuclear E receptor than estradiol and that serum albumin can modulate DES binding to uterine E receptor. 相似文献
3.
Rat vitamin D binding protein. Determination of the full-length primary structure from cloned cDNA 总被引:6,自引:0,他引:6
N E Cooke 《The Journal of biological chemistry》1986,261(7):3441-3450
Vitamin D binding protein (DBP) is an abundant serum glycoprotein secreted by the liver which transports vitamin D sterols, binds to actin, and is found on the surface of B-lymphocytes and subpopulations of T-lymphocytes. In the current study, a cDNA to rat DBP mRNA was cloned from a bacteriophage lambda gt 11 rat liver expression library. This DBP cDNA clone was identified by immunoblotting and its identity was confirmed by immunoprecipitation of a 54-kDa protein after hybrid-assisted translation. Northern analysis and primer extension mapping of rat liver mRNA indicated that the full-length DBP mRNA contains 1700 bases. By DNA sequence analysis this 1655-base pair clone contains a single open reading frame encoding the 476-amino acid containing full-length DBP and includes its 16-amino acid signal sequence. Analysis of the sequence reveals about 40% nucleotide and 23% amino acid homology to both albumin and alpha-fetoprotein. The encoded DBP contains a characteristic placement of cysteine residues, identical to that in albumin, suggesting a similar secondary folding structure. Albumin and alpha-fetoprotein are composed of three internally homologous domains. DBP mRNA terminates 122 amino acids before the larger albumin mRNA in the third internal domain, but retains the characteristic homology among the first two domains and the truncated portion of the third domain. These data support the conclusion that DBP is a member of a multigene family which includes albumin and alpha-fetoprotein. 相似文献
4.
P A Nygren C Ljungquist H Tr?mborg K Nustad M Uhlén 《European journal of biochemistry》1990,193(1):143-148
The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions. 相似文献
5.
Hybrids formed by the fusion of mouse hepatoma (BWTG3) and rat fibroblast (JF1) cells exhibit the extinction of mouse albumin and alpha-fetoprotein synthesis. Karyotype analyses suggest that all parental chromosomes are present in the hybrids. The extinction, therefore, of mouse hepatocyte genes is attributed to the inhibitory action of the rat genome. In these studies, we show that these hybrids possess and express the mouse beta-glucuronidase gene (which is encoded on the same chromosome as the mouse albumin and alpha-fetoprotein gene), and we present data of Southern blot analysis which demonstrate that such hybrids have indeed retained both mouse and rat albumin DNA sequences. In addition, using mouse albumin cDNA, we have shown by cDNA-RNA reassociation kinetics that albumin mRNA is virtually absent in these hybrids. We conclude from these studies that the extinction of albumin synthesis involves a mechanism which results in the loss of cytoplasmic albumin mRNA. 相似文献
6.
Mouse alpha 1-fetoprotein and albumin. A comparison of their binding properties with estrogen and fatty acid ligands 总被引:6,自引:0,他引:6
L Savu C Benassayag G Vallette N Christeff E Nunez 《The Journal of biological chemistry》1981,256(18):9414-9418
The binding of estradiol-17 beta (E2), diethylstilbestrol (DES), and polyene fatty acids, in particular arachidonate (C20:4), to alpha 1-fetoprotein (alpha-FP) and albumin purified from mouse embryo sera was studied using equilibrium dialysis and electrophoretic techniques. E2, arachidonate, and DES all bind to alpha-FP, but with decreasing strength. E2 is a high affinity, low capacity ligand (Ka approximately 0.8 X 10(8) M-1 and approximately 0.3 sites/mol of alpha-FP at 25 degrees C); arachidonate is a weaker ligand disposing of more sites (Ka approximately 0.3 X 10(7) M-1 and 4-5 sites/mol of alpha-FP); the binding of DES is of comparatively low affinity and capacity (Ka approximately 0.2 X 10(7) M-1 and n approximately 0.7/mol of alpha-FP). In spite of different structures and equilibrium parameters, E2, DES, and arachidonate are able to compete with each other for binding to the fetoprotein. The C22:4 and C22:6 fatty acids are also efficient concentration-dependent inhibitors of E2 or DES binding. Albumin binds the fatty acids and DES, but equilibrium parameters are different from those of alpha-FP. In particular, arachidonate is a better ligand for albumin, where it interacts with at least two classes of apparent sites (Ka1 approximately 0.3 X 10(8) M-1 and n1 approximately 1; Ka2 approximately 0.2 X 10(7) M-1 and n2 approximately 30). In contrast to alpha-FP, albumin virtually does not bind E2. Also, no competition could be demonstrated between DES and fatty acid ligands for binding to albumin. None of the studied interactions, with either albumin or alpha-FP, was modified even by high doses of bilirubin. The possible functions of the various binding activities present in fetal sera in the process of growth are discussed. 相似文献
7.
Rat alpha-fetoprotein contains a site that both binds serine enzyme inhibitors and substrates and regulates estrogen binding. We report that mM concentrations of the histidine selective reagent, diethylpyrocarbonate, inhibit estrogen binding to rat alpha-fetoprotein and that this inhibition is reversed by hydroxylamine. We suggest that rat alpha-fetoprotein contains one or more histidine residues that regulate estrogen binding. We also find that either estrone or the chymotrypsin substrate, acetyl-tryptophan methyl ester, protects rat alpha-fetoprotein from diethyl-pyrocarbonate-mediated inhibition of estrogen binding. We infer that the protease substrate and estrogen binding sites contain histidine residue(s) essential for estrogen binding by alpha-fetoprotein. 相似文献
8.
Funmei Yang Judith M. Bergeron Leslie A. Linehan Peter A. Lalley Alan Y. Sakaguchi Barbara H. Bowman 《Genomics》1990,7(4):509-516
The group-specific component (GC), also known as the vitamin D-binding protein, transports vitamin D and its metabolites in plasma to target tissues throughout the body. The GC gene shares an evolutionary origin with genes encoding albumin (ALB) and alpha-fetoprotein (AFP). All three genes are descendants of an evolutionary ancestor that arose from an intragenic triplication. As a result, each gene is composed of three homologous domains. The study described here characterizes and compares mouse GC to the corresponding nucleotide and amino acid sequences of GC from human and rat. The deduced amino acid sequence of mouse GC was 78% identical to human and 91% identical to rat GC. The results suggest that, unlike the corresponding sequences in the ALB and AFP genes, chromosomal sequences encoding the first domain and the leader sequence of the GC gene have specifically been conserved throughout vertebrate evolution. Protection of domain I during evolution may correlate with an important functional aspect of its sequence. The mouse GC gene was mapped to chromosome 5, where the ALB and AFP genes are also located, demonstrating conservation of the three genes in vertebrate species. 相似文献
9.
Selective detection of rat and mouse specific albumin and alpha-fetoprotein mRNA molecules under highly stringent hybridization conditions 总被引:2,自引:0,他引:2
The molecular analysis of some important interactions observed between the parental genomes in interspecific cell hybrids requires the availability of highly specific hybridization assays to selectively quantitate mRNA sequences coding for the same protein but transcribed from the two different genomes. Specific hybridization techniques which should permit the selective detection of rat and mouse albumin and alpha-fetoprotein (AFP) mRNA molecules in a mixture of the two types of mRNAs are presented here. The high degree of homology existing between the AFP mRNA sequences coding for mouse and rat AFP, and, presumably, albumin, results in extensive cross-hybridization with the cDNA probes under standard hybridization conditions. No size differences could be detected between the two types of mRNA molecules from the two species. A Tm difference of 7 degrees C between the intra- and interspecific mRNA:rat cDNA hybrids allowed the establishment of highly stringent solution hybridization conditions necessary to measure separately the contents of rat albumin and AFP mRNAs. Mouse albumin and AFP cDNA clones were then isolated from mouse liver and yolk sac cDNA libraries, and used to show the usefulness of highly stringent washing conditions to discriminate between rat and mouse albumin and AFP mRNA molecules in conventional "Northern blotting" techniques. In combination with the solution hybridization assay, these filter hybridization techniques can be used to specifically quantitate the content of rat and mouse albumin and AFP mRNA molecules in interspecific cell hybrids. 相似文献
10.
11.
Multiple estrogen binding sites in the uterus: stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites 总被引:1,自引:0,他引:1
K Chae S H Johnston K S Korach 《The Journal of steroid biochemistry and molecular biology》1991,38(1):35-42
Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [3H]IA enantiomers (IA-R and IA-S) and [3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [3H]ligand; DES, IA-Rac, IA-R and IA-S. IA-S enantiomer (Kd = 0.67) binds to the estrogen receptor with the same affinity as DES (Kd = 0.71) and four times higher affinity than IA-R (Kd = 2.56). The number of binding sites for IA-S is approximately the same as estradiol, DES and IA-Rac while IA-R binds far fewer sites than the other ligands. Saturation binding assays indicated that [3H]DES and [3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [3H]DES and [3H]IA compounds bound to the 8S form of the receptor, the same as E2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displaced by the addition of excess unlabeled E2 and DES. Further characterization of the 4S component is described. 相似文献
12.
Conditions required for activation of the mouse albumin or α-fetoprotein gene in hybrids between mouse lymphoblastoma and rat hepatoma cells 总被引:1,自引:0,他引:1
Carole H. Sellem Mary C. Weiss Doris Cassio 《Differentiation; research in biological diversity》1988,39(1):66-77
Activation of two previously silent mouse hepatic genes has been investigated in hybrid cells between pseudodiploid mouse lymphoblastoma cells and hyperdiploid or hypertetraploid rat hepatoma cells. In this material, activation of the mouse albumin gene is a frequent event, whereas activation of mouse alpha-fetoprotein (AFP) occurs only in those cells that produce large amounts of albumin. Quantitative tests of hybrid populations for the activated proteins and their mRNAs revealed the expected sizes and structures: moreover, as in hepatoma cells, the amount of both rat and mouse albumin produced was directly proportional to the intracellular concentration of the corresponding mRNA. The cellular environment required for activation of the liver-specific genes was investigated by cell-by-cell analysis of each hybrid clone. Immunostaining for the presence of rat and mouse albumin and mouse AFP revealed unexpected heterogeneity in the phenotypes of the hybrid populations, which were found to contain cells that: (a) failed to express either of the proteins; (b) produced all three; (c) produced both rat and mouse albumin; or (d) produced rat albumin only. Karyotypic analysis indicated that the hybrid-cell phenotype depended on parental chromosome ratios rather than absolute numbers of chromosomes. It was found for albumin and mouse AFP that the fraction of immunostained cells was equal to the fraction of metaphases that contained a minimal rat-to-mouse chromosome ratio of 2.5 and 9, respectively. It is concluded that in those hybrids, expression of liver-specific genes is regulated by extinguishers, but in a dose-dependent fashion, suggesting the intervention of antagonistic activators from the rat hepatoma chromosomes. 相似文献
13.
Rat skeletal muscle cytosol proteins bound 3H-diethylstilbestrol (3H-DES). More than 90% of this binding was high capacity and low affinity. Serum albumin accounted for roughly 50–60% of the binding, as evidenced by its precipitation with anti-rat albumin IgG. About half of the binding was distinguishable from albumin and other serum proteins by its precipitation in 40% saturated ammonium sulfate. This material sedimented at 4–5S in high-salt sucrose gradients, and resolved into two components (8S and 4–5S) in low-salt. Following incubation at 23–27°C for one hour, 2% of the bound 3H-DES in whole cytosol (approximately 2 fmole/mg cytosol protein) was retained by DNA-cellulose, and was eluted with 0.6 M KCl. This small fraction of the total binding was inhibited by estrogens and DES analogues: estradiol-17β, DES, dienestrol, and hexestrol were strong inhibitors; isodienestrol, dimethylstilbestrol, estradiol-17α, estrone, tamoxifen, MER-25, CI-628, and nafoxidine were weak inhibitors; dihydrotestosterone, testosterone, and prednisone did not compete. These observations indicate that specific estrogen-binding sites exist in rat skeletal muscle. 相似文献
14.
The synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE2), are more potent than 17 beta-estradiol (E2) in inducing uterine weight gain in the neonatal rat, due to the binding of E2 to serum alpha-fetoprotein (AFP). However, all three hormones are equipotent in inducing neonatal uterine ornithine decarboxylase (ODC) activity. The present study assessed estrogen potency in fetal rats. Pregnant CD rats were injected sc daily on gestation days (GD) 16-20 with DES, EE2, or E2 in sesame oil. Both DES and EE2, but not E2, significantly increased uterine weight at birth, to more than twice that of controls. In addition, implants which continuously release E2 only slightly increased uterine weight at birth. Alternatively, dams were given a single estrogen injection on GD 20 and were sacrificed at various times after injection. Peak fetal uterine ODC activity occurred at 6-8 hours after maternal injection for all three estrogens. E2 had a relative potency about tenfold less than either DES or EE2 in stimulating fetal ODC activity, in contrast to equal potencies of the three estrogens in the postnatal rat uterus. Similar patterns were found following direct fetal injection with E2 or DES. In summary, these data demonstrate a transplacental induction of fetal uterine ODC activity and uterine weight gain by both DES and EE2. In addition, the lack of correlation between these endpoints in response to E2 suggests that they may be useful as selective indicators of potential toxicity of both natural and synthetic estrogens. 相似文献
15.
We report the equilibrium binding parameters for the interactions of the estrogen analogue diethylstilbestrol (DES) with highly purified rat alpha 1-fetoprotein (AFP) and serum albumin preparations. At 25 degrees C and pH 7.4, an association constant (Ka) of about 1.5 X 10(6)M-1 and 2 sites/mole are measured with the DES-AFP system, whereas for the DES-albumin interaction, we find a Ka of approximately 2 X 10(5)M-1 and about 11 sites/mole of protein. The removal of fatty acids from pure AFP causes a reversible 3 fold increase of the number of DES binding sites; the same delipidation procedure applied to albumin slightly diminishes its DES binding parameters. We also demonstrate the capability of DES to displace competitively estradiol-17 beta (E2) from its high affinity sites on the estrophilic rat AFP. Finally, the binding behaviour of the two serum proteins towards the synthetic estrogen is compared to their interaction with the natural hormones. The physiological and pharmacological relevance of these data is discussed. 相似文献
16.
Dimitrova MN Matsumura H Dimitrova A Neitchev VZ 《International journal of biological macromolecules》2000,27(3):187-194
The interactions of three serum albumin species (rat, human, and bovine) with liposomes containing dimyristoylphosphatidylcholine, distearoylphosphatidylcholine or mixtures of both under different membrane fluidity conditions have been investigated using isothermal titration calorimetry and steady-state fluorescence anisotropy. Calorimetric titration studies of the binding of liposomes to the albumin species indicate in all cases exothermic processes with multiple sites of binding in the albumin molecules. Distinct saturation of the protein-lipid binding processes was observed at low or high molar lipid/protein ratio depending on the particular system. The thermodynamic parameters, including the association enthalpy and entropy, and the optimal values for the binding constants were thoroughly varied as a function of the number of identical binding sites, defining the best value of the parameter. Our experimental results, obtained using complementary biophysical techniques, provide experimental evidence for a significant difference in the association of the three protein species to phospholipid membranes. These observations also suggest a close relation between the binding parameters of the protein/lipid association and the lipid state of the phospholipid membranes. 相似文献
17.
The binding of zinc to both human alpha-fetoprotein (AFP) and albumin isolated from cord serum was studied by Sephadex G-50 gel-filtration chromatography. We found that the total number of binding sites for zinc on AFP and albumin were approximately 16 and 12, respectively. Both graphical analysis and the computer program 'LIGAND' indicate that there are at least two major classes of binding sites for both proteins. Both methods of analysis suggested that there are four to five high-affinity sites for zinc on AFP and only two to three similar sites on albumin. The affinity of zinc for AFP (dissociation constant, Kd, 6-8 X 10(-6) mol/l) was higher than for albumin (Kd, 1-3 X 10(-5) mol/l) for the high-affinity sites. The estimates for the zinc low-affinity binding sites were more uncertain, and several classes of low-affinity binding sites of different affinities might be present in both proteins. The results of our inhibition studies suggest that calcium, copper and lead might also bind with AFP at the zinc-binding sites. 相似文献
18.
oxidation of diethylstilbestrol (DES) by peroxidase preparations from horse radish or mouse uterus in the presence of hydrogen peroxide yields β-dienestrol, which is also a major metabolite of DES in several mammalian species. The oxidation reaction appears to involve reactive intermediates, presumably the semiquinone and quinone of DES, since nonextractable binding to salmon sperm deoxyribonucleic acid and bovine serum albumin was found. The peroxidase-catalyzed oxidation of DES to reactive metabolites in estrogen target organs may be related to the organ toxicity of this synthetic estrogen. 相似文献
19.
The methods of somatic cell genetics and molecular hybridization were applied to a panel of mouse X rat hepatocyte hybrids segregating rat chromosomes to assign the rat genes coding for two serum proteins, albumin and alpha-fetoprotein (Alb and Afp). The molecular hybridization of DNAs from different hybrids with cloned DNA probes showed that all the hybrid clones possessing the rat Alb gene and expressing it also retained the rat Afp locus, which is not expressed in these hybrids. So the Alb and Afp genes are syntenic in the rat, as in the mouse. Furthermore, the cytogenetic analysis allowed the assignment of these two loci to rat chromosome 14. 相似文献
20.
The binding of bilirubin and the polyene fatty acids cis-parinaric acid and cis-eleostearic acid to human alpha-fetoprotein was studied using fluorescence quenching and fluorescence enhancement techniques. alpha-Fetoprotein has three fatty acid binding sites of decreasing affinity (association constants 2.1 x 10(7) M-1 9.1 X 10(5) M-1, and 1.4 x 10(5) M-1) and one relatively strong and one relatively weak bilirubin binding site (association constants 1.1 x 10(7) M-1 and 1.8 x 10(5) M-1). These association constants are slightly weaker than the corresponding association constants for binding to human albumin. Competition experiments failed to show preferential binding of polyunsaturated fatty acids. Fluorescence quenching was used to determine 11 ligand-ligand and ligand-tryptophanyl residue distances. Each of these 11 calculated distances (ranging from 19 A to 32 A) was within 5 A of the corresponding distances measured previously for human albumin (Berde, C.B., Hudson, B.S., Simoni, R.D., and Sklar, L.A. 1979, J. Biol. Chem. 254, 391-400). Thus, in addition to previously described sequence homology, immunologic cross-reactivity, and other similarities, human albumin and human alpha-fetoprotein have functional and geometric homologies. 相似文献