Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Estrogen receptor (ER) was purified from calf uterus by immunoaffinity chromatography in the absence of the ligand. The purified ER consists of a mixture of monomer and homodimer forms of 67-kDa hormone-binding subunit (no 90-kDa heat shock protein is present). The purified ER was incubated with a 32P-labeled 61-basepair oligonucleotide containing the sequence of the estrogen response element (ERE) of the Xenopus laevis A2 vitellogenin gene. DNA mobility shift assays showed formation of specific complexes of the ERE containing oligonucleotide with ER, formation which did not require and was not affected by estradiol or antiestrogenic molecules. Both the monomer and the dimer were equally able to interact with the ERE-containing oligonucleotide. Sucrose gradient experiments showed that only the ER monomer is able to interact with an oligonucleotide in which a single mutation destroyed the dyad symmetry of ERE. Multiple symmetric mutations which did not alter the dyad symmetry of ERE nevertheless totally destroyed the ability of the oligonucleotide to form complexes with either the monomeric or dimeric form of ER. These results suggest that ER is able to bind to ERE independently of the presence of estradiol or other proteins and, therefore, that estradiol does not act by modulating the ability of ER to bind to ERE on DNA.
Mol Endocrinol 1991 Apr
PMID:In vitro binding of the purified hormone-binding subunit of the estrogen receptor to oligonucleotides containing natural or modified sequences of an estrogen-responsive element. 192 88

Transforming growth factor-beta (TGF beta) is produced by most tissues, including bone, as a complex that is biologically inert. Release of TGF beta homodimer from this latent complex is necessary for TGF beta to exert effects on target cells. Thus, the nature of the latent complex and the mechanisms responsible for TGF beta release are the key to understanding TGF beta actions. We have found that murine calvarial bone cultures secrete multiple latent forms of TGF beta. Using analytical chromatography and Western blot analysis, we have compared bone latent TGF beta with the previously characterized latent complex present in platelets and with simian TGF beta precursor, which is stably expressed in a latent form by Chinese hamster ovarian (CHO) cells. A major component of the bone material appears to be a latent complex of 100 kDa, consisting of mature TGF beta (25-kDa homodimer). Like the recombinant TGF beta precursor, it elutes from a Mono-Q fast pressure liquid chromatography anion exchange column at 0.2 M NaCl and shows a very similar banding pattern on Western blots. Thus, this bone complex closely resembles recombinant TGF beta precursor expressed in a latent form by CHO cells and differs from the naturally occurring platelet complex, which has an additional 135-kDa binding protein that is bound through disulfide bonds to the precursor proregion. Western blot analysis also indicates that, like CHO cells, which express recombinant TGF beta precursor, but unlike other cell types, the bone cultures secrete detectable amounts of uncleaved TGF beta precursor. The bone calvarial culture is the first example of a naturally occurring system that expresses the 100-kDa latent TGF beta complex.
Mol Endocrinol 1991 Jun
PMID:Latent forms of transforming growth factor-beta (TGF beta) derived from bone cultures: identification of a naturally occurring 100-kDa complex with similarity to recombinant latent TGF beta. 192 93

Platelet-derived growth factor (PDGF)-like molecules secreted by alveolar macrophages have been postulated to be mediators of lung fibrogenesis since these cytokines stimulate the proliferation and chemotaxis of lung fibroblasts. We are studying the biology and biochemistry of rat macrophage-derived PDGF and have identified for the first time the specific isoforms of PDGF (-AA, -AB, and -BB) that these macrophages secreted in vitro following activation with either chrysotile asbestos or carbonyl iron spheres. Subsequently, the proliferative response of rat lung fibroblasts (RLF) to the different PDGF isoforms was established. Using several antibodies raised against the distinct isoforms, we established that two different PDGF-like factors with molecular masses of 30 to 34 kD and 16 to 18 kD were contained in alveolar macrophage-conditioned medium. Within each of these molecular mass regions was a mixture of all three PDGF isoforms. We estimated that the 30- to 34-kD PDGF was mainly PDGF-BB (approximately 50%), while the remaining consisted of PDGF-AA (approximately 13%) and PDGF-AB (approximately 37%). Purified recombinant PDGF isoforms were tested for their ability to stimulate the growth of early-passage RLF and Swiss 3T3 cells in a 3-day cell proliferation assay. PDGF-BB and PDGF-AB were the most potent inducers of RLF proliferation and stimulated growth half-maximally at approximately 1 ng/ml and approximately 7 ng/ml, respectively. While these two B-chain-containing dimers stimulated lung fibroblast growth by as much as 150% above control medium, the PDGF-AA homodimer stimulated lung fibroblast proliferation less than 25% above control medium at the highest concentrations tested (20 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1991 Dec
PMID:Differential proliferation of rat lung fibroblasts induced by the platelet-derived growth factor-AA, -AB, and -BB isoforms secreted by rat alveolar macrophages. 195 75

The petunia nuclear gene which encodes the chloroplast isozyme of superoxide dismutase, SOD-1, has been fused with an efficient rbcS promoter fragment and 3' flanking region and introduced into tobacco and tomato cells. Transformed plants carrying this chimeric gene have up to 50-fold the levels of SOD-1 which occur in wild-type plants. However, tobacco plants with 30- to 50-fold the normal SOD-1 activity do not exhibit resistance to the light-activated herbicide paraquat. Similarly, tomato plants with 2- to 4-fold increases in SOD-1 do not exhibit tolerance to photoinhibitory conditions known to increase superoxide levels (high light, low temperatures and low CO2 concentrations). Our data indicate that increasing the chloroplastic SOD level in a plant cell is not sufficient to reduce the toxicity of superoxide.
Plant Mol Biol 1990 Apr
PMID:Transformed plants with elevated levels of chloroplastic SOD are not more resistant to superoxide toxicity. 196 84

Upon activation, T lymphocytes accumulate high levels of the neuropeptide enkephalin which correlate with high levels of proenkephalin mRNA in the cells. Here we investigated the transcriptional basis for these changes. The proenkephalin promoter contains a sequence GGGGACGTCCCC, named B2, which is similar to the kappa B sequence GGGGACTTTCC, the binding site of the transcription factor nuclear factor (NF)-kappa B. Activation of T lymphocytes induces an NF-kappa B-like binding activity to the B2 site, concomitant with activation of the proenkephalin promoter. Mutations at the B2 site abolish this transcriptional activation. The purified homodimer (two p50s) of the DNA-binding subunit of NF-kappa B binds the B2 site of proenkephalin relatively better than does the heterotetramer (two p65s plus two p50s) form of the factor. Thus, it appears that the T-cell-specific activation of the proenkephalin promoter is mediated by NF-kappa B. However, as NF-kappa B is ubiquitous and the transcriptional activation through the B2 site is T cell specific, yet another T-cell-specific factor which synergizes with NF-kappa B should be considered.
Mol Cell Biol 1991 Feb
PMID:Nuclear factor kappa B activates proenkephalin transcription in T lymphocytes. 199 Feb 63

The nucleotide sequence of the gene cysH from Escherichia coli K12 was determined. The open reading frame was 735 nucleotides in length; it was flanked by a repetitive palindromic sequence centred 36 nucleotides upstream of cysH and a terminator-like structure located 20 nucleotides downstream. CysH encoded a polypeptide of Mr 27927 consisting of 244 amino acids. The gene product was isolated as a homodimer exhibiting phospho-adenylylsulphate reductase (PAPS reductase) activity. The active enzyme was devoid of electron transferring cofactors and contained only one cysteine per subunit. Reduction of the enzyme by dithiols resulted in a shift of the apparent molecular weight from 44,000 to 62,000 without formation of an enzyme-thioredoxin complex.
Mol Gen Genet 1991 Feb
PMID:Characterisation of the gene cysH and of its product phospho-adenylylsulphate reductase from Escherichia coli. 200 73

Alkaline phosphatase (AP) is a widely distributed non-specific phosphomonoesterase that functions through formation of a covalent phosphoseryl intermediate (E-P). The enzyme also catalyzes phosphoryl transfer reaction to various alcohols. Escherichia coli AP is a homodimer with 449 residues per monomer. It is a metalloenzyme with two Zn2+ and one Mg2+ at each active site. The crystal structure of native E. coli AP complexed with inorganic phosphate (Pi), which is a strong competitive inhibitor as well as a substrate for the reverse reaction, has been refined at 2.0 A resolution. Some parts of the molecular have been retraced, starting from the previous 2.8 A study. The active site has been modified substantially and is described in this paper. The changes in the active site region suggest the need to reinterpret earlier spectral data, and suggestions are made. Also presented are the structures of the Cd-substituted enzyme complexed with inorganic phosphate at 2.5 A resolution, and the phosphate-free native enzyme at 2.8 A resolution. At pH 7.5, where the X-ray data were collected, the Cd-substituted enzyme is predominantly the covalent phosphoenzyme (E-P) while the native Zn/Mg enzyme exists in predominantly noncovalent (E.P) form. Implication of these results for the catalytic mechanism of the enzyme is discussed. APs from other sources are believed to function in a similar manner.
J Mol Biol 1991 Mar 20
PMID:Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis. 201 Sep 19

The human osteoinductive proteins BMP-2a and BMP-2b have been cloned and expressed in mammalian cells. In order to improve expression levels we examined the role of the proregion in assembly and export. Use of the BMP-2a proregion combined with the mature region of BMP-2b leads to dramatically improved expression of mature BMP-2b. Mature BMP-2b has been purified to near homogeneity from the BMP-2a/2b hybrid, and its structural properties and biological activity determined. Recombinant mature BMP-2b homodimer elicits bone formation in vivo.
Mol Endocrinol 1991 Jan
PMID:Bone-inducing activity of mature BMP-2b produced from a hybrid BMP-2a/2b precursor. 201 89

The structure of Mn(III) superoxide dismutase (Mn(III)SOD) from Thermus thermophilus, a tetramer of chains 203 residues in length, has been refined by restrained least-squares methods. The R-factor [formula: see text] for the 54,056 unique reflections measured between 10.0 and 1.8 A (96% of all possible reflections) is 0.176 for a model comprising the protein dimer and 180 bound solvents, the asymmetric unit of the P4(1)2(1)2 cell. The monomer chain forms two domains as determined by distance plots: the N-terminal domain is dominated by two long antiparallel helices (residues 21 to 45 and 69 to 89) and the C-terminal domain (residues 100 to 203) is an alpha + beta structure including a three-stranded sheet. Features that may be important for the folding and function of this MnSOD include: (1) a cis-proline in a turn preceding the first long helix; (2) a residue inserted at position 30 that distorts the helix near the first Mn ligand; and (3) the locations of glycine and proline residues in the domain connector (residues 92 to 99) and in the vicinity of the short cross connection (residues 150 to 159) that links two strands of the beta-sheet. Domain-domain contacts include salt bridges between arginine residues and acidic side chains, an extensive hydrophobic interface, and at least ten hydrogen-bonded interactions. The tetramer possesses 222 symmetry but is held together by only two types of interfaces. The dimer interface at the non-crystallographic dyad is extensive (1000 A2 buried surface/monomer) and incorporates 17 trapped or structural solvents. The dimer interface at the crystallographic dyad buries fewer residues (750 A2/monomer) and resembles a snap fastener in which a type I turn thrusts into a hydrophobic basket formed by a ring of helices in the opposing chain. Each of the metal sites is fully occupied, with the Mn(III) five-co-ordinate in trigonal bipyramidal geometry. One of the axial ligands is solvent; the four protein ligands are His28, His83, Asp166 and His170. Surrounding the metal-ligand cluster is a shell of predominantly hydrophobic residues from both chains of the asymmetric unit (Phe86A, Trp87A, Trp132A, Trp168A, Tyr183A, Tyr172B, Tyr173B), and both chains collaborate in the formation of a solvent-lined channel that terminates at Tyr36 and His32 near the metal ion and is presumed to be the path by which substrate or other inner-sphere ligands reach the metal.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1991 May 20
PMID:Manganese superoxide dismutase from Thermus thermophilus. A structural model refined at 1.8 A resolution. 203 60

The centromere DNA element I (CDEI) is an important component of Saccharomyces cerevisiae centromere DNA and carries the palindromic sequence CACRTG (R = purine) as a characteristic feature. In vivo, CDEI is bound by the helix-loop-helix protein CPF1. This article describes the in vivo analysis of all single-base-pair substitutions in CDEI in the centromere of an artificial chromosome and demonstrates the importance of the palindromic sequence for faithful chromosome segregation, supporting the notion that CPF1 binds as a dimer to this binding site. Mutational analysis of two conserved base pairs on the left and two nonconserved base pairs on the right of the CDEI palindrome revealed that these are also relevant for mitotic CEN function. Symmetrical mutations in either half-site of the palindrome affect centromere activity to a different extent, indicating nonidentical sequence requirements for binding by the CPF1 homodimer. Analysis of double point mutations in CDEI and in CDEIII, an additional centromere element, indicate synergistic effects between the DNA-protein complexes at these sites.
Mol Cell Biol 1991 Jul
PMID:In vivo characterization of the Saccharomyces cerevisiae centromere DNA element I, a binding site for the helix-loop-helix protein CPF1. 204 68


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