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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human leukocyte interferon-A1 (IFN-alpha A) structure in solution was investigated by fluorescence polarization, circular dichroism and scanning microcalorimetry techniques. Using gel-filtration it was established that at neutral pH values and at concentration not exceeding 0.3 mg/ml IFN-alpha A has a dimeric configuration in solution. At pH below 5, IFN-alpha A exists as a monomer. Using circular dichroism technique the IFN-alpha A molecule was shown to preserve a native structure upon decreasing pH to 3.5. The rotational correlation time of IFN-alpha A molecule in dimeric and monomeric form was measured using fluorescence of DNS, conjugated with the protein, and fluorescence of tryptophan residues. Our data indicate that the shape of IFN-alpha A molecule may be approximated by the rigid ellipsoid of revolution with the axis ratio = 4:1. The intramolecular melting of IFN-alpha A was studied by scanning microcalorimetry and circular dichroism in the acidic pH range. Thermodynamic analysis reveals two independent cooperative transitions. These transitions can be explained by assuming that the IFN-alpha A molecule consists of two structural domains.
Mol Biol (Mosk)
PMID:[Study of the structural properties of recombinant leukocyte interferon A by means of fluorescence polarization, circular dichroism, and differential microcalorimetry]. 179

Citrate synthase (EC 4.1.3.7), which is present in all living organisms as a key enzyme in aerobic energy metabolism, is one of the most highly phylogenetically conserved enzymes known in terms of its primary and active site structure. However, in terms of other parameters such as in vitro stability, tolerance to changes in pH, degree of self-polymerization, etc., citrate synthases from different sources are markedly different. These divergences can be observed even between isoforms of the enzyme within the same species. Data documenting these diversities suggest that a high degree of difference in tertiary structures may occur. Therefore, the surface profiles of citrate synthase enzymes from yeast, pig, rat, tomato and Escherichia coli were investigated with immunological methods using monoclonal antibody families generated against either pig citrate synthase (alpha-PCS) or yeast citrate synthase-2 (alpha-YCS-2). A high degree of homology of enzyme epitopes was detected on the mitochondrial citrate synthases originating from yeast, tomato, pig and rat cells. Major differences were found between the hexameric citrate synthase originating from E. coli compared with those dimeric forms prepared from eukaryotic cells. Only modest similarities were detected between the highly homologous peroxisomal and mitochondrial yeast citrate synthases. Furthermore, a point mutation of one of the catalytic residues (H274R on recombinant pig and H313R on yeast enzyme) of mitochondrial citrate synthase (CS-1) resulted in a significant increase in immunological similarity with the peroxisomal isoenzyme (CS-2). These findings are discussed in terms of the possible mechanism of evolution of CS-2 in yeast.
J Mol Recognit
PMID:Immunological mapping of fine molecular surface structures of citrate synthase enzymes from different cell types. 181 Mar 49

Bisphosphoglycerate mutase (EC 2.7.5.4) catalyzes the synthesis and breakdown of 2,3-diphosphoglycerate in red cells. The human enzyme, cloned and expressed in Escherichia coli has been crystallized in the rhombohedral space group R32 with a = b = c = 100.4 A and alpha = beta = gamma = 81.2 degrees. The asymmetric unit contains either a dimeric enzyme molecule, or a monomer.
J Mol Biol 1991 Mar 20
PMID:Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase. 182 31

Human C3b bound to the ghost of sheep erythrocytes (E*) via activation of the alternative complement pathway (E*AC3b) consists of four major constituents on SDS-PAGE of 350, 260, 210 and 180 kDa. 350 kDa C3b is a dimeric form of C3b in which the alpha' chain of one C3b binds covalently to that of the other C3b. This complex is presumed to serve as a core for the alternative pathway C5 convertase. The other C3b populations are monomers complexed with membrane proteins or sugars. Using E*AC3b (C3b labeled) as a substrate, we have investigated functional properties of membrane cofactor protein (MCP), which is an integral membrane protein with C3b-binding and factor I-dependent cofactor activities. In conjunction with factor I, MCP was found to degrade the protein-bound C3b preferentially including the 350 kDa dimer. There was a similar but lesser tendency of this selective cleavage of C3b-dimer by CR1 but not by factor H or C4bp. In contrast to CR1 and factor H, detergent solubilization of EAC3b was required for MCP to fully express its cofactor activity for this selective degradation of C3b. We next separated the C3b dimer from the monomers and assessed their ability to assemble the alternative C5 convertase. The C3b dimer but not the monomers expressed C5 convertase activity following the addition of factors B and D, C5 and Ni2+. Kinetic analysis of the degradation of the C3b dimer by MCP and factor I suggested that only one C3b was efficiently converted to C3bi and this occurred concomitant with a decrease in C5 convertase activity. These results suggest that MCP has the ability to more efficiently interact with protein-bound C3b and that this may relate as well to its preferential ability to irreversibly inactivate the C5 convertase.
Mol Immunol 1991 Oct
PMID:Preferential inactivation of the C5 convertase of the alternative complement pathway by factor I and membrane cofactor protein (MCP). 183 39

The recombinant plasmids have been constructed encoding the synthesis of a full-sized diphtheria toxin from its own or PR, PL-promoters of bacteriophage lambda in Escherichia coli cells. The high level constitutive synthesis of toxin results in slow cell growth and plasmid elimination. The toxin was mainly detected in the periplasm, partially in the membrane and to a less extent in the cytoplasm and culturing medium. The dimeric form of toxin was found in the cytoplasm. Participation of toxin B-subunit in secreting of the toxin into culturing medium is discussed. Proteolytic degradation of the synthesized toxin in different Escherichia coli strains was demonstrated. The process takes place in cytoplasm and periplasm mainly. The main enzyme participating in the process is a La-protease. The data on proteolysis obtained by immunoprecipitation immunoblotting, affinity chromatography and in mini-cells of Escherichia coli are summarized.
Mol Gen Mikrobiol Virusol 1991 Oct
PMID:[Synthesis, secretion, and proteolytic degradation of diphtheria toxin in Escherichia coli]. 183 36

In Rhizobium meliloti, expression of the nodulation genes (nod and nol genes) is under both positive and negative controls. These genes are activated by the products of the three related nodD genes, in conjunction with signal molecules from the host plants. We showed that negative regulation is mediated by a repressor protein, binding to the overlapping nodD1 and nodA as well as to the nodD2 promoters. The encoding gene, termed nolR, was identified and cloned from strain 41. By subcloning, deletion and Tn5 mutagenesis, a region of 594 base-pairs was found to be necessary and sufficient for repressor production in strains of R. meliloti lacking the repressor or in Escherichia coli. Sequence analysis revealed that nolR encodes a 13,349 Da protein, which is in agreement with the molecular weight of the NolR protein, determined after purification by affinity chromatography, utilizing long synthetic DNA multimers of the 21 base-pair conserved repressor-binding sequence. Our data suggest that the native NolR binds to the operator site in dimeric form. The NolR contains a helix-turn-helix motif, which shows homology to the DNA-binding sequences of numerous prokaryotic regulatory proteins such as the repressor XylR or the activator NodD and other members of the LysR family. Comparison of the putative DNA-binding helix-turn-helix motifs of a large number of regulatory proteins pointed to a number of novel regularities in this sequence. Hybridizations with an internal nolR fragment showed that sequences homologous to the nolR gene are present in all R. meliloti isolates tested, even in those that do not produce the repressor. In another species, such as Rhizobium leguminosarum, where NodD is autoregulated, however, such sequences were not detected.
J Mol Biol 1991 Dec 20
PMID:Identification of NolR, a negative transacting factor controlling the nod regulon in Rhizobium meliloti. 184 Jun 15

1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.
Cell Mol Neurobiol 1991 Feb
PMID:Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme. 184 51

1. We analyzed the mode of attachment of 16 S tailed acetylcholinesterase (AChE; EC 3.1.1.7) to rat superior cervical ganglion (SCG) neuronal membranes. Using extractions by high-salt (HS) and nonionic detergent (Triton X-100), we found two pools of 16 S AChE. 2. The detergent-extracted (DE) 16 S AChE was tightly bound to membranes through detergent-sensitive, high-salt insensitive interactions and was distinct from high-salt-soluble 16 S AChE. The detergent-extracted (DE) 16 S AChE constituted a significant proportion of about one-third of the total 16 S AChE. 3. Treatment of the neuronal membranes by a phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the release of some, but not all DE 16 S AChE, indicating that a significant amount of the neuronal DE 16 S AChE, about one-third, is anchored to membranes through a phosphatidylinositol containing residue. Thus, a covalent association of a glycolipid and catalytic or structural AChE polypeptidic chains occurs not only for dimeric AChE but also for the asymmetric species of AChE. 4. The complex polymorphism of AChE is due not only to different globular or asymmetric associations of catalytic and structural subunits but also to the alternative existence of a transmembrane domain or a glycolipid membrane anchor.
Cell Mol Neurobiol 1991 Feb
PMID:Phosphatidylinositol is involved in the attachment of tailed asymmetric acetylcholinesterase to neuronal membranes. 184 54

Cytochrome c-oxidase type aa3 (EC 1.9.3.1) was purified to homogeneity from vegetative Bacillus cereus by ion-exchange and hydroxylapatite chromatography in the presence of Triton X-100. Gel filtration analysis suggested a dimeric structure apparently 172 kDa in size; however, only a monomer of 81 kDa was detected when analysed by non-denaturing gel electrophoresis. Denaturing gel electrophoresis analysis of the protein showed the presence of two subunits (51 and 30 kDa). Atomic absorption and visible spectroscopy showed typical aa3 redox centres with haem a iron and copper in a ratio of 22 nmol and 35 ng-atom per mg protein, respectively. No haem c was found associated with the purified enzyme in the conditions reported here. Oxidase activity was fully reconstituted by phospholipids in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine or reduced yeast cytochrome c (but not horse cytochrome c) as electron donors. This activity was abolished by cyanide and carbon monoxide.
Mol Microbiol 1991 Jan
PMID:Purification and characterization of two-subunit cytochrome aa3 from Bacillus cereus. 184 7

We have used a previously described cross-linking assay and half-FRT site substrates to examine the requirements for synapsis, strand exchange, and strand scission. The cross-linking assay showed that the minimum functional FRT site needed for synapsis contains two inverted FLP-binding elements surrounding an 8-bp core. This indicates that four FLP molecules interact with four binding elements in a synaptic complex. The analysis using half-sites showed that the enzyme can catalyze efficient strand exchange between a half-site and the intact FRT site. The reaction occurred only if the half-site had at least 2 bp but no more than 4 bp of the adjoining core sequence. The exchange occurred exclusively at the regions of limited core homology between the respective half-site and the FRT site. The absence of strand exchange between an intact site and a half-site bearing regions of core nonhomology indicates that 1 bp of homology is not sufficient for the formation of stable recombinant structures. Qian et al. (X.-H. Qian, R. B. Inman, and M. M. Cox, J. Biol. Chem. 265:21779-21788, 1990) have recently shown that the FLP protein can catalyze the formation of dimeric, trimeric, and tetrameric complexes with half-FRT sites. We show that only half-sites that contained at least 2 bp of adjacent core could form stable dimer products and be cleaved by the enzyme. Stable dimers were formed between a noncleavable half-site and a cleavable half-site, suggesting that only a single cleavage event is needed for the formation of the dimer.
Mol Cell Biol 1991 Sep
PMID:Synapsis, strand scission, and strand exchange induced by the FLP recombinase: analysis with half-FRT sites. 187 35


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