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Soluble Fragment (soluble + fragment)
Selected AbstractsSoluble LDL-R are formed by cell surface cleavage in response to phorbol estersFEBS JOURNAL, Issue 3 2004Michael J. Begg A 140-kDa soluble form of the low density lipoprotein (LDL) receptor has been isolated from the culture medium of HepG2 cells and a number of other cell types. It is produced from the 160-kDa mature LDL receptor by a proteolytic cleavage, which is stimulated in the presence of 4,-phorbol 12-myristate 13-acetate (PMA), leading to the release of a soluble fragment that constitutes the bulk of the extracellular domain of the LDL receptor. By labeling HepG2 cells with [35S]methionine and chasing in the presence of PMA, we demonstrated that up to 20% of LDL-receptors were released into the medium in a 2-h period. Simultaneously, the level of labeled cellular receptors was reduced by 30% in those cells treated with PMA compared to untreated cells, as was the total number of cell surface LDL-receptors assayed by the binding of 125I-labeled antibody to whole cells. To determine if endocytosis was required for cleavage, internalization-defective LDL-receptors were created by mutagenesis or deletion of the NPXY internalization signal, transfected into Chinese hamster ovary cells, and assayed for cleavage in the presence and absence of PMA. Cleavage was significantly greater in the case of the mutant receptors than for wild-type receptors, both in the absence and presence of PMA. Similar results were seen in human skin fibroblasts homozygous for each of the internalization-defective LDL receptor phenotypes. LDL receptor cleavage was inhibited by the hydoxamate-based inhibitor TAPI, indicating the resemblance of the LDL receptor cleavage mechanism to that of other surface released membrane proteins. [source] Solution structure of the functional domain of Paracoccus denitrificans cytochrome c552 in the reduced stateFEBS JOURNAL, Issue 13 2000Primo, Pristov In order to determine the solution structure of Paracoccus denitrificans cytochrome c552 by NMR, we cloned and isotopically labeled a 10.5-kDa soluble fragment (100 residues) containing the functional domain of the 18.2-kDa membrane-bound protein. Using uniformly 15N-enriched samples of cytochrome c552 in the reduced state, a variety of two-dimensional and three-dimensional heteronuclear double-resonance NMR experiments was employed to achieve complete 1H and 15N assignments. A total of 1893 distance restraints was derived from homonuclear 2D-NOESY and heteronuclear 3D-NOESY spectra; 1486 meaningful restraints were used in the structure calculations. After restrained energy minimization a family of 20 structures was obtained with rmsd values of 0.56 ± 0.10 Å and 1.09 ± 0.09 Å for the backbone and heavy atoms, respectively. The overall topology is similar to that seen in previously reported models of this class of proteins. The global fold consists of two long helices at the N-terminus and C-terminus and three shorter helices surrounding the heme moiety; the helices are connected by well-defined loops. Comparison with the X-ray structure shows some minor differences in the positions of the Trp57 and Phe65 side-chain rings as well as the heme propionate groups. [source] Purification, crystallization and preliminary X-ray diffraction analysis of the lytic transglycosylase MltF from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Pramod K. Madoori The lytic transglycosylase MltF from Escherichia coli is an outer-membrane-bound periplasmic protein with two domains: a C-terminal catalytic domain with a lysozyme-like fold and an N-terminal domain of unknown function that is homologous to the periplasmic substrate-binding proteins of ABC transporters. In order to investigate its structure and function, a soluble form of full-length MltF (sMltF) containing both domains and a soluble fragment containing only the N-terminal domain (sMltF-NTD) were purified and crystallized. Crystals of sMltF belonged to space group P43212 or P41212, with unit-cell parameters a = b = 110.8, c = 163.5,Å and one or two molecules per asymmetric unit. A complete data set was collected to 3.5,Å resolution. Crystals of sMltF-NTD belonged to space group P3121, with unit-cell parameters a = b = 82.4, c = 75.2,Å and one molecule per asymmetric unit. For sMltF-NTD, a complete native data set was collected to 2.20,Å resolution. In addition, for phasing purposes, a three-wavelength MAD data set was collected to 2.5,Å resolution using a bromide-soaked sMltF-NTD crystal. Using phases derived from the Br-MAD data, it was possible to build a partial model of sMltF-NTD. [source] Effect of endo -xylanase-containing enzyme preparations and laccase on the solubility of rye bran arabinoxylanJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 7 2003Roger Andersson Abstract Three commercial enzyme preparations with endo -xylanase activity, namely Bio-Feed Wheat, Bio-Feed Plus and Grindamyl H 640, and laccase have been tested for their effects on the solubilisation of arabinoxylan (AX) in rye bran or autoclaved rye bran. Autoclaving efficiently increased the availability of AX for enzymatic degradation. Both Bio-Feed Wheat (a monocomponent enzyme) and Bio-Feed Plus (a multicomponent preparation with different enzymatic activities) efficiently degraded the autoclaved rye bran AX into lower-molecular-weight fragments. As much as 70% of the xylose residues and 58% of the arabinose residues in the autoclaved bran were soluble after treatment with Bio-Feed Plus; the weight,average molecular weight of the detectable portion of these soluble polymers was 104,000,Da. Grindamyl H 640 solubilised only a small fraction of the AX in autoclaved rye bran; the molecular weight of these soluble fragments was higher than that of those released by the Bio-Feed xylanases. Addition of laccase during treatment with Bio-Feed Wheat or Grindamyl H 640 decreased the yield of water-soluble AX. © 2003 Society of Chemical Industry [source] | ||||||||||