| 1. |
- Hillarp, A, et al.
(författare)
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Novel subunit in C4b-binding protein required for protein S binding
- 1988
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 263:25, s. 64-12759
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Tidskriftsartikel (refereegranskat)abstract
- C4b-binding protein (C4BP) is a multimeric protein with regulatory functions in the complement system. It also interacts with vitamin K-dependent protein S, which is involved in the regulation of the coagulation system. It has been demonstrated that C4BP consists of seven disulfide-linked, identical 70-kDa subunits, which are arranged to give the molecule a spider-like structure. We now have evidence for the presence of a new subunit in C4BP. On sodium dodecyl sulfate-poly-acrylamide gel electrophoresis it appears as a weakly stainable band with a molecular weight of approximately 45,000. The subunit was isolated by gel filtration in 6 M guanidine hydrochloride of reduced and carboxymethylated C4BP. Its amino-terminal sequence is distinct from previously known protein sequences. The stoichiometry of 45- to 70-kDa subunits was estimated to be 1:9, indicating the presence of one 45-kDa subunit per C4BP molecule. The new subunit was demonstrated to be a disulfide-linked component of the central core of C4BP. It was sensitive to proteolysis by chymotrypsin, and when cleaved the protein S binding ability of C4BP was lost. With protein S bound to C4BP, the 45-kDa subunit was protected from degradation by chymotrypsin, and the protein S binding site remained intact. These data suggest that the new subunit is directly involved in protein S binding.
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| 2. |
- Hillarp, A, et al.
(författare)
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The protein S-binding site localized to the central core of C4b-binding protein
- 1987
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 262:23, s. 7-11300
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Tidskriftsartikel (refereegranskat)abstract
- Human C4b-binding protein (C4BP) is a regulator of the classical pathway of the complement system. It appears in two forms in plasma, as free protein and in a noncovalent complex with the vitamin K-dependent coagulation protein, protein S. In the electron microscope C4BP has a spider-like structure with a central core and seven extended tentacles, each of which has a binding site for C4b, although the protein S-binding site has not been unequivocally pinpointed. C4BP was subjected to chymotrypsin digestion which yielded two major fragments, one of 160 kDa representing the central core, and one of 48 kDa representing the cleaved-off tentacles. We have now localized the protein S-binding site to the 160-kDa central core fragment. Using immunoblotting with a panel of polyclonal antisera, the isolated central core was shown to be completely devoid of 48-kDa fragments. The protein S-binding site was susceptible to proteolysis by chymotrypsin, but was protected by a molar excess of protein S included during the proteolysis. The 160-kDa central core fragment consisted of identical, disulfide-linked 25-kDa peptides and a proper disulfide bond arrangement was crucial to protein S binding. Using a direct binding assay it was shown that the isolated central core had the same affinity for protein S as intact C4BP.
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| 3. |
- Connolly, Eamonn, et al.
(författare)
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Norepinephrine-induced Na+ influx in brown adipocytes is cyclic AMP-mediated
- 1986
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 261:31, s. 14377-14385
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Tidskriftsartikel (refereegranskat)abstract
- To examine the involvement of Na+ ions in adrenergic responses in brown adipose tissue, a method is described for measuring Na+ influx into isolated brown adipocytes, using short (30 s) incubations with 22Na+, followed by a two-step centrifugation recovery procedure. Using this method, a clear norepinephrine-stimulated accumulation of intracellular 22Na+ was observed, which was enhanced by the addition of ouabain, was insensitive to amiloride (a Na+/H+ exchange blocker), and could not be mimicked by the total removal of oxygen from the incubation medium. The norepinephrine-stimulated Na+ influx was dose-dependent for the hormone with an EC50 of 250 nM, was blocked by the beta-antagonist propranolol but not by the alpha 1-antagonist prazosin, and could be induced by adrenergic agonists with the order of potency: isoproterenol greater than norepinephrine greater than phenylephrine, indicating a beta-receptor-mediated process. The Na+ influx was found to be cAMP-dependent since it could be induced by both theophylline (a phosphodiesterase inhibitor) and forskolin (an adenylate cyclase activator), but it was independent of other known cellular cAMP-dependent responses since neither addition of fatty acid substrates (octanoate or palmitate), nor of the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone could induce the phenomenon, despite having significant stimulatory effects on cellular respiration. Furthermore, total respiratory inhibition with rotenone, or total oxygen depletion of the medium with dithionite, did not prevent the normal norepinephrine-induced Na+ influx. The possibility that this beta-mediated norepinephrine-stimulated Na+ influx plays an important physiological role in brown adipose tissue activity is discussed, perhaps as one of the, as yet undefined, signals initiating tissue growth in the chronically beta-stimulated tissue of animals facing long-term increases in thermogenic demands.
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| 4. |
- Björk, S, et al.
(författare)
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Structures of blood group glycosphingolipids of human small intestine. A relation between the expression of fucolipids of epithelial cells and the ABO, Le and Se phenotype of the donor.
- 1987
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 262:14, s. 6758-65
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Tidskriftsartikel (refereegranskat)abstract
- Small intestinal epithelial cells (enterocytes) were isolated from specimens obtained at operation from four human individuals with different blood group ABO, Lewis, and secretor phenotypes. The non-acid glycolipids were isolated and characterized by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy and for reactivity with monoclonal antibodies on thin-layer chromatograms. Monohexosylceramides and blood group ABH (type 1 chain) and Lewis glycolipids with 5-7 sugar residues were the major compounds present in all cases, and the expression of the major blood group glycolipids was in agreement with the ABO, Lewis, and secretor phenotype of the individual donors. Small amounts of more complex glycolipids with up to 10 sugar residues were identified by mass spectrometry in all cases. In addition, small amounts of lactotetraosylceramide, a blood group H-active triglycosylceramide with the structure of Fuc alpha 1-2Gal-Hex-Cer (where Fuc is fucose, Hex is hexose, and Cer is ceramide), and dihexosylceramides were identified in some cases. Globotriaosyl- and globotetraosylceramides were absent from the epithelial cells. Small amounts of Leb-active glycolipids in blood group OLe(a+b-), non-secretor and OLe(a-b-), secretor individuals as well as trace amounts of type 2 carbohydrate chain compounds in all individuals were detected by specific monoclonal antibodies.
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| 5. |
- Bock, K, et al.
(författare)
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Specificity of binding of a strain of uropathogenic Escherichia coli to Gal alpha 1----4Gal-containing glycosphingolipids.
- 1985
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 260:14, s. 8545-51
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Tidskriftsartikel (refereegranskat)abstract
- A strain of Escherichia coli originally isolated from urine of a patient with acute pyelonephritis was studied in detail for binding to glycosphingolipids. Bacteria labeled metabolically with [14C]glucose were layered over a glycolipid chromatogram and bound bacteria were detected by autoradiography. The detection was down to a few ng of glycolipid (pmol level) under these assay conditions. At a test level of 500 ng all glycolipids (more than a dozen molecular species analyzed) with Gal alpha 1----4Gal as an internal or terminal part bound the bacteria strongly while glycolipids known to lack this sequence were negative. Conformational analysis using hard sphere calculations including the exo-anomeric effect showed a bend in the saccharide chain at this disaccharide with a largely hydrophobic surface of the convex side, probably being part of the binding epitope. Mixtures of glycolipids isolated from a human ureter scraping and from urinary sediments bound bacteria in the 2- to 7-sugar interval. Thus, this infectious strain of E. coli recognizes glycolipids being present in epithelial cells lining the urinary tract.
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| 6. |
- Brodelius, Peter, et al.
(författare)
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A Phosphorus-31 Nuclear Magnetic Resonance Study of Phosphate Uptake and Storage in Cultured Catharanthus roseus and Daucus carota Plant Cells
- 1985
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 260, s. 3556-3560
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Tidskriftsartikel (refereegranskat)abstract
- High resolution "P NMR spectra (103.2 MHz) ofoxygenated Catharanthus roseu8 and Daucus carotacells grown in suspension cultures were obtained usinga solenoidal perfusion probe. The spectra showed resonancesfor various phosphorylated metabolites suchas ATP, ADP, NAD(P)(H), nucleoside diphosphoglucose,and sugar phosphates. The relative levels ofthe phosphorylated metabolites remained constantthroughout the growth curve. No resonances for storagecompounds such as polyphosphates, pyrophosphate,or phytates were observed. Two resolved resonancesfor Pi indicated an intracellular pH of 7.3 and5.7 (or below) for the cytoplasm and vacuoles, respectively.The time course of Pi uptake and storage duringgrowth in fresh culture medium was followed by studyingthe level of vacuolar Pi with 31PN MR (145.7 MHz).Simultaneously, the level of Pi in the culture mediumwas followed with radioactive s2P. C. roseus quicklytakes up all the Pi from the culture medium (maximumrate 1.7 pmol min" g" (dry weight of cells)). The Pi isfirst stored in the vacuoles; subsequently, one part ofthis pool is used to keep a constant cytoplasmic Pi levelwhile another part is apparently accumulated as anNMR invisible Pi store, probably in another cell organelle.In contrast, D. carota does not accumulate Pi inthe vacuoles and consequently it takes up Pi from themedium at a much slower rate (0.05 pmol min" g"(dry weight of cells)).
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| 7. |
- Dahlbäck, Björn, et al.
(författare)
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Localization of thrombin cleavage sites in the amino-terminal region of bovine protein S
- 1986
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 261:11, s. 5-5111
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Tidskriftsartikel (refereegranskat)abstract
- Protein S is a vitamin K-dependent plasma protein. It functions as a cofactor to activated protein C in the inactivation of factors Va and VIIIa by limited proteolysis. Protein S is very sensitive to proteolysis by thrombin which reduces its calcium ion binding and leads to a loss of its cofactor activity. We have now determined the sequence of the 100 amino-terminal amino acid residues and localized the thrombin cleavage sites. Protein S contains 11 gamma-carboxyglutamic acid residues in the amino-terminal region (residues 1-36). This part of protein S is highly homologous to the corresponding parts in the other vitamin K-dependent clotting factors, whereas the region between residues 45 and 75 is not at all homologous to the other clotting factors. Thrombin cleaves two peptide bonds in this part of protein S, first at arginine 70 and then at arginine 52. The peptide containing residues 53-70 is released from protein S after thrombin cleavage. The amino-terminal fragment, residues 1-52, is linked to the large carboxyl-terminal fragment by a disulfide bond, which involves cysteine 47. After residue 78, protein S is again homologous to factors IX and X and to proteins C and Z, but not to prothrombin. Position 95 is occupied by a beta-hydroxyaspartic acid residue.
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| 8. |
- Eytan, G D, et al.
(författare)
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Energy-linked nicotinamide-nucleotide transhydrogenase. Characterization of reconstituted ATP-driven transhydrogenase from beef heart mitochondria.
- 1987
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 262, s. 5008-5014
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between pure transhydrogenase and ATPase (Complex V) from beef heart mitochondria was investigated with transhydrogenase-ATPase vesicles in which the two proteins were co-reconstituted by dialysis or dilution procedures. In addition to phosphatidylcholine and phosphatidylethanolamine, reconstitution required phosphatidylserine and lysophosphatidylcholine. Transhydrogenase-ATPase vesicles catalyzed a 20-30-fold stimulation of the reduction of NADP+ or thio-NADP+ by NADH and a 70-fold shift of the apparent equilibrium expressed as the nicotinamide nucleotide ratio [NADPH][NAD+]/[NADP+][NADH]. In both of these respects, the transhydrogenase-ATPase vesicles were severalfold more efficient than beef heart submitochondrial particles. By measuring the ATP-driven transhydrogenase and the oligomycin-sensitive ATPase activities simultaneously and under the same conditions at low ATP concentrations, i.e. below 15 microM, the ATP-driven transhydrogenase/oligomycin-sensitive ATPase activity ratio was found to be about 3. This value is consistent with the stoichiometries of three protons translocated per ATP hydrolyzed and one proton translocated per NADPH formed and with a mechanism where the two enzymes interact through a delocalized proton-motive force.
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| 9. |
- Finne, J, et al.
(författare)
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Novel polyfucosylated N-linked glycopeptides with blood group A, H, X, and Y determinants from human small intestinal epithelial cells.
- 1989
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 264:10, s. 5720-35
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Tidskriftsartikel (refereegranskat)abstract
- A novel type of N-linked glycopeptides representing a major part of the glycans in human small intestinal epithelial cells from blood group A and O individuals were isolated by gel filtrations and affinity chromatography on concanavalin A-Sepharose and Bandeiraea simplicifolia lectin I-Sepharose. Sugar composition, methylation analysis, 1H NMR spectroscopy of the underivatized glycopeptides and FAB-mass spectrometry and electron impact-mass spectrometry of the permethylated glycopeptides indicated a tri- and tetra-antennary structure containing an intersecting N-acetylglucosamine and an alpha (1----6)-linked fucose residue in the core unit for the majority of the glycans. In contrast to most glycopeptides of other sources, the intestinal glycopeptides were devoid of sialic acid, but contained 6-7 residues of fucose. The outer branches contained the following structures: Fuc alpha 1-2Gal beta 1-3GleNAc beta 1- (H type 1) Fuc alpha 1-2Gal beta 1-4GleNAc beta 1- (H type 2) Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1- (X) Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GleNAc beta 1- (Y) GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GleNAc beta 1- (A type 1) GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GleNAc beta 1- (monofucosyl A type 2) GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1- (trifucosyl A type 2) The blood group determinant structures were mainly of type 2, whereas glycolipids from the same cells contained mainly type 1 determinants. The polyfucosylated glycans represent a novel type of blood group active glycopeptides. The unique properties of the small intestinal glycopeptides as compared with glycopeptides of other tissue sources may be correlated with the specialized functional properties of the small intestinal epithelial cells.
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| 10. |
- Kastern, W, et al.
(författare)
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Developmental and tissue-specific expression of alpha 1-microglobulin mRNA in the rat
- 1986
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 261:32, s. 4-15070
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Tidskriftsartikel (refereegranskat)abstract
- A rat liver cDNA library was constructed in the lambda gt11 expression vector. Three clones expressing alpha 1-microglobulin, an immunosuppressive plasma protein, were detected by screening with rabbit antiserum against rat alpha 1-microglobulin. The alpha 1-microglobulin activity from one of the clones, 6b, was confirmed with monoclonal antibodies in a solid phase radioimmunoassay. The nucleotide sequence of the fragment (165 base pairs) was determined, and the translated amino acid sequence (55 amino acids) showed a 75% homology to human alpha 1-microglobulin (position 122-176). Southern blots of restriction endonuclease-digested rat DNA indicated two distinct genes with alpha 1-microglobulin homology when probed with radioactive cDNA fragment from clone 6b. Northern blots showed the presence of a single mRNA species in rat liver, and the level was low in 1-month-old animals, increased to reach a maximum during adulthood (3 months), and decreased with aging (12 months). The alpha 1-microglobulin concentration in rat serum showed the same age dependence between 1 and 12 months, with the highest values at 3 months. Embryonic development (8.5-day to 17.5-day) was studied using total fetal RNA, and expression of alpha 1-microglobulin mRNA was detected in low amounts only at day 15.5. alpha 1-Microglobulin mRNA levels, studied by an RNA dot blot assay, were high in liver and kidney, low in brain and testis, and none were found in hypothalamus and spleen cells.
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