scholarly journals Complexing of heparin with phosphatidylcholine. A possible supramolecular assembly of plasma heparin

1985 ◽  
Vol 227 (1) ◽  
pp. 57-65 ◽  
Author(s):  
S Vannucchi ◽  
M Ruggiero ◽  
V Chiarugi
Keyword(s):  
Heparan Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Supramolecular Assembly ◽  
Membrane Phospholipids ◽  
Anionic Phospholipids ◽  
High Yields ◽  
Plasma Heparin

In a series of attempts to reveal plasma heparin, we found that high ionic strength and modification of protein amino groups were not effective in extracting endogenous heparin (or, indeed, a large percentage of exogenous labelled heparin), whereas delipidation in the presence of 4M-guanidinium chloride gave high yields, indicating that plasma heparin may be assembled with compounds other than proteins, in a form making it inaccessible to water and ions. During the extraction of lipids, a paradoxical entry of heparin into the organic phase was observed. Detergents, including sodium dodecyl sulphate, did not shift heparin into the aqueous phase, whereas repeated chloroform/methanol extraction did so. Using purified compounds we were able to reproduce in vitro both the scavenging of heparin from water as well as the formation of heparin-phosphatidylcholine complexes soluble in organic solvents. Evidence for complexing of heparin with phosphatidylcholine was also obtained by electrophoretic and ultracentrifugation assays. The quaternary-ammonium-containing phosphatidylcholine was the more effective phospholipid in binding heparin; anionic phospholipids did not bind. Only heparin-like glycosaminoglycans bound phosphatidylcholine, but less-sulphated compounds (heparan sulphate and dermatan sulphate) were weaker ligands. Gel-filtration experiments showed that heparin was not bound to liposome vesicles, but that a measurable percentage of the phospholipids was stripped off from vesicles and was found in the form of a complex separable from liposomes by gel filtration. The molecular basis as well as the biological role of the interaction of heparin with major membrane phospholipids are discussed.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

Schistosoma mansoni: evidence for a role of serum factors in protecting artificially transformed schistosomula against antibody-mediated killing in vitro

Parasitology ◽  
1978 ◽  
Vol 77 (2) ◽  
pp. 225-233 ◽  
Author(s):  
C. A. P. Tavares ◽  
Rita C. Soares ◽  
P. M. Z. Coelho ◽  
G. Gazzinelli
Keyword(s):  
Schistosoma Mansoni ◽  
Protective Factor ◽  
Gel Filtration ◽  
Defined Medium ◽  
Rabbit Serum ◽  
Chemically Defined Medium ◽  
Lethal Effects ◽  
Serum Factors

SummaryArtificially transformed schistosomula of Schistosoma mansoni develop a consistent but small protection against the lethal effects of antibody plus complement when cultured for 24 h in a chemically defined medium. In contrast, they become rapidly resistant to antibody plus complement, when cultured in the presence of a complex medium consisting of equal parts of heat-inactivated rabbit serum and Earle's/lactalbumin or in defined medium supplemented with small amounts of heat-inactivated rabbit serum. Sephadex G-200 gel filtration revealed that the protective factor in rabbit serum is a macromolecule with a molecular weight between 7 and 19S. Parasites cultured at 10 °C or in the presence of 200 μg of puromycin show less serum-induced protection against the lethal effects of antibody plus complement than do controls.

scholarly journals Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids

2018 ◽  
Vol 115 (33) ◽  
pp. E7720-E7727 ◽  
Author(s):  
Spencer T. Glantz ◽  
Erin E. Berlew ◽  
Zaynab Jaber ◽  
Benjamin S. Schuster ◽  
Kevin H. Gardner ◽  
...  
Keyword(s):  
Electrostatic Interaction ◽  
Signal Transmission ◽  
Model Systems ◽  
Membrane Localization ◽  
Diffusion Limit ◽  
Dependent Manner ◽  
Membrane Phospholipids ◽  
Anionic Phospholipids ◽  
Domain Of Unknown Function

We report natural light–oxygen–voltage (LOV) photoreceptors with a blue light-switched, high-affinity (KD ∼ 10−7 M), and direct electrostatic interaction with anionic phospholipids. Membrane localization of one such photoreceptor, BcLOV4 from Botrytis cinerea, is directly coupled to its flavin photocycle, and is mediated by a polybasic amphipathic helix in the linker region between the LOV sensor and its C-terminal domain of unknown function (DUF), as revealed through a combination of bioinformatics, computational protein modeling, structure–function studies, and optogenetic assays in yeast and mammalian cell line expression systems. In model systems, BcLOV4 rapidly translocates from the cytosol to plasma membrane (∼1 second). The reversible electrostatic interaction is nonselective among anionic phospholipids, exhibiting binding strengths dependent on the total anionic content of the membrane without preference for a specific headgroup. The in vitro and cellular responses were also observed with a BcLOV4 homolog and thus are likely to be general across the dikarya LOV class, whose members are associated with regulator of G-protein signaling (RGS) domains. Natural photoreceptors are not previously known to directly associate with membrane phospholipids in a light-dependent manner, and thus this work establishes both a photosensory signal transmission mode and a single-component optogenetic tool with rapid membrane localization kinetics that approaches the diffusion limit.

scholarly journals Isolation and characterization of lung connective-tissue glycoproteins

1982 ◽  
Vol 203 (3) ◽  
pp. 593-601 ◽  
Author(s):  
C Lafuma ◽  
M Moczar ◽  
L Robert
Keyword(s):  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Lung Parenchyma ◽  
Isolation And Characterization ◽  
Isoelectric Points ◽  
And Function

1. Glycoproteins of hamster, rat and baboon lung parenchyma were investigated by using [14C]glucosamine incorporation in vitro followed by sequential extraction of the macromolecular components and characterization of the glycoproteins in the extracts. 2. Slices of lung parenchyma maintained in vitro incorporated [U-14C]glucosamine linearly with time into non-diffusible macromolecules for up to 5h. All the macromolecule-associated 14C label was present as [14C]glucosamine. 3. These 14C-labelled macromolecules were extracted from previously delipidated and salt-extracted lung by 5M-guanidinium chloride in the presence of dithiothreitol and proteinase inhibitors before (extract A1) and after (extract A2) hydrolysis of the collagen by collagenase. The [14C]glucosamine-labelled glycoproteins in extracts A1 and A2 contained 55 and 5% respectively of the total [14C]glucosamine incorporated in the lung of all three species studied. 4. The [14C]glucosamine-labelled glycoproteins were analysed by gel-filtration chromatography, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing. The major [14C]glucosamine-labelled glycoproteins of baboon lung parenchyma had apparent mol.wts. of about 400 000, 140 000 and 65 000 with isoelectric points respectively of 4.8, 5.4 and 5.4. The hamster lung glycoproteins with isoelectric points of 4.1 and 5.8 were devoid of hydroxyproline and contained galactose, mannose and N-acetylglucosamine. These experiments indicate that several distinct glycoproteins are synthesized in situ by the cells of pulmonary parenchyma and may well play a role in its structure and function.

Biosynthesis of N-Acetylneuraminic Acid in Cells Lacking UDP-N-Acetylglucosamine 2-Epimerase/ N-Acetylmannosamine Kinase

2001 ◽  
Vol 382 (2) ◽  
pp. 291-297 ◽  
Author(s):  
Stephan Hinderlich ◽  
Markus Berger ◽  
Oliver T. Keppler ◽  
Michael Pawlita ◽  
Werner Reutter
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Kinase Activity ◽  
Gel Filtration ◽  
Acetylneuraminic Acid ◽  
Biological Recognition ◽  
Functionally Impaired ◽  
Cell Surface Glycoconjugates

Abstract The first two steps in mammalian biosynthesis of Nacetylneuraminic acid, an important carbohydrate moiety in biological recognition systems, are performed by the bifunctional enzyme UDPNacetylglucosamine 2-epimerase/Nacetylmannosamine kinase. A subclone of the human B lymphoma cell line BJAB K20, lacking UDPNacetylglucosamine 2- epimerase/Nacetylmannosamine kinase mRNA as well as epimerase activity, displayed hyposialylated, functionally impaired cell surface glycoconjugates. Here we show that this cell line surprisingly still retains Nacetylmannosamine kinase activity. A gel filtration analysis of BJAB K88 control cells, which express UDPNacetylglucosamine 2-epimerase/Nacetylmannosamine kinase, revealed two Nacetylmannosamine kinase activity peaks, one coeluting with UDPNacetylglucosamine 2-epimerase activity and one coeluting with Nacetylglucosamine kinase. For this enzyme previous studies already showed ManNAc kinase activity in vitro. In contrast, the hyposialylated BJAB K20 subclone displayed only the Nacetylmannosamine kinase peak, comigrating with Nacetylglucosamine kinase. The CMPNacetylneuraminic acid content of both K88 and K20 cells and the sialylation of cell surface glycoconjugates of K20 cells could be significantly increased by supple menting the medium with Nacetylmannosamine. This Nacetylmannosamineinduced increase was drastically reduced by cosupplementation with Nacetylglucosamine only in K20 cells. We therefore propose the phosphorylation of Nacetylmannosamine as a hitherto unrecognized role of Nacetylglucosamine kinase in living cells.

scholarly journals Role of plasmin in the degradation of the stroma-derived fibrin in human ovarian carcinoma

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1673-1678 ◽  
Author(s):  
O Wilhelm ◽  
R Hafter ◽  
A Henschen ◽  
M Schmitt ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
High Performance ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fibrin Degradation Products ◽  
Sds Page ◽  
Human Ovarian Carcinoma

Abstract The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2- mercaptoethanol, the peptide chain components were separated by reverse- phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.

scholarly journals Stimulation of interleukin-6 and prostaglandin E2 secretion from peritoneal macrophages by polymers of albumin

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2853-2864 ◽  
Author(s):  
E Shacter ◽  
GK Arzadon ◽  
JA Williams
Keyword(s):  
Prostaglandin E2 ◽  
Interleukin 6 ◽  
Acute Infection ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polymyxin B ◽  
Peritoneal Macrophages ◽  
Stimulatory Activity

Abstract Interleukin-6 (IL-6) is a multifunctional cytokine that is elevated in vivo during acute infection, chronic inflammation, and some hematopoietic malignancies. To understand how IL-6 becomes elevated in vivo, it is important to identify factors that can stimulate its secretion from effector cells. We found that commercial preparations of bovine serum albumin (BSA) stimulated murine macrophages to secrete high levels of IL-6. In fact, BSA was at least as potent as bacterial lipopolysaccharide (LPS) in stimulating IL-6 production. Stimulation was clearly visible at concentrations as low as 20 micrograms/mL and reached saturation at 0.5 to 1 mg/mL albumin, at which concentration 1.1 x 10(6) oil-elicited macrophages produced 6,000 +/- 700 B9 units of IL-6 in an overnight incubation. Prostaglandin E2 production was induced by the same concentrations of BSA. Both resident and oil- elicited peritoneal cells were responsive to the albumin. The stimulatory activity did not derive from contamination of the protein with Escherichia coli LPS; when compared directly with LPS, the response to BSA was more rapid, had a higher amplitude, and was not inhibitable by polymyxin B. In addition, macrophages isolated from C3H/HeJ mice, which have an inherited defect in their ability to respond to LPS, secreted IL-6 in response to BSA but not to LPS. The stimulatory activity was stable to heat, mild acid, and reduction/alkylation and copurified with albumin on Cibachron Blue agarose (Sigma, St Louis, MO) and anti-albumin immunoaffinity chromatography. Comparison of different sources and preparations of albumin showed differences in the levels of IL-6-inducing activity; three different lots of commercial fatty acid-free BSA and one lot of polymer-enhanced BSA stimulated IL-6 secretion by more than 100-fold over basal levels whereas other preparations showed more limited activity. A sample of BSA that was active in vitro caused a marked elevation of IL-6 when injected into BALB/c mice, thus demonstrating inflammatory activity in vivo. When the albumin preparations were fractionated by ion exchange and gel filtration chromatography and then analyzed by sodium dodecyl sulfate-gel electrophoresis and Western blot immunoassay, it was found that the IL-6-inducing activity resided in high molecular weight polymers of albumin. The ability of albumin polymers to stimulate IL-6 production represents a novel mechanism for modulation of this cytokine.

scholarly journals The Response of Leukemic Lymphocytes to Cortisol: A Suggested Role of Transcortin

Blood ◽  
1971 ◽  
Vol 37 (4) ◽  
pp. 463-472 ◽  
Author(s):  
SEYMOUR WERTHAMER ◽  
LEONARD AMARAL
Keyword(s):  
Protein A ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Absolute Requirement ◽  
Plasma Factor

Abstract Lymphocytes obtained from patients with chronic lymphocytic leukemia (CLL) respond to the in vitro presence of cortisol by depressed incorporation of precursors into RNA and protein. The decreased incorporation of uridine into RNA is the sum of (1) an inhibition in the synthesis of RNA and (2) an enhanced destruction of newly synthesized RNA. Whereas cortisol was not dependent upon plasma for the manifestation of the above effects, the presence of plasma was an absolute requirement in order for cortisol to have an inhibitory effect on the synthesis of protein. A comparison of leukemic and normal lymphocytes demonstrated that the magnitude of inhibition of precursors into RNA and protein was greater in leukemic cells. Because it is believed that the plasma factor required is transcortin, determination of transcortin levels by cortisolbinding gel filtration technics were performed. These indicated that transcortin levels of CLL plasma were about 50 per cent lower than that of the normal. Consequently, further experiments involving type-specific plasma substitutions were performed. The results obtained from these experiments indicated that the magnitude of the effect of cortisol on the synthesis of lymphocyte protein was directly related to the transcortin level of the plasma employed.

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