scholarly journals Physicochemical behaviour and structural characteristics of membrane-bound acetylcholinesterase from Torpedo electric organ. Effect of phosphatidylinositol-specific phospholipase C

1985 ◽  
Vol 226 (2) ◽  
pp. 369-377 ◽  
Author(s):  
A H Futerman ◽  
R M Fiorini ◽  
E Roth ◽  
M G Low ◽  
I Silman
Keyword(s):  
Phospholipase C ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Electric Organ ◽  
Catalytic Subunit ◽  
Proteinase K ◽  
Short Amino Acid Sequence ◽  
Torpedo Electric Organ

Quantitative solubilization of the phospholipid-associated form of acetylcholinesterase (AChE) from Torpedo electric organ can be achieved in the absence of detergent by treatment with phosphatidylinositol-specific phospholipase C (PIPLC) from Staphylococcus aureus [Futerman, Low & Silman (1983) Neurosci. Lett. 40, 85-89]. The sedimentation coefficient on sucrose gradients of AChE solubilized in detergents (DSAChE) varies with the detergent employed. However, the coefficient of AChE directly solubilized by PIPLC is not changed by detergents. Furthermore, PIPLC can abolish the detergent-sensitivity of the sedimentation coefficient of DSAChE purified by affinity chromatography, suggesting that one or more molecules of phosphatidylinositol (PI) are co-solubilized with DSAChE and remain attached throughout purification. DSAChE binds to phospholipid liposomes, whereas PIPLC-solubilized AChE and DSAChE treated with PIPLC do not bind even to liposomes containing PI. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows that PIPLC-solubilized AChE, like unmodified DSAChE, is a catalytic subunit dimer; electrophoresis in the presence of reducing agent reveals no detectable difference in the Mr of the catalytic subunit of unmodified DSAChE, of AChE solubilized by PIPLC and of AChE solubilized by Proteinase K. The results presented suggest that DSAChE is anchored to the plasma membrane by one or more PI molecules which are tightly attached to a short amino acid sequence at one end of the catalytic subunit polypeptide.

scholarly journals Weissellicin 110, a Newly Discovered Bacteriocin from Weissella cibaria 110, Isolated from Plaa-Som, a Fermented Fish Product from Thailand

2007 ◽  
Vol 73 (7) ◽  
pp. 2247-2250 ◽  
Author(s):  
Sirinat Srionnual ◽  
Fujitoshi Yanagida ◽  
Li-Hsiu Lin ◽  
Kuang-Nan Hsiao ◽  
Yi-sheng Chen
Keyword(s):  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Proteinase K ◽  
Mass Spectrometry Analysis ◽  
Fermented Fish ◽  
Spectrometry Analysis ◽  
Weissella Cibaria ◽  
Fish Product

ABSTRACT Weissella cibaria 110, isolated from the Thai fermented fish product plaa-som, was found to produce a bacteriocin active against some gram-positive bacteria. Bacteriocin activity was not eliminated by exposure to high temperatures or catalase but was destroyed by exposure to the proteolytic enzymes proteinase K and trypsin. The bacteriocin from W. cibaria 110 was purified, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified bacteriocin contained one protein band that was approximately 2.5 kDa in size. Mass spectrometry analysis showed the mass of the peptide to be approximately 3,487.8 Da. N-terminal amino acid sequence analysis was performed, and 27 amino acids were identified. Because it has no similarity to other known bacteriocins, this bacteriocin was defined as a new bacteriocin and termed weissellicin 110.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

Germin: physicochemical properties of the glycoprotein which signals onset of growth in the germinating wheat embryo

1987 ◽  
Vol 65 (12) ◽  
pp. 1039-1048 ◽  
Author(s):  
William C. McCubbin ◽  
Cyril M. Kay ◽  
Theresa D. Kennedy ◽  
Byron G. Lane
Keyword(s):  
Circular Dichroism ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Helical Structure ◽  
Random Coil ◽  
Sedimentation Equilibrium ◽  
Cross Linking ◽  
Wheat Embryo ◽  
Circular Dichroïsm

The size and structure of germin, the homooligomeric glycoprotein which marks the onset of growth in germinating wheat embryos, has been examined by gel filtration, ultracentrifugation, electron microscopy, chemical cross-linking, and optical techniques (circular dichroism). Germin has a sedimentation coefficient (S20,w) of 7.3S, and a Stokes' radius (RS) of 4.5 nm, the latter value being compatible with the dimensions of the particle observed by negative staining in the electron microscope. By three methods (sedimentation equilibrium, sodium dodecyl sulphate (SDS) – polyacrylamide electrophoresis, S20,w/RS), the mean particle mass of the two closely related forms of germin (G and G′) is ca. 130 kilodaltons (kDa). Cross-linking with dimethyl suberimidate indicates that the oligomer is homopentameric, compatible with the molecular mass of the protomer (ca. 26 kDa) as determined by SDS–polyacrylamide gel electrophoresis. Using the Provencher and Glockner analysis to interpret circular dichroism measurements (in the far ultraviolet), both forms of germin contain about 10–20% α-helical structure, 50–60% β-sheet/turn structure, and 20–30% random coil. In a structure-inducing environment (45% trifluoroethanol), the α-helical structure increases to a value (35–40%) similar to that predicted by Chou–Fasman analysis of the protein sequence deduced by cDNA sequencing.

Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization

1982 ◽  
Vol 152 (1) ◽  
pp. 239-245
Author(s):  
R M Berka ◽  
M L Vasil
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phospholipase C ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Tetradecyltrimethylammonium Bromide ◽  
Heat Labile ◽  
Hydrophobic Moiety ◽  
Culture Supernatants

Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free.

scholarly journals Comparison of the Structural Characteristics of Native Collagen Fibrils Derived from Bovine Tendons Using Two Different Methods: Modified Acid-Solubilized and Pepsin-Aided Extraction

Materials ◽  
2020 ◽  
Vol 13 (2) ◽  
pp. 358 ◽  
Author(s):  
Haiyan Ju ◽  
Xiuying Liu ◽  
Gang Zhang ◽  
Dezheng Liu ◽  
Yongsheng Yang
Keyword(s):  
Type I Collagen ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dry Weight ◽  
Collagen Fibrils ◽  
Type I ◽  
X Ray Diffraction ◽  
Aggregation Structure ◽  
Native Collagen

Native collagen fibrils (CF) were successfully extracted from bovine tendons using two different methods: modified acid-solubilized extraction for A-CF and pepsin-aided method for P-CF. The yields of A-CF and P-CF were up to 64.91% (±1.07% SD) and 56.78% (±1.22% SD) (dry weight basis), respectively. The analyses of both amino acid composition and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that A-CF and P-CF were type I collagen fibrils. Both A-CF and P-CF retained the intact crystallinity and integrity of type I collagen’s natural structure by FTIR spectra, circular dichroism spectroscopy (CD) and X-ray diffraction detection. The aggregation structures of A-CF and P-CF were displayed by UV–Vis. However, A-CF showed more intact aggregation structure than P-CF. Microstructure and D-periodicities of A-CF and P-CF were observed (SEM and TEM). The diameters of A-CF and P-CF are about 386 and 282 nm, respectively. Although both A-CF and P-CF were theoretically concordant with the Schmitt hypothesis, A-CF was of evener thickness and higher integrity in terms of aggregation structure than P-CF. Modified acid-solubilized method provides a potential non-enzyme alternative to extract native collagen fibrils with uniform thickness and integral aggregation structure.

Isolation and Preliminary Characterization of a Bacteriocin Produced by Lactobacillus plantarum N014 Isolated from Nham, a Traditional Thai Fermented Pork

2006 ◽  
Vol 69 (8) ◽  
pp. 1937-1943 ◽  
Author(s):  
PONGSAK RATTANACHAIKUNSOPON ◽  
PARICHAT PHUMKHACHORN
Keyword(s):  
Lactobacillus Plantarum ◽  
Proteolytic Enzymes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteinase K ◽  
Exponential Growth Phase ◽  
Plasmid Isolation ◽  
Inhibitory Spectrum ◽  
Lipase A

Lactobacillus plantarum N014 was isolated from nham, a traditional Thai fermented pork, and exhibited antimicrobial activity against Listeria monocytogenes. Its bacteriocin had a broad inhibitory spectrum toward both gram-positive and gram-negative bacteria. The bacteriocin activity was sensitive to all proteolytic enzymes used in this study, including papain, pepsin, pronase E, proteinase K, and trypsin, but was resistant to the other enzymes, such as α-amylase, lipase A, and lysozyme. Furthermore, activity was stable over various heat treatments and pH values. The bacteriocin exerted a bacteriolytic mode of action. It was produced during the exponential growth phase and reached its highest level as producer cells entered the stationary phase. Adsorption of the bacteriocin onto producer cells was pH-dependent. No bacteriocin adsorption was detected at pH 1 to 3, whereas 100% bacteriocin adsorption was found at pH 7. Plasmid isolation revealed that L. plantarum N014 contained no plasmids. From Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and growth inhibition testing against L. monocytogenes, the estimated molecular mass of L. plantarum N014 bacteriocin was 8 kDa.

scholarly journals Structural characteristics of Tla products.

1985 ◽  
Vol 162 (3) ◽  
pp. 781-789 ◽  
Author(s):  
M J Chorney ◽  
J S Tung ◽  
Y Bushkin ◽  
F W Shen
Keyword(s):  
Peptide Mapping ◽  
Biochemical Study ◽  
Structural Characteristics ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chromosome 17 ◽  
Leukemia Cells ◽  
Mouse Strains ◽  
Chymotryptic Peptide ◽  
Sds Page

Biochemical study of thymus leukemia antigen (TL) from thymocytes of various Tla genotypes and from leukemia cells revealed features that, given present evidence, are peculiar to TL among class I products of the H-2:Qa:Tla region of chromosome 17. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of TL from thymocytes of all TL+ mouse strains, precipitated by anti-TL antiserum or monoclonal antibodies, showed two closely migrating bands of equal intensity in the heavy (H) chain position (45-50,000 mol wt). Comparison of these two bands by two-dimensional isoelectric focusing (2D IEF)-SDS-PAGE and 2D chymotryptic peptide mapping showed no differences indicative of protein dissimilarity. Thus, the two components of the H chain doublet may differ only in a feature of glycosylation that does not affect charge. The two leukemias studied gave only a single band in the H chain position. On 2D peptide mapping and 2D IEF-SDS-PAGE, the patterns for TL of Tlaa and Tlae thymocytes, which are closely related serologically, were broadly similar, but clearly different from the pattern typical of Tlac and Tlad thymocytes. 2D peptide maps of TL from Tlaa thymocytes and Tlaa leukemia cells did not differ. Leukemia cells of Tlab origin (thymocytes TL-) gave 2D peptide and 2D IEF-SDS-PAGE patterns of a third type. With the exception of Tlaa, thymocytes of TL+ mice yielded additional TL products of higher molecular weight than the TL H chain.

The kinetics of in vitro reoxidation and reduction of the inter heavy–light chain disulfide bond in an unusual murine immunoglobulin G myeloma protein lacking inter-heavy chain disulfide bonds

1979 ◽  
Vol 57 (3) ◽  
pp. 279-285 ◽  
Author(s):  
Maire E. Percy ◽  
Lebe Chang ◽  
Catherine Demoliou ◽  
Reuben Baumal
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Disulfide Bridge ◽  
Myeloma Protein ◽  
Kinetics Of

After 5 years of subcutaneous transfer in Balb/C mice, our MOPC 173 myeloma tumour line (originally an IgG2a,κ H2L2-producer) exclusively synthesized an unusual IgG2b,κ protein lacking inter-heavy (H) chain disulfide bonds. This protein was designated MOPC 173B. On sodium dodecyl sulfate – polyacrylamide gel electrophoresis, it migrated with an apparent molecular weight of 77 000; following complete reduction and alkylation, the mobilities of its constituent H and light (L) chains were found to differ slightly from those of MOPC 173 H2L2. MOPC 173B was serologically identical to another typical IgG2b,κ myeloma protein, MOPC 195, and peptide mapping studies showed that it possessed only the inter H–L disulfide bond characteristic of typical IgG2b,κ proteins. In a nondissociating solvent, the sedimentation coefficient of the protein was 6.3S even at concentrations as low as 0.2 mg/ml, indicating that noncovalent interactions existed between two half-molecule subunits. Since this unusual IgG myeloma protein contained only a single category of interchain disulfide bridge, the inter H–L bond, it was an ideal model system for characterization of the kinetics of formation and reduction of interchain disulfide bonds. The kinetics of the glutathione-catalyzed reoxidation of the inter H–L disulfide bridge in MOPC 173B followed an apparent second-order rate equation. In contrast, reduction of its inter H–L bridge under anaerobic conditions with dithioerythritol in excess, was strictly a first-order process and not a simple reversal of the reoxidation. These studies provide the basis for the more complex mathematical models that describe the reoxidation and reduction of typical immunoglobulin molecules.

scholarly journals Identification of the mRNA and polypeptide subunit for prostaglandin endoperoxide synthase from mouse mastocytoma P-815 cells

1983 ◽  
Vol 212 (1) ◽  
pp. 219-222 ◽  
Author(s):  
M Kondo ◽  
Y Koshihara ◽  
M Kawamura ◽  
S Murota
Keyword(s):  
Polypeptide Chain ◽  
De Novo ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Prostaglandin Endoperoxide ◽  
Single Polypeptide Chain ◽  
Free Translation ◽  
Prostaglandin Endoperoxide Synthase ◽  
Single Polypeptide

Cloned mouse mastocytoma P-815.2-E-6 cells are barely able to synthesize prostaglandins because of a lack of prostaglandin endoperoxide synthase activity. However, the addition of sodium n-butyrate at 1 mM induces synthesis de novo of prostaglandins in this cell line. Employing this system, we could isolate an mRNA for prostaglandin endoperoxide synthase by a combination of cell-free translation and immunoprecipitation. The antibody, prepared in rabbit by injecting purified prostaglandin endoperoxide synthase from bovine vesicular gland, was shown to cross-react with the corresponding enzyme from 2-E-6 cells. The poly(A)-containing mRNA has a sedimentation coefficient of 17S and codes for a single polypeptide chain of Mr 62 000 as estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr of the mouse polypeptide chain appears very similar to that of the purified carbohydrate-free prostaglandin endoperoxide synthase from sheep vesicular gland. These findings are a contribution to the isolation of the gene for prostaglandin endoperoxide synthase.

scholarly journals Purification and properties of succinyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney

1978 ◽  
Vol 173 (3) ◽  
pp. 759-765 ◽  
Author(s):  
J A Sharp ◽  
M R Edwards
Keyword(s):  
Isoelectric Focusing ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Coenzyme A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Transferase Activity ◽  
Coa Transferase

CoA-transferase (succinyl-CoA-3-oxo acid CoA-transferase, EC 2.8.3.5) isolated from sheep kidney was purified to homogeneity. The purified enzyme has a specific activity of approx. 200 units/mg. A mol.wt. of 110000 was obtained by gel filtration on Sephadex G-200, and a lower mol.wt. of 102000 was determined by analytical ultracentrifugation. A sedimentation coefficient of 5.6S was also determined. A subunit mol.wt. of 56000 was obtained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing of sheep kidney extracts indicated the presence of a single band of CoA-transferase activity with pI9.0. However, isoelectric focusing of purified CoA-transferase showed the presence of two peaks of CoA-transferase activity with pI values of 8.7 and 8.4, suggesting the presence of proteolytic activity during purification. Evidence for sheep kidney CoA-transferase being a dimer of two identical subunits has been obtained from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the amino acid composition, peptide ‘mapping’ and N-terminal analysis.

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