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.

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 Molecular weight and subunit size of fatty acid synthetase from rabbit mammary gland

1976 ◽  
Vol 159 (1) ◽  
pp. 181-184 ◽  
Author(s):  
N Paskin ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Fatty Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fatty Acid Synthetase ◽  
Subunit Size

Fatty acid synthetase purified from the mammary gland of the rabbit has a mol. wt. of 968000 as determined by gel filtration. The enzyme gave one band, corresponding to a mol.wt. of approx. 35000, on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate and phenylmethanesulphonyl fluoride.

scholarly journals The molecular size of N-methylglutamate dehydrogenase of Pseudomonas aminovorans

1977 ◽  
Vol 167 (2) ◽  
pp. 509-512 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Partial Specific Volume ◽  
Triton X

N-Methylglutamate dehydrogenase, purified to a specific activity of 0.29 unit/mg of protein, gave one band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a molecular weight of 130 000. Enzyme-Triton complexes were found to have a partial specific volume of 0.73 cm3/g, suggesting that the protein binds less than 0.1 g of Triton/g of protein. A molecular weight for the intact enzyme in the presence of 1% (w/v) Triton X-100 of 550 000 suggested that the enzyme may be a tetramer.

scholarly journals Tamm–Horsfall urinary glycoprotein. The subunit structure

1970 ◽  
Vol 120 (2) ◽  
pp. 425-432 ◽  
Author(s):  
A. P. Fletcher ◽  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Disulphide Bonds ◽  
Terminal Amino ◽  
Urinary Glycoprotein

1. Subunit molecular weights of 76000–82000 were obtained for native and alkylated Tamm–Horsfall glycoprotein by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. 2. A further estimate of the subunit molecular weight of 79000±4000 was obtained by disc gel electrophoresis in sodium dodecyl sulphate. 3. A minimum value of the chemical molecular weight of 79000±6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 4. Similar values were obtained for the subunit molecular weight of Tamm–Horsfall glycoprotein from patients with cystic fibrosis. 5. On ultracentrifugation both in 1.0% sodium dodecyl sulphate and in 70% formic acid, Tamm–Horsfall glycoprotein sedimented as a single component, slightly faster than serum albumin. 6. On reduction of the disulphide bonds the same subunit molecular weight was obtained, which suggested that these bonds are intrachain.

scholarly journals The purification and properties of Factor X from pig serum and its role in hypercoagulability in vivo

1973 ◽  
Vol 133 (2) ◽  
pp. 311-321 ◽  
Author(s):  
R. J. Dupe ◽  
R. M. Howell
Keyword(s):  
Molecular Weight ◽  
Thrombus Formation ◽  
Gel Filtration ◽  
Factor Xa ◽  
Factor X ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Initial Adsorption

The molecular weights or shapes of Factor X preparations determined by gel filtration were dependent on the density of the BaSO4 used for the initial adsorption from serum. One form obtained with BaSO4 of density 2g/ml behaved as if it had a molecular weight of 39000 and possessed preformed clotting activity (Factor Xa), whereas that of the form adsorbed with BaSO4 of density 1g/ml had a molecular weight of 69000 and consisted of inactive Factor X precursor. Thus degradation accompanied by activation seems to occur as a result of surface adsorption on high-density BaSO4 and is associated with an interchange of protein between the two bands observed electrophoretically. The clotting and esterase activities measurable in vitro after complete activation were not matched by a corresponding ability to induce thrombus formation and ‘lethality’ in vivo. The most effective preparations of Factor X in this respect possessed preformed activity, which was enhanced in the presence of phospholipid. Factor X lost activity more rapidly in dilute solution, and its concentration at the surface of phospholipid micelles probably decreases loss by dilution in circulating blood.

scholarly journals Variable major proteins of Borrellia hermsii.

1982 ◽  
Vol 156 (5) ◽  
pp. 1312-1324 ◽  
Author(s):  
A G Barbour ◽  
S L Tessier ◽  
H G Stoenner
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Antigenic Variation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Protein ◽  
Relapsing Fever ◽  
Western Blots

Borrelia hermsii, a relapsing fever agent, manifests antigenic variation in vivo and in vitro. We studied three mouse-passaged serotypes of strain HS1 (7, 14, and 21) and a HS1 derivative obtained after multiple in vitro passages (C serotype). All four serotypes had two major proteins in whole cell lysates fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One major protein species (pII) had the same apparent subunit molecular weight (or approximately 3.9 X 10(4) in all the serotypes. In contrast, the other abundant protein in lysates, pI, had a different apparent molecular weight in each serotype. In one gel the molecular weights of pIc, pI7, pI14, and pI21 were 1.9, 4.2, 4.1, and 4.0 X 10(4), respectively. Serotype-specific mouse antisera bound to both hemologous and heterologous pIIs, to homologous pI, but not to heterologous pI in Western blots. Hybridomas were raised from spleens of mice infected with B. hermsii. Monoclonal antibodies were identified by immunofluorescence assays using whole organisms. Monoclonal antibodies specific for serotype 7 (H1826) or for serotype 21 (H3326) bound only to pI7 or pI21, respectively, in Western blots. The surface location of the pI was suggested not only by the immunofluorescence studies but also by the labeling of pI7 and pI21 when whole cells of serotypes 7 and 21 were incubated with 125I in the presence of Iodogen. Under the same circumstances, pII was relatively poorly labeled. These studies have identified the variable pI proteins of B. hermsii as serotype-specific antigens. A change from one pI to another may be the basis of antigenic variation of Borrelia species during relapsing fever.

scholarly journals The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border

1974 ◽  
Vol 137 (3) ◽  
pp. 489-495 ◽  
Author(s):  
M. A. Kerr ◽  
A. J. Kenny
Keyword(s):  
Molecular Weight ◽  
Brush Border ◽  
Sodium Dodecyl Sulphate ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Atomic Absorption Spectrophotometry ◽  
Sedimentation Equilibrium ◽  
Rabbit Kidney

1. Some properties of a brush-border neutral endopeptidase purified from rabbit kidney were investigated. The peptidase was assayed by its ability to hydrolyse [125I]iodoinsulin B chain. 2. The enzyme was found to be homogeneous when studied in the analytical ultracentrifuge and stained as a single glycoprotein band after electrophoresis in polyacrylamide gels. 3. The molecular weight was estimated by gel filtration in columns of Sephadex G-200, by polyacrylamide-gel electrophoresis in the presence of 2-mercapto-ethanol and sodium dodecyl sulphate and by sedimentation equilibrium in the ultra-centrifuge. The estimates fell within the range 87000–96000. The mean from two sedimentation equilibrium experiments was 93000, though this estimate may be slightly inflated because of the carbohydrate component of the enzyme. No evidence of dissociation into smaller subunits was obtained in the presence of thiol, sodium dodecyl sulphate or guanidine hydrochloride. 4. The endopeptidase was maximally active at pH6.0, although in phosphate buffer, which was strongly inhibitory, an optimum above pH8 was observed. 5. The enzyme was not affected by di-isopropyl phosphofluoridate nor by several thiol reagents. It was, however, strongly inhibited by many thiols and by EDTA and other chelating agents. 6. Although activity of the EDTA-treated enzyme could be partially restored by various bivalent metal ions, the optimum concentration for its reactivation by Zn2+ was lower than that for other ions. This metal was detected in the enzyme preparation by atomic absorption spectrophotometry in an amount equivalent to approximately one atom/mol. 7. The enzyme is the only endopeptidase shown to be located in the kidney brush border and is the first mammalian example of a neutral Zn2+- activated endopeptidase to be characterized.

Radiolabelling of the excretory-secretory and somatic antigens of Anisakis simplex larvae

1992 ◽  
Vol 66 (4) ◽  
pp. 305-309 ◽  
Author(s):  
Kazuo Sugane ◽  
Shu-Han Sun ◽  
Tadashi Matsuura
Keyword(s):  
Molecular Weight ◽  
Staphylococcus Aureus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anisakis Simplex ◽  
Molecular Weights ◽  
Human Sera ◽  
Somatic Antigens

ABSTRACTAnisakis simplex larvae were cultured in vitro in medium containing 35-methionine for ten days. The medium and the larval tissues were analysed for biosynthetically labelled polypeptide by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Immunoprecipitates with positive and negative human antisera were similarly analysed, using Staphylococcus aureus to absorb immunocomplexes. ES products of Anisakis larvae contained many polypeptides with molecular weights of less than 200 K. 180 KDa and 40 KDa polypeptides in ES products reacted with IgG in Anisakis-infected human sera. Somatic extracts also contained many polypeptides with molecular weights of less than 200 K. One of these polypeptides with a molecular weight of 130 K reacted with IgG in Anisakis-infected human sera. These polypeptides did not react with other nematode-infected human sera.

scholarly journals Size heterogeneity of rat pituitary prolactin

1980 ◽  
Vol 189 (3) ◽  
pp. 605-614 ◽  
Author(s):  
M Wallis ◽  
M Daniels ◽  
S A Ellis
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Freezing And Thawing ◽  
Molecular Weights ◽  
Size Heterogeneity ◽  
Very High

The occurrence of multiple forms of rat prolactin with different molecular weights (size heterogeneity) was studied with anterior pituitary extracts, purified rat prolactin and 125I-labelled rat prolactin. In each case, three main forms of the hormone were detected by gel filtration on Sephadex G-100: a major one (80–90%) corresponding to monomeric prolactin (mol.wt. 22000–25000), a peak (8–20%) that could be a dimer (mol.wt. 45000–50000) and a small quantity (1–5%) of a component of much greater molecular weight. On freezing and thawing of 125I-labelled rat prolactin, there was little interconversion of monomer and ‘dimer’ peaks, but both were converted substantially to very high-molecular-weight material. All three peaks of 125I-labelled rat prolactin could be precipitated by anti-(rat prolactin) serum and all three gave similar patterns of radioactive peptides after digestion with chymotrypsin followed by high-voltage paper electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the monomer peak of 125I-labelled prolactin migrated as a single component of mol.wt. 22000, the very high-molecular-weight peak largely dissociated to a component running in the same position as the monomer, and the ‘dimer’ peak migrated partly as a component of mol.wt. 45000 and partly as a component migrating with monomeric prolactin. No treatment was found that could dissociate the ‘dimer’ peak completely to monomeric prolactin.

scholarly journals Synthesis of δ-aminolaevulinate synthase by isolated liver polyribosomes

1976 ◽  
Vol 158 (2) ◽  
pp. 391-400 ◽  
Author(s):  
M J Whiting
Keyword(s):  
Protein Synthesis ◽  
Chick Embryo ◽  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Synthesis ◽  
Pulse Labelling ◽  
General Protein Synthesis

1. Postmitochondrial supernatants were prepared from the livers of chick embryos and were incubated under conditions that supported protein synthesis. delta-Aminolaevulinate synthase (EC 2.3.1.37) was synthesized by supernatants from livers treated with the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and/or 3,5-diethoxycarbonyl-1,4-dihydrocollidine, but synthesis by supernatants from normal livers could not be detected. Synthesis of enzyme released from polyribosomes was measured by immunoprecipitation with specific antibody to the mitochondrial enzyme, and the specificity of the reaction was established by electrophoresis of dissociated immunoprecipitates on sodium dodecyl sulphate/polyacrylamide gels. 2. The relative synthesis of delta-aminolaevulinate synthase in vitro was comparable with that previously measured in vivo, and was correlated with the enzyme activity of the liver. 3. Enzyme synthesis in vitro occurred predominantly on free rather than membrane-bound polyribosomes. 4. The mol.wt. of the product synthesized in vitro was 7000 +/- 7000 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, pulse-labelling of the enzyme in vivo confirmed its mol.wt. to be 49000 +/- 5000 when isolated from the mitochondrion. A small amount of immunoprecipitable enzyme of mol.wt. 70000 was detected in the cytosol in vivo. In chick embryo liver, delta-aminolaevulinate synthase therefore appears to be synthesized on cytoplasmic polyribosomes as a polypeptide of mol.wt. 70000, which in vivo is rapidly incorporated into the mitochondrion, and is then extracted as a lower-molecular-weight form. 5. Haemin added to the postmitochondrial supernatant-containing incubation mixture at concentrations up to 10 muM had no effect on general protein synthesis or the synthesis of delta-aminolaevulinate synthase. On the other hand, haemin treatment of induced chick embryo livers in vivo for 3h markedly decreased the relative synthesis of delta-aminolaevulinate synthase in vitro. These results suggest that haemin represses the synthesis of delta-aminolaevulinate synthase by decreasing the amount of mRNA for the enzyme available for translation.

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