scholarly journals Aggregation equilibria of tyrosinase of Harding-Passey mouse melanoma

1985 ◽  
Vol 228 (1) ◽  
pp. 95-101 ◽  
Author(s):  
J C Garcia-Borron ◽  
F Solano ◽  
J L Iborra ◽  
J A Lozano
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Aqueous Environment ◽  
Fluorescence Studies ◽  
Mouse Melanoma ◽  
Low Protein ◽  
Tryptophan Residues ◽  
Sample Dilution

The purification of two isoenzymes of tyrosinase has been carried out in Harding-Passey mouse melanoma. One is found in the cytosol and the other one bound to melanosomes. Both migrate as single bands on sodium dodecyl sulphate/polyacrylamide gels, having an apparent Mr of 58 000. Solubilized particulate tyrosinase showed an aggregation equilibrium involving a monomer, tetramer, octamer and a high-Mr micellar form with Brij 35, the solubilizing agent. H.p.l.c. studies indicated a interconversion between those species, the monomer contribution increasing with the sample dilution. The tetramer and the octamer probably represent the predominant forms in vivo. Soluble tyrosinase showed a simpler aggregation equilibrium, involving two forms, monomer and tetramer, with the same interconversion pattern. Fluorescence studies suggested that tryptophan residues were exposed to the aqueous environment when tyrosinase was dissociated by dilution. Tyrosinase shows a tendency to aggregate, at low protein concentration, and a resistance to dissociation by urea or SDS so remarkable that gel-permeation chromatography in 4M-urea does not affect the equilibrium, and the band obtained on SDS/polyacrylamide-gel electrophoresis is a dimer.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

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.

scholarly journals The interrelations between high- and low-molecular-weight forms of normal and mutant (Krabbe-disease) galactocerebrosidase

1980 ◽  
Vol 189 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Yoav Ben-Yoseph ◽  
Melinda Hungerford ◽  
Henry L. Nadler
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Krabbe Disease ◽  
Low Molecular Weight ◽  
Molecular Weight Form

Galactocerebrosidase (β-d-galactosyl-N-acylsphingosine galactohydrolase; EC 3.2.1.46) activity of brain and liver preparations from normal individuals and patients with Krabbe disease (globoid-cell leukodystrophy) have been separated by gel filtration into four different molecular-weight forms. The apparent mol.wts. were 760000±34000 and 121000±10000 for the high- and low-molecular-weight forms (peaks I and IV respectively) and 499000±22000 (mean±s.d.) and 256000±12000 for the intermediate forms (peaks II and III respectively). On examination by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the high- and low-molecular-weight forms revealed a single protein band with a similar mobility corresponding to a mol.wt. of about 125000. Antigenic identity was demonstrated between the various molecular-weight forms of the normal and the mutant galactocerebrosidases by using antisera against either the high- or the low-molecular-weight enzymes. The high-molecular-weight form of galactocerebrosidase was found to possess higher specific activity toward natural substrates when compared with the low-molecular-weight form. It is suggested that the high-molecular-weight enzyme is the active form in vivo and an aggregation process that proceeds from a monomer (mol.wt. approx. 125000) to a dimer (mol.wt. approx. 250000) and from the dimer to either a tetramer (mol.wt. approx. 500000) or a hexamer (mol.wt. approx. 750000) takes place in normal as well as in Krabbe-disease tissues.

scholarly journals Expression of a functional laminin receptor (alpha 6 beta 1, very late activation antigen-6) on human eosinophils

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2872-2879 ◽  
Author(s):  
SN Georas ◽  
BW McIntyre ◽  
M Ebisawa ◽  
JL Bednarczyk ◽  
SA Sterbinsky ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Basement Membranes ◽  
Laminin Receptor ◽  
Receptor Interaction ◽  
Inflammatory Reactions ◽  
Adhesive Interactions ◽  
Activation Antigen

Abstract The mechanisms responsible for the accumulation of eosinophils at sites of allergic and other inflammatory reactions are unknown, but recent studies have implicated both eosinophil and endothelial adhesion molecules in this process. However, less well studied have been the adhesive interactions between eosinophils and the subendothelial basement membrane and interstitial connective tissues. To test the hypothesis that eosinophils might interact with extracellular matrix proteins, we analyzed purified human eosinophils for the expression and function of various beta 1 integrins. Using indirect immunofluorescence and flow cytometry, purified eosinophils from mildly allergic donors were found to consistently express the integrin subunits beta 1 (CD29), alpha 4 (CD49d, very late activation antigen [VLA]-4 alpha), and alpha 6 (CD49f, VLA-6 alpha). No significant expression of the alpha 1, alpha 2, alpha 3, alpha 5, or beta 4 subunits was detected. Platelet contamination of the eosinophil preparations was excluded by light microscopy and by the inability to detect expression of platelet glycoproteins alpha v, CD41b, and CD42b. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of eosinophils confirmed the expression of cell-surface beta 1, alpha 4, and alpha 6. Furthermore, eosinophils purified from allergic donors were shown to adhere to plate-bound laminin, but not to type 1 or type 4 collagen. Adhesion to laminin was concentration-dependent, required divalent cations, and was completely and specifically inhibited by the anti-alpha 6 monoclonal antibody (MoAb) GoH3 and by the anti-beta 1 MoAb 33B6. Interestingly, the anti-beta 1 MoAb 18D3 (which like 33B6 blocks eosinophil binding to VCAM-1) did not inhibit eosinophil adhesion to laminin, suggesting that there are functionally distinct epitopes on the beta 1 subunit. Eosinophils purified from 4 healthy, nonallergic donors also showed alpha 6-dependent adhesion to laminin, although these cells adhered less well. These studies establish the expression of alpha 6 beta 1 on human eosinophils and document its function as a laminin receptor. Interaction of eosinophil alpha 6 beta 1 with laminin, eg, in basement membranes, may contribute to the localization of these cells at inflammatory sites in vivo.

scholarly journals The Largest Open Reading Frame (pks12) in the Mycobacterium tuberculosis Genome Is Involved in Pathogenesis and Dimycocerosyl Phthiocerol Synthesis

2003 ◽  
Vol 71 (7) ◽  
pp. 3794-3801 ◽  
Author(s):  
Tatiana D. Sirakova ◽  
Vinod S. Dubey ◽  
Hwa-Jung Kim ◽  
Michael H. Cynamon ◽  
Pappachan E. Kolattukudy
Keyword(s):  
Fatty Acids ◽  
Mycobacterium Tuberculosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Open Reading Frame ◽  
Pcr Analysis ◽  
Reading Frame ◽  
Sds Page

ABSTRACT The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12, is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12-encoded sequence. Biochemical analysis using [1-14C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.

scholarly journals Isolation and characterization of the native glycoprotein from pig small-intestinal mucus

1981 ◽  
Vol 195 (1) ◽  
pp. 267-275 ◽  
Author(s):  
M Mantle ◽  
A Allen
Keyword(s):  
Nucleic Acid ◽  
Analytical Ultracentrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intestinal Mucus ◽  
Isolation And Characterization ◽  
Average Maximum ◽  
Small Intestinal ◽  
Covalently Bound

Glycoprotein from pig small-intestinal mucus was isolated free of non-covalently bound protein and nucleic acid with a yield of over 60%. No non-covalently bound protein could be detected by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis or by equilibrium centrifugation in a density gradient of CsCl with 4 M-guanidinium chloride. The intrinsic viscosity and reduced viscosity of the glycoprotein preparations rose with the removal of non-covalently bound protein and nucleic acid from the glycoprotein, evidence that non-covalently bound protein does not contribute to the rheological properties of the glycoprotein in the mucus. The pure glycoprotein, in contrast with impure preparations, gelled at the same concentration of glycoprotein as that present in the gel in vivo. The glycoprotein was a single component, as judged by gel filtration and analytical ultracentrifugation. The distribution of sedimentation coefficients was polydisperse but unimodal with an s025,w of 14.5S and a molecular weight of 1.72 X 10(6). The chemical composition of the glycoprotein was 77% carbohydrate and 21% protein, 52% of which was serine, threonine and proline. The glycoprotein had a strong negative charge and contained 3.1% and 18.3% by weight ester sulphate and sialic acid respectively. The molar proportion of N-acetylgalactosamine was nearly twice that of any of the other sugars present, the glycoprotein had A and H blood-group activity and the average maximum length of the carbohydrate chains was deduced to be six to eight sugar residues.

scholarly journals Purification and Biochemical Characterization of the VIM-1 Metallo-β-Lactamase

2000 ◽  
Vol 44 (11) ◽  
pp. 3003-3007 ◽  
Author(s):  
Nicola Franceschini ◽  
Berardo Caravelli ◽  
Jean-Denis Docquier ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Metal Removal ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Mediterranean Area ◽  
Exchange Chromatography ◽  
Amino Terminal ◽  
Chromatography Step

ABSTRACT VIM-1 is a new group 3 metallo-β-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla VIM-1 gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of β-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-β-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/Km ratios (>106M−1 · s−1) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different β-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these β-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-β-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.

Isolation and preliminary characterization of histone H1.b allelic variants from quail erythrocytes

Genome ◽  
1998 ◽  
Vol 41 (5) ◽  
pp. 709-719 ◽  
Author(s):  
Jan Palyga ◽  
James M Neelin
Keyword(s):  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cation Exchange Resin ◽  
Gel Permeation Chromatography ◽  
Histone H1 ◽  
Linker Histone ◽  
Allelic Variants ◽  
Limited Digestion

Our goal was to purify and characterize the allelic variants H1b1 and H1b2 of histone H1.b, one of the seven subtypes of this linker histone extracted from Japanese quail erythrocyte nuclei. These variants are revealed phenotypically as band H1.3 or part of band H1.4 by polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS). All H1 subtypes together were separated from H5 by gel-permeation chromatography through Bio-Gel P-150. H1 was then fractionated on a column of the cation-exchange resin Amberlite CG-50 by using a shallow guanidine hydrochloride gradient, which enriched subtype H1.b together with H1.z and overlapping with subtypes H1.a and H1.b. Alternatively purification of subtypes was achieved electrophoretically: total H1 fractions from quail with different H1 phenotypes were first resolved into sub-types by PAGE in acetic acid - urea; after staining, the appropriate H1.b bands from several parallel gel pieces were excised and the histone was concentrated by PAGE in SDS. After fragmentation of H1.b in the gel pieces with N-bromosuccinimide (NBS), PAGE in SDS indicated no difference between H1b1 and H1b2 in the C-terminal "half" of the polypeptides. In contrast, limited digestion with endoprotease V8 from Staphylococcus aureus has shown that differences, probably by a few residues in length, reside in the N-terminal part of the molecule, close to the amino-terminus.Key words: quail histone H1.b, electrophoretic fractionation, linker histone variant.

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