scholarly journals Surface labelling for human tumour KB cells. Iodination and fractionation of membrane glycoproteins

1975 ◽  
Vol 150 (1) ◽  
pp. 59-69 ◽  
Author(s):  
T D Butters ◽  
R C Hughes
Keyword(s):  
Molecular Weight ◽  
Plasma Membranes ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Membrane Glycoproteins ◽  
Kb Cells ◽  
Human Tumour ◽  
Molecular Weights ◽  
Surface Labelling ◽  
Enzyme Markers

1. Human tumour KB cells growing in suspension culture were labelled by lactoperoxidase-catalysed iodination. Several major radioactively labelled proteins were detected by poly-acrylamide-gel electrophoresis in sodium dodecyl sulphate. 2. After reduction with 2-mercaptoethanol the major radioactive electrophoretic bands migrated as substances with apparent molecular weights of about 90,000, 70,000, 60,000, 50,000 and 34,000 and corresponded closely to the positions at which the major glycosylated polypeptide subunits of KB-cell homogenates migrated during electrophoresis under the same conditions. 3. All the iodinated protein bands except one were present in purified preparations of KB plasma membranes. 4. Most of the 50,000-molecular-weight species, supposedly a surface protein component labelled during iodination of intact and viable KB cells by a non-penetrating enzyme reagent, appeared in a crude nuclear pellet during fractionation. 5. The glyco-protein nature of the major external iodinated species of KB cells was confirmed by adsorption chromatography of these substances, dissolved in low concentrations of Triton X-100, on a lectin-Sepharose column. Two major enzyme markers of the KB plasma membrane, 5′-nucleotidase and alkaline phosphatase were also found to be glycoproteins. 6. Enzyme-catalysed incorporation of radioactive iodine into a fraction of low molecular weight and soluble in chloroform-methanol mixtures also occurred during lactoperoxidase treatment of intact KB cells. The partial characterization of this fraction is briefly described.

The outer membrane of Haemophilus influenzae type b: cell envelope associations of major proteins

1983 ◽  
Vol 29 (2) ◽  
pp. 280-287 ◽  
Author(s):  
J. W. Coulton ◽  
D. T. F. Wan
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Cell Envelope ◽  
Sodium Dodecyl ◽  
Haemophilus Influenzae Type ◽  
Major Protein ◽  
Haemophilus Influenzae Type B ◽  
Type B ◽  
Molecular Weights ◽  
Triton X

Membrane proteins fom the cell envelope of Haemophilus influenzae type b ATCC 9795 were examined by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. When envelopes were extracted with a phosphate-based buffer containing 2% Triton X-100, a major protein of molecular weight 43 000 was detected in fractions containing cytoplasmic membrane proteins. The cell wall material which was Triton X-100 insoluble contained six major proteins of molecular weights 46 000, 40 000, 36 000, 30 000, 27 000, and 16 000. One of these proteins showed a shift in molecular weight from 27 000 to 36 000 when it was heated over a temperature range from 50 °C to 100 °C in buffer containing 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol. This alteration in mobility could be demonstrated either by the membrane-bound form of the protein or by a detergent-soluble form of the protein. Enriched preparations of the 36 000 molecular weight form were obtained by a series of purification steps. Extraction of the Triton X-100 insoluble material with buffer containing 2% Triton X-100, 5.0 mM EDTA yielded chiefly one major protein molecular weight 30 000 and many minor protein species. Pretreatment of the Triton X-100 insoluble fraction with lysozyme followed by extraction with buffer containing 2% Triton X-100, 5.0 mM EDTA released two proteins of molecular weights 16 000 and 27 000 and few minor proteins. By these operational manipulations, the proteins of molecular weights 16 000 and 27 000 may be considered as peptidoglycan-associated proteins.

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 Transformation-deficient mutants of Bacillus subtilis impaired in competence-specific nuclease activities

1982 ◽  
Vol 152 (1) ◽  
pp. 166-174
Author(s):  
J A Mulder ◽  
G Venema
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Magnesium Ions ◽  
Polyacrylamide Gels ◽  
Wild Type ◽  
Molecular Weights ◽  
Defective Mutant ◽  
Mutant Strains

A comparison of the nucleolytic activities in competent and physiologically low-competent wild-type cultures of Bacillus subtilis in DNA-containing sodium dodecyl sulfate-polyacrylamide gels revealed the existence of three competence-associated nuclease activities with apparent molecular weights of 13,000, 15,000, and 26,000. The three activities, which were dependent on manganese or magnesium ions, were specifically present in the competent fraction of a competent culture. The competence-associated nucleolytic activities of eight transformation-defective mutant strains were assayed, resulting in the following three classes of mutants: (i) four strains which, according to this assay, were not impaired in any of the nucleolytic activities mentioned above; (ii) one strain which was strongly impaired in the 13,000- and 26,000-molecular-weight activities, but showed a considerable level of the 15,000-molecular-weight activity; and (iii) three strains which were severely impaired in all three activities. The results indicated that the 26,000-molecular-weight activity was a dimer of the 13,000-molecular-weight activity and that this nuclease was involved in the entry of DNA.

Purification, Characterization, and Heterologous Expression in Fusarium venenatum of a Novel Serine Carboxypeptidase from Aspergillus oryzae

1999 ◽  
Vol 65 (8) ◽  
pp. 3298-3303 ◽  
Author(s):  
Alexander M. Blinkovsky ◽  
Tony Byun ◽  
Kimberly M. Brown ◽  
Elizabeth J. Golightly
Keyword(s):  
Molecular Weight ◽  
Aspergillus Oryzae ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Peptide Sequence ◽  
Sequence Information ◽  
Serine Carboxypeptidase ◽  
Recombinant Enzymes ◽  
Molecular Weights ◽  
Fusarium Venenatum

ABSTRACT A novel serine carboxypeptidase (EC 3.4.16.1 ) was found in anAspergillus oryzae fermentation broth and was purified to homogeneity. This enzyme has a molecular weight of ca. 67,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its specific activity is 21 U/mg for carbobenzoxy (Z)-Ala-Glu at pH 4.5 and 25°C. It has a ratio of bimolecular constants for Z-Ala-Lys and Z-Ala-Phe of 3.75. Optimal enzyme activity occurs at pH 4 to 4.5 and 58 to 60°C for Z-Ala-Ile. The N terminus of this carboxypeptidase is blocked. Internal fragments, obtained by cyanogen bromide digestion, were sequenced. PCR primers were then made based on the peptide sequence information, and the full-length gene sequence was obtained. An expression vector that contained the recombinant carboxypeptidase gene was used to transform aFusarium venenatum host strain. The transformed strain ofF. venenatum expressed an active recombinant carboxypeptidase. In F. venenatum, the recombinant carboxypeptidase produced two bands which had molecular weights greater than the molecular weight of the native carboxypeptidase from A. oryzae. Although the molecular weights of the native and recombinant enzymes differ, these enzymes have very similar kinetic parameters.

Macrozin, a Novel Storage Globulin From Seeds of Macrozamia communis L. Johnson

1984 ◽  
Vol 11 (2) ◽  
pp. 69 ◽  
Author(s):  
RJ Blagrove ◽  
GG Lilley ◽  
TJV Higgins
Keyword(s):  
Molecular Weight ◽  
Broad Band ◽  
Sodium Dodecyl ◽  
Optical Rotatory Dispersion ◽  
Molecular Weights ◽  
Neutral Ph ◽  
Cellulose Acetate Membranes ◽  
Dodecyl Sulfate ◽  
Α Helix ◽  
Polypeptide Chains

The isolation, characterization and amino acid composition are reported for macrozin, the major storage globulin found in seeds of Macrozamia communis. Electrophoresis of macrozin on cellulose acetate membranes at neutral pH resulted in a single broad band indicating limited charge heterogeneity. Isoelectric focusing under dissociating and reducing conditions showed this globulin to be composed of a family of polypeptide chains with apparent isoelectric points in the range pH 6.0-7.5. Sedimentation equilibrium studies showed that the main component purified by gel filtration in aqueous buffers at neutral pH has a molecular weight of 260 000 and a sedimentation coefficient S020.w = 10.9 S. This component dissociates in 8 M urea to yield subunits of molecular weight 126 000. Each subunit is composed of disulfide-bonded polypeptide chains of approximate molecular weight 44 000. The apparent molecular weights for the macrozin subunit and its constituent polypeptides were 130 000 and 46 000 from dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dimeric nature of the main oligomer in aqueous solution was confirmed by crosslinking the subunits with dithiobis(succinimidylpropionate); the presence of three polypeptide chains per subunit is inferred from the molecular weights. Optical rotatory dispersion and circular dichroism measurements suggest that macrozin is devoid of α-helix in its native conformation, but contains some 25% α-helix after incubation with sodium dodecyl sulfate.

Effect Of Storage On Surface Glycoproteins And Cyto-Skeletons Of Platelets

1981 ◽  
Author(s):  
A K Rao ◽  
G P Tuszynski ◽  
L Knight ◽  
J Willis ◽  
C Beckett
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Blue Staining ◽  
In Vitro Tests ◽  
Membrane Glycoproteins ◽  
Autologous Plasma ◽  
Coomassie Blue ◽  
Functional Defect

Platelets stored as concentrates (PC) at 22° C for 72 hours develop a functional defect in vitro tests. Alterations in membrane glycoproteins of platelets have been shown to effect platelet function. We have investigated the effect of storage on membrane glycoproteins (GP) and cytoskeletons (cyto.) of platelets. Gel filtered platelets from fresh PC were labeled with 125Iodine by Iodogen technique and gel filtered again to remove free iodide. Platelets were concentrated by albumin density gradient centrifugation, resuspended in autologous plasma and stored for 72 hours at 22° C. Aliquots of fresh and stored PC were solubilized with 2% sodium dodecyl sulphate (SDS) containing 5% mercaptoethanol and subjected to polyacrylamide gel electrophoresis (PAGE). In one experiment, separate aliquots of fresh and stored platelets were labeled and similarly analyzed. Gels were stained with Coomassie blue and subjected to autoradiography. Coomassie blue staining did not reveal major differences between fresh and stored platelets. Autoradiography revealed a decrease in the 170,000 dalton surface protein (GP-I) of platelets after storage. Triton insoluble cyto. of thrombin activated fresh and stored platelets were solubilized with SDS and analyzed by PAGE and autoradiography. Cytoskeletons from fresh PC revealed the presence of a 110,000 dalton surface protein (GP-III). However, cyto. from similarly treated stored platelets showed a markedly decreased amount of this protein. Thus stored platelets have decreased amounts of the 170,000 dalton surface protein (GP-I) along with decreased amounts of the 110,000 dalton protein (GP-III) associated with the cyto. of thrombin activated platelets. These changes may contribute to the functional defect reported in stored platelets.

Further Characterization Of Platelet Membrane Glycoproteins

1981 ◽  
Author(s):  
P Clezardin ◽  
J L McGregor ◽  
K J Clemetson ◽  
M Dechavanne ◽  
E F Lüscher
Keyword(s):  
Ricinus Communis ◽  
Lectin Binding ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Galactose Oxidase ◽  
Membrane Glycoproteins ◽  
Neuraminidase Treatment ◽  
Platelet Surface ◽  
Reducing Conditions ◽  
Surface Labelling

The binding of 125I-labelled lectins to major and minor platelet glycoproteins (GP) and their subunits has been investigated. Human platelets were isolated, washed, solubilized in sodium dodecyl sulphate (SDS) under non-reducing conditions and separated on 5, 7.5 and 10 % non-reduced/reduced 2-D polyacrylamide gels. The gels were incubated with 125I-labelled lectins; Lens culinaris lectin (LCL), concanavalin A (ConA) wheat germ agglutinin (WGA) or Ricinus communis agglutinin (RCA-120), then washed extensively dried and exposed to X-ray film by indirect autoradiography. Surface-labelled platelets were similarly separated. WGA and RCA bound predominantly to GPIbα but also to two minor bands above and below it which were affected by neuraminidase treatment. One of them bound two 125I-lectins (LCL and ConA) while GPIbα did not. Additional GP bands were detected by lectin binding and by surface-labelling beneath GPIIIβ (IV). With platelets labelled by the neuraminidase/galactose oxidase/NaB3H4 method a GP was detected between Ila and Ilia which was not found with periodate/ NaB3H4 labelling (not affected by reduction). Two spots on the diagonal bound LCL and ConA. GP Ibβ bound LCL more strongly than IIbp. GPIbp also bound WGA and RCA. GPIcβ apparently bound only ConA. GPIbβ and IIbβ were labelled equally strongly by surface labelling techniques, Icβ was apparently not labelled. Further GP subunits were detected one below Ibβ and IIbβ and another which originated in the GPVII region. These techniques demonstrate that the platelet surface is even more complex than previously thought.

scholarly journals ISOLATION AND REACTIVATION OF THE AXOSTYLE

1973 ◽  
Vol 56 (1) ◽  
pp. 13-26 ◽  
Author(s):  
Mark S. Mooseker ◽  
Lewis G. Tilney
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Atpase Activity ◽  
Adenosine Triphosphate ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gels ◽  
Molecular Weights ◽  
Dodecyl Sulfate ◽  
Cilia And Flagella ◽  
Triton X

The contractile axostyle is a ribbon-shaped organelle present in certain species of flagellates found in the hindgut of wood eating insects. This organelle propagates an undulatory wave whose motion, like flagella and cilia, is related to microtubules. Unlike the axoneme of cilia and flagella, however, the axostyle is composed of singlet microtubules linked together in parallel rows. Axostyles were isolated from Cryptocercus gut protozoa with Triton X-100. Normal motility of the isolated axostyle could be restored with adenosine triphosphate (ATP); the specific conditions necessary for this reactivation were essentially identical with those reported for the reactivation of isolated flagella or whole sperm. ATPase activity of the isolated axostyle was comparable to the values reported for ciliary or flagellar axonemes. The axostyle was reasonably specific for ATP. Most of the proteins of the isolated axostyle comigrated with proteins of the ciliary axoneme on sodium dodecyl sulfate (SDS) polyacrylamide gels (i e. equivalent molecular weights). These included the following: the higher molecular weight component of dynein, tubulin, linkage protein (nexin), and various secondary proteins. Evidence for dynein in the axostyle is presented and a model proposed to explain how repeated propagated waves can be generated.

IDENTIFICATION OF CANADIAN AND AMERICAN WHEAT CULTIVARS BY SDS GRADIENT PAGE ANALYSIS OF GLIADIN AND GLUTENIN SUBUNITS

1987 ◽  
Vol 67 (4) ◽  
pp. 945-952 ◽  
Author(s):  
B. A. MARCHYLO
Keyword(s):  
Molecular Weight ◽  
Spring Wheat ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Low Molecular Weight ◽  
Wheat Cultivars ◽  
Glutenin Subunits ◽  
Molecular Weights ◽  
Additional Protein ◽  
Hmw Glutenin

Sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis (SDSGPAGE) was used to resolve gliadin and high- and low-molecular-weight glutenin subunits from 19 registered Canadian spring wheat cultivars eligible for Canada Western Red Spring (CWRS) and Canada Prairie Spring (CPS) wheat grades and eight nonregistered spring wheat cultivars from the U.S.A. Reproducible molecular weight estimates were obtained for wheat proteins of apparent molecular weights ranging from 34 238 to 136 174 (avg. CV = 0.72%). Eight different patterns of HMW glutenin subunits consisting of 7–11 protein bands were observed for the 27 cultivars and their biotypes. SDSGPAGE was able to discriminate among the majority of cultivars with all non-registered cultivars and their biotypes distinguishable from registered cultivars. Separation of glutenin subunits along with gliadins provided additional protein bands which assisted in the discrimination of cultivars.Key words: SDS gradient PAGE, wheat cultivar identification, gliadin, glutenin subunits

scholarly journals Purification and subunit structure of legumin of Vicia faba L. (broad bean)

1974 ◽  
Vol 141 (2) ◽  
pp. 413-418 ◽  
Author(s):  
David J. Wright ◽  
Donald Boulter
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Broad Bean ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Isoelectric Precipitation ◽  
Molecular Weights

Zonal isoelectric precipitation was shown to be an effective method for the preparation of legumin which was homogeneous as judged by ultracentrifugation and polyacrylamide-gel electrophoresis. The subunit structure of legumin was investigated by preparative sodium dodecyl sulphate–polyacrylamide-gel electrophoresis and ion-exchange chromatography in urea. Five distinct subunits, of which two were acidic (α) and had a molecular weight of 37000, and three were basic (β) with molecular weights of 20100, 20900 and 23800, were identified. The α and β subunits were present in equimolar amounts in the legumin molecule and, in view of this and molecular-weight considerations, an α6β6 subunit model was proposed for legumin.

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