scholarly journals A re-examination of the interaction of vinculin with actin.

1986 ◽  
Vol 102 (3) ◽  
pp. 1085-1092 ◽  
Author(s):  
J A Wilkins ◽  
S Lin
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Shear Viscosity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Active Protein ◽  
High Affinity ◽  
Low Shear Viscosity ◽  
Low Shear

Vinculin prepared by published procedures (i.e., Feramisco, J. R., and K. Burridge, 1980, J. Biol. Chem., 255:1194-1199) contains contaminants that have been shown by Evans et al. (Evans, R. R., R. M. Robson, and M. H. Stromer, 1984, J. Biol. Chem., 259:3916-3924) to reduce the low-shear viscosity of F-actin solutions. In this study we separated contaminants from conventional vinculin preparations by hydroxylapatite chromatography. We found that although the contaminants represented a small fraction (less than or equal to 5%) of the total protein in the conventional vinculin preparations, they were responsible for practically all of the filament capping and bundling activities previously attributed to vinculin. In addition, we examined the size of the molecule(s) responsible for the observed capping activity and found that its apparent molecular weight under denaturing conditions in sodium dodecyl sulfate (SDS) polyacrylamide gels fell within a broad range of 23,000-33,000. These results contrast with the observation that under nondenaturing conditions, the activity migrated in gel filtration columns at a position that corresponded to the Stoke's radius of a much bigger molecule. Since the migration of the activity in these chromatographic experiments is independent of the presence of vinculin, it is unlikely that the active protein associates with vinculin with high affinity under the conditions examined.

scholarly journals Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain

1976 ◽  
Vol 156 (1) ◽  
pp. 143-150 ◽  
Author(s):  
R H Quarles
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Weight Class ◽  
Developmental Difference ◽  
Adult Rats ◽  
Neuraminidase Treatment

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.

Variation in goat fibre protein

1989 ◽  
Vol 40 (3) ◽  
pp. 675 ◽  
Author(s):  
DJ Tucker ◽  
AHF Hudson ◽  
A Laudani ◽  
RC Marshall ◽  
DE Rivett
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Wool Fibre ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Cashmere Goats

The proteins from a range of cashmere, mohair, angoratcashmere crossbred and wool fibre samples were extracted at pH 8 with 8 M urea containing dithiothreitol, and were then radiolabelled by S-carboxymethylation using iodo(2-14C) acetate. The proteins from each sample were examined by two dimensional polyacrylamide gel electrophoresis in which the separation in the first dimension was according to charge at pH 8.9 and in the second dimension according to apparent molecular weight in the presence of sodium dodecyl sulfate. After electrophoresis the proteins were detected by fluorography. Protein differences in keratin samples from some individual goats existed, although the overall protein patterns were similar. None of the differences were consistent with any one goat fibre type. The protein patterns obtained for fibre samples from individual cashmere goats showed some differences when compared to those found for commercial blends from the same country of origin, indicating that blending can mask any animal-to-animal variation. While the electrophoretic technique does not unequivocally distinguish between cashmere, mohair and angora/cashmere crossbred fibres it does differentiate between wool and goat fibres.

Purification of Ristocetin Willebrand Factor (RWF)

1975 ◽  
Author(s):  
J. D. Olson ◽  
D. N. Fass ◽  
W. J. Brockway ◽  
E. J. W. Bowie ◽  
K. G. Mann
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Amino Sugar ◽  
Procoagulant Activity ◽  
Factor Viii Inhibitor ◽  
Induced Aggregation

A component required for the ristocetin-induced aggregation of platelets was isolated in yields of 20-30% from pooled human and porcine plasma by cryoethanol concentration of the RWF, after removal of the vitamin K dependent coagulation factors on barium citrate. The concentrate was further purified using gel filtration (4% agarose) and ion exchange (DEAE-cellulose) chromatography. Sodium dodecyl sulfate polyacrylamide gels of the isolated factor indicated an apparent molecular weight of greater than 500,000. After reduction of RWF with mercaptoethanol, a single band is resolved with an apparent molecular weight of 230,000. The purified component had no factor VIII procoagulant activity and did not compete for the activity of naturally occurring factor VIII inhibitor (human). Antisera raised in rabbits directed against the purified component inhibited the RWF activity but not the factor VIII procoagulant activity of plasma. Amino acid analysis indicated the presence of all normal amino acids and failed to detect any amino sugar. Analysis of lipid revealed a significant amount of lipid composed of mono, di and triglycerides, cholesterol, cholesterol esters and free fatty acid, with small portions of phospholipids.

scholarly journals Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes

1975 ◽  
Vol 151 (3) ◽  
pp. 685-697 ◽  
Author(s):  
M Letarte-Muirhead ◽  
A N Barclay ◽  
A F Williams
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Lentil Lectin ◽  
Polypeptide Chains

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.

Binding specificity and reactivity studies on a broad-specificity β-glycosidase from porcine kidney

1988 ◽  
Vol 66 (8) ◽  
pp. 830-838 ◽  
Author(s):  
R. E. Huber ◽  
R. L. Brockbank
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Porcine Kidney ◽  
Hydrolysis Of ◽  
Broad Specificity ◽  
Native Molecular Weight

A broad-specificity β-glycosidase from porcine kidney was purified to homogeneity. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis showed that it had a monomeric molecular weight of 55 000–60 000. Gel filtration showed a native molecular weight of about 115 000. These data imply that the native enzyme is a dimer. The enzyme can catalyze the hydrolysis of β bonds between glycosides and 4-methylumbelliferone or nitrophenol yielding D-fucopyranose, D-galactopyranose, D-glucopyranose, D-xylopyranose, and D-mannopyranose and of α bonds to yield L-arabinopyranose. This is the first study that shows a mammalian broad-specificity cytosolic β-glycosidase carrying out a reaction with a β-D-mannopyranoside. The nature of the broad specificity was studied with inhibitors. Similar inhibitor constants were found regardless of whether the substrate was a β-D-glucopyranoside or a β-D-galactopyranoside, so the enzyme probably has only one binding site with a broad specificity. The enzyme prefers to bind compounds with an axial hydroxyl at the 2 position and an equatorial hydroxyl at the 4 position; the 3 position does not affect binding significantly. The hydroxyl at the 6 position affects binding, but binding at that position depends on the configurations at the 2 and 4 positions. Thus, there must be some interactions between these three positions (2, 4, and 6). Lactones are also good inhibitors and this may relate to strain effects.

Identification of high-affinity calmodulin-binding proteins in rat liver

1987 ◽  
Vol 252 (3) ◽  
pp. C277-C284 ◽  
Author(s):  
R. M. Hanley ◽  
J. R. Dedman ◽  
S. Shenolikar
Keyword(s):  
Rat Liver ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Apparent Molecular Weight ◽  
Convincing Evidence ◽  
Calmodulin Binding ◽  
High Affinity

The Ca2+-dependent binding of [125I]calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or “acceptor” proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised greater than 80% of the Ca2+-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding “subunit” of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver.

Identification of the glpT -Encoded sn -Glycerol-3-Phosphate Permease of Escherichia coli , an Oligomeric Integral Membrane Protein

1982 ◽  
Vol 152 (3) ◽  
pp. 1008-1021
Author(s):  
Timothy J. Larson ◽  
Günter Schumacher ◽  
Winfried Boos
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gene Product ◽  
Active Transport ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Wild Type ◽  
Dodecyl Sulfate ◽  
Active Transport System

A collection of hybrid plasmids carrying either the wild-type or mutated glpT gene was generated in vitro and used to characterize the glpT -dependent active transport system for sn -glycerol-3-phosphate in Escherichia coli K-12. Restriction endonuclease analysis and recloning of DNA fragments localized glpT to a 3-kilobase pair Pst I- Hpa I segment of DNA. Comparison of DNA carrying glpT-lacZ fusions with DNA carrying intact glpT allowed determination of the direction of transcription. Through characterization of the proteins synthesized by strains harboring hybrid plasmids carrying amber, missense, or deletion mutations in glpT , it was shown that glpT is a promoter-proximal gene in an operon consisting of at least two genes. The gene product of glpT , the sn -glycerol-3-phosphate permease, was found associated with the inner membrane. It could be solubilized by treatment with sodium dodecyl sulfate at 50°C. Its molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was dependent upon sample treatment before electrophoresis. The apparent molecular weight was 44,000 when membrane fractions were heated to 50°C; subsequent treatment at 95°C modified the protein such that it migrated faster (apparent molecular weight = 33,000). Several missense mutations in glpT were negatively dominant over wild-type glpT , indicating that the active form of the permease is multimeric. A gene (named glpQ ) promoter distal to glpT codes for a periplasmic protein. This protein had previously been named GLPT protein to indicate its relationship to the glpT gene. The present report demonstrates that it is not the gene product of glpT and is not required for active transport of sn -glycerol-3-phosphate.

Cell-free synthesis of cod preproinsulin

1978 ◽  
Vol 56 (8) ◽  
pp. 791-793 ◽  
Author(s):  
Choy L. Hew
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Wheat Germ ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Insulin Antibodies ◽  
Translation System

Poly(A)-containing ribonucleic acid from cod islet greatly stimulated the incorporation of radioactive amino acids into proteins when assayed in a wheat germ translation system. The translation products were examined by specific immunoprecipitation with guinea pig anti cod insulin antibodies and by extraction with acid–ethanol. These measurements revealed at least a fivefold increase in incorporation of labelled amino acid over the nonprogrammed system. Sodium dodecyl sulfate disc gel electrophoresis and gel filtration chromatography showed a product of molecular weight 12 500, a size considerably larger than cod proinsulin (9000). It is concluded that cod proinsulin is synthesized via a larger precursor, preproinsulin.

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