scholarly journals The Isolation of Aggregates of Spectrin From Bovine Erythrocyte Membranes

1975 ◽  
Vol 28 (3) ◽  
pp. 259 ◽  
Author(s):  
GB Ralston
Keyword(s):  
Ionic Strength ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Sedimentation Equilibrium ◽  
Erythrocyte Membranes ◽  
Bovine Erythrocyte ◽  
Molecular Weights ◽  
Wide Range

Aggregated states of spectrin from bovine erythrocyte membranes can be detected in sedimentation velocity experiments. These aggregates have been isolated by means of gel filtration on columns of 4 % agarose. They appear to be stable over a wide range of pH and ionic strength, although they are dissociated by sodium dodecyl sulphate. Sedimentation equilibrium measurements yielded values of 960000 and 480000 for the molecular weights of the major aggregates, corresponding to a tetramer and dimer, respectively. The presence of different aggregated states in spectrin preparations may explain the wide variation in the reported physica~ properties of spectrin.

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 Subunit composition and structure of subcomponent C1q of the first component of human complement

1976 ◽  
Vol 155 (1) ◽  
pp. 19-23 ◽  
Author(s):  
K B M Reid ◽  
R R Porter
Keyword(s):  
Ionic Strength ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Helical Structure ◽  
Guanidinium Chloride ◽  
Molecular Weights ◽  
Composition And Structure ◽  
Covalently Linked ◽  
Low Ionic Strength ◽  
Expected Position

1. Unreduced human subcomponent C1q was shown by electrophoresis on polyacrylamide gels run in the presence of sodium dodecyl sulphate to be composed of two types of non-covalently linked subunits of apparent mol.wts. 69 000 and 54 000. The ratio of the two subunits was markedly affected by the ionic strength of the applied sample. At a low ionic strength of applied sample, which gave the optimum value for the 54 000-apparent mol.wt. subunit, a ratio of 1.99:1.00 was obtained for the ratio of the 69 000-apparent mol.wt. subunit to the 5400-apparent-mol.wt. subunit. The amount of the 54 000-apparent-mol.wt. subunit detected in the expected position on the gel was found to be inversely proportional to increases in the ionic strength of the applled sample. 2. Human subcomponent C1q on reduction and alkylation, or oxidation, yields equimolar amounts of three chains designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. The results obtained by Yonemasu & Stroud [(1972) Immunochemistry 9, 545-554], which showed that the 69 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the A and B chains and that the 54 000-apparent-mol.wt. subunit was a disulphide-linked dimer of the C chain, were confirmed. 3. Gel filtration on Sephadex G-200 in 6.0M-guanidinium chloride showed that both types of unreduced subunit were eluted together as a single symmetrical peak of apparent mol.wt. 49 000-50 000 when globular proteins were used as markers. The molecular weights of the oxidized or reduced A, B and C chains have been shown previously to be very similar all being in the range 23 000-24 000 [Reid et al. (1972) Biochem. J. 130, 749-763; Reid (1974) Biochem. J. 141, 189-203]. 4. It is proposed that subcomponent C1q (mol.wt. 410000) is composed of nine non-covalently linked subunits, i.e. six A-B dimers and three C-C dimers. 5. A structure for subcomponent C1q is proposed and is based on the assumption that the collagen-like regions of 78 residues in each of the A, B and C chains are combined to form a triple-helical structure of the same type as is found in collagens.

scholarly journals The fractionation of histones isolated from Euglena gracilis

1978 ◽  
Vol 169 (1) ◽  
pp. 103-111 ◽  
Author(s):  
N J Jardine ◽  
J L Leaver
Keyword(s):  
Aspartic Acid ◽  
Euglena Gracilis ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Histone H1 ◽  
Histone H2a ◽  
Molecular Weights ◽  
Compositional Properties

1. The histones of Euglena gracilis were separated by gel filtration into five fractions. 2. Each fraction was characterized in terms of its electrophoretic, solubility and compositional properties. 3. Euglena gracilis clearly contains histones corresponding to vertebrate H1, H2B, H3 and H4 fractions, although they all differ in containing more lysine. 4. The remaining Euglena histone is considered to be homologous to vertebrate histone H2A, but it differs in having a much higher ratio of lysine to arginine. 5. The Euglena histone H1 appears to be lacking in aspartic acid. 6. Electrophoresis in the presence of sodium dodecyl sulphate indicates that the molecular weights of the Euglena histones are close to those of the homologous vertebrate histones.

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 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.

scholarly journals Restoration of β-galactosidase to Escherichia coli M15. Complementation studies

1977 ◽  
Vol 165 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Dobrivoje V. Marinkovic ◽  
Jelka N. Marinkovic
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Galactosidase Activity ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Molecular Weights ◽  
Terminal Amino

Carboxymethylated β-galactosidase from Escherichia coli was dissociated at 100°C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore β-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ‘complementable fractions’. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the β-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented β-galactosidase can exist.

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.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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.

Isomerization of Peroxidizing Thiadiazolidine Herbicides Is Catalyzed by Glutathione S-Transferase

1996 ◽  
Vol 51 (5-6) ◽  
pp. 342-354 ◽  
Author(s):  
Beate Nicolaus ◽  
Yukiharu Sato ◽  
Ko Wakabayashi ◽  
Peter Böger
Keyword(s):  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Total Activity ◽  
Naphthalic Anhydride ◽  
Molecular Weights ◽  
Isomerase Activity ◽  
Ammonium Sulfate Precipitate ◽  
Catalyzed Reactions

Abstract Thiadiazolidine-converting activity (isomerase), detected in a 45-75% ammonium sulfate precipitate from corn seedlings extracts, was purified by chromatography on hydroxyapatite and by anion exchange on Mono Q Sepharose. Two fractions 1 and 2 with isomerase activity were separated on Mono Q by combination of a stepwise elution and continuous salt gradient; fraction 2 eluting at higher salt concentrations was found the most active. Total activity could be enhanced by treatment of seedlings with naphthalic anhydride. Both fractions containing isomerase activity were further purified by glutathione-(GSH) agarose affinity chromatography and characterized by their specificity for different thiadiazolidines. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration revealed that the isomerase of fraction 2 consists either of a homodimer or a heterodimer of two proteins with apparent molecular weights of 28 and 31 kDa, respectively. The protein pattern as well as the strict dependence of activity on thiol groups (GSH or dithiothreitol) suggested a glutathione Stransferase (GST) catalyzing the thiadiazolidine conversion. Further evidence was obtained by measuring reactions specific for GSTs in both purified fractions, namely the conjugating activity for l-chloro-2,4-dinitrobenzene (CDNB ). atrazine and metazachlor. While no atrazine turnover was found, metazachlor and CDNB conjugation occurred rapidly. Both fractions differed in their activities to several GST substrates with fraction 2 being more effective in metazachlor but less active in C DN B conjugation. Inhibitors specific for GST-catalyzed reactions also inhibited thiadiazolidine conversion confirming that isomerizing activity is attributed to a GST form. We conclude that GST isoforms with different affinities towards thiadiazolidines have been isolated. CDNB activity, molecular weight, the protein pattern on SDS-PAGE as well as the amino acid sequence of one of its polypeptides suggest that fraction 1, less active in thiadiazolidine isomerization, is identical to GST I. The second peptide of this fraction was resistant to Edman degradation probably due to N-terminal blockage. The properties of the high isomerase activity found in fraction 2 are in agreement with characteristics of a GST previously termed as isoform II.

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