scholarly journals Studies on the heterogeneity of subfragment-1 preparations. Isolation of a new proteolytic fragment of the heavy chain of myosin

1973 ◽  
Vol 131 (1) ◽  
pp. 127-137 ◽  
Author(s):  
D. Stone ◽  
S. V. Perry
Keyword(s):  
Molecular Weight ◽  
Heavy Chain ◽  
Light Chain ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Active Components ◽  
Proteolytic Fragment ◽  
Molecular Weights ◽  
Chain Fraction ◽  
Light Components

1. The physical, chemical and enzymic properties of subfragment 1 prepared from myosin of rabbit skeletal muscle by using two different concentrations of insoluble papain were compared. 2. Subfragment 1 prepared by using a myosin/papain ratio of 2000: 1 (by wt.) migrated on electrophoresis in non-dissociating conditions as a single enzymically active band. When prepared with a myosin/papain ratio of 200: 1 the preparation consisted of two enzymically active components of slightly different electrophoretic mobility. 3. The two types of preparation were obtained in similar yield and possessed similar specific adenosine triphosphatase activities when determined in the presence of Ca2+. 4. Gel electrophoresis in the presence of 8m-urea showed that both preparations contained three light components. The component of molecular weight 15500 was apparently identical with one of the light-chain components of myosin (Ml1). The other two light-chain components of subfragment 1 were not identical with any of the light-chain components of myosin. 5. The heavy-chain fraction of subfragment 1 prepared by using low concentrations of papain dissociated into components with molecular weights of 87000, 69000 and 26000 on electrophoresis in sodium dodecyl sulphate. The heavy-chain fraction of subfragment 1 prepared by using higher concentrations of papain contained components with molecular weights of 69000 and 53000 and relatively increased amounts of the component of molecular weight 26000. 6. The isolated 26000 dalton component had an amino acid composition similar to that of the heavy-chain fraction of subfragment 1 and contained 3-methylhistidine and mono-and tri-Nε-methyl-lysine. It was homogeneous on electrophoresis in the presence of sodium dodecyl sulphate but gave two bands on electrophoresis in 8m-urea.

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 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 effect of cross-links on the mobility of proteins in dodecyl sulphate–polyacrylamide gels

1972 ◽  
Vol 126 (3) ◽  
pp. 553-560 ◽  
Author(s):  
I. P. Griffith
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Cross Linking ◽  
Polyacrylamide Gels ◽  
Gel Method ◽  
Molecular Weights ◽  
Before And After ◽  
Cross Links ◽  
Polyacrylamide Gel Method

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate–polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate–polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.

scholarly journals A phosphorylated light-chain component of myosin from skeletal muscle

1973 ◽  
Vol 135 (1) ◽  
pp. 151-164 ◽  
Author(s):  
W. T. Perrie ◽  
L. B. Smillie ◽  
S. V. Perry
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Amino Acid ◽  
Light Chain ◽  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Low Molecular Weight ◽  
Obvious Similarity

1. The low-molecular-weight components of myosin from rabbit skeletal muscle migrated as four bands on polyacrylamide-gel electrophoresis in 8m-urea but only as three in systems containing sodium dodecyl sulphate. The two bands of intermediate mobility in 8m-urea (Ml2 and Ml3) had identical mobilities in sodium dodecyl sulphate. 2. The isolation of pure samples of all four low-molecular-weight components by DEAE-Sephadex chromatography is described. 3. The amino acid compositions of components Ml2 and Ml3 were identical. Further analyses showed the presence of 1 mol of phosphate/18500g of component Ml2 and less than 10% of this amount in component Ml3. Neither light component contained ribose. 4. Alkaline phosphatase from Escherichia coli converted component Ml2 into Ml3. Incubation with crude preparations of phosphorylase b kinase or protein kinase in the presence of ATP converted component Ml3 into Ml2. 5. Phosphorylation of component Ml3 with the kinases isolated from skeletal muscle and [γ-32P]ATP gave incorporation of 32P only into component Ml2 whether whole myosin or separated low-molecular-weight components were used. 6. High-voltage electrophoresis at pH6.5 and pH1.8 of a chymotryptic digest of 32P-labelled component Ml2 yielded one major radioactive peptide containing serine phosphate. 7. The amino acid sequence of this peptide was shown to be: Arg-Ala-Ala-Ala-Glu-Gly-Gly-(Ser,Ser(P))-Asn-Val-Phe. This sequence shows no obvious similarity to the site phosphorylated in the conversion of phosphorylase b into phosphorylase a by phosphorylase b kinase. 8. Evidence suggests that in vivo all the 18500-molecular-weight light chain is in the phosphorylated form. The extent of dephosphorylation that occurred during myosin extraction depended on the conditions employed.

INHIBITION OF HUMAN BLOOD COAGULATION FACTOR Xa BY α2-MACROGLO-BULIN

1987 ◽  
Author(s):  
Joost C M Meijers ◽  
Pim N M Tijburg ◽  
Bonno N Bouma
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Sodium Dodecyl Sulphate ◽  
Enzyme Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor X ◽  
Order Rate

The inactivation of activated factor X (factor Xa) by α2 macroglobulin (α2M) was studied. Irreversible inhibition was observed with the initial formation of a reversible enzyme-inhibitor complex The secopd-order rate constant for the reaction was 8.4 × 104 M−1 min−1. The binding ratio was found to be 2 mol factor Xa/ mol α2M. Interaction of factor Xa with α2M resulted in the appearance of four thiolgroups/molecule α2. The apparent second-order rate constants for the appearance of thiolgroups were dependent on the factor Xa concentration. Sodium dodecyl sulphate gradient polyacrylamide gel electrophoresis was used to study complex formation between α2M and factor Xa. Under non-reducing conditions four factor Xa - α2M complexes were observed. Reduction of these complexes showed the formation of two new bands. One complex (Mr 225000) consisted of the heavy chain of the factor Xa molecule covalently bound to a subunit of α2M, while the second complex (Mr A00000) consisted of the heavy chain of factor Xa molecule and two subunits of α2M. Factor Xa was able to form a bridge between two subunits or α2M, either within one molecule of α2M, or by linking two molecules of The role of the light chain of factor Xa in this process remains to be elucidated. For this purpose, monoclonal antibodies specific for the light chain of factor Xa were prepared. Sodium dodecyl sulphate agarose electrophoresis studies showed that complexes involving more than two molecules of α2M were not formed.

scholarly journals The binding of sodium dodecyl sulphate to various proteins

1968 ◽  
Vol 109 (5) ◽  
pp. 825-830 ◽  
Author(s):  
Rosalind Pitt-Rivers ◽  
F. S. Ambesi Impiombato
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Equilibrium Dialysis ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Dye Solubilization

1. The binding of sodium dodecyl sulphate to proteins by equilibrium dialysis was investigated. 2. Most of the proteins studied bound 90–100% of their weight of sodium dodecyl sulphate. 3. The glycoproteins studied bound 70–100% of their weight of sodium dodecyl sulphate, calculated in terms of the polypeptide moiety of the molecule. 4. Proteins not containing S·S groups bound about 140% of their weight of sodium dodecyl sulphate. 5. Reduction of four proteins containing S·S groups caused a rise in sodium dodecyl sulphate binding to 140% of the weight of protein. 6. The apparent micellar molecular weights of the protein–sodium dodecyl sulphate complexes were measured by the dye-solubilization method; they were all found to have approximately the same micellar molecular weight (34000–41000) irrespective of the molecular weight of the protein to which they were attached.

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 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.

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