scholarly journals PROPERTIES OF PHYSARUM MYOSIN PURIFIED BY A POTASSIUM IODIDE PROCEDURE

1974 ◽  
Vol 62 (1) ◽  
pp. 54-65 ◽  
Author(s):  
V. T. Nachmias
Keyword(s):  
Potassium Iodide ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Molar Ratio ◽  
Heavy Chains ◽  
Molecular Weights ◽  
Magnesium Atpase ◽  
Low Ionic Strength ◽  
Actin Concentration

Myosin has been purified free of actin from Physarum actomyosin by a two step adaptation of the classical potassium iodide method for depolymerizing actin. On 12% sodium dodecyl sulfate (SDS) gels, the single major slowly moving protein band present in the calcium activated adenosine triphosphatase peak (90% pure) is associated with two fast moving bands of molecular weights of approximately 17,000 and 21,000 daltons, respectively. Densitometry shows the molar ratio of heavy chains to the 21,000 and 17,000 dalton chains on the gels to be 1:2:1. The highly purified myosin forms filaments up to 2.5 µm long in the presence of 5 mM magnesium and 0.05 M KCl. Calcium ions were not required for the formation of long filaments from this highly purified myosin. At low ionic strength (0.05 M KCl) the magnesium ATPase of the highly purified myosin is activated four- to tenfold by muscle actin. The extent of activation is a function of the actin concentration and levels off at high levels of actin. In 0.1 mM calcium salts the ATPase activity is approximately 60% of that in 1 mM EGTA. In summary, Physarum myosin is similar to a number of muscle myosins as well as to platelet and fibroblast myosin, which all possess light chains of two different molecular weights associated with the heavy chains. Under ionic conditions close to those in vivo, highly purified Physarum myosin aggregates into long filaments.

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.

Synthesis of acid-soluble spore proteins by Bacillus subtilis

1982 ◽  
Vol 152 (3) ◽  
pp. 1117-1125
Author(s):  
J M Leventhal ◽  
G H Chambliss
Keyword(s):  
Bacillus Subtilis ◽  
Maximum Rate ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Spore Proteins ◽  
Major Acid

The major acid-soluble spore proteins (ASSPs) of Bacillus subtilis were detected by immunoprecipitation of radioactively labeled in vitro- and in vivo-synthesized proteins. ASSP synthesis in vivo began 2 h after the initiation of sporulation (t2) and reached its maximum rate at t7. This corresponded to the time of synthesis of mRNA that stimulated the maximum rate of ASSP synthesis in vitro. Under the set of conditions used in these experiments, protease synthesis began near t0, alkaline phosphatase synthesis began at about t2, and refractile spores were first observed between t7 and t8. In vivo- and in vitro-synthesized ASSPs comigrated in sodium dodecyl sulfate-polyacrylamide gels. Their molecular weights were 4,600 (alpha and beta) and 11,000 (gamma). The average half-life of the ASSP messages was 11 min when either rifampin (10 micrograms/ml) or actinomycin D (1 microgram/ml) was used to inhibit RNA synthesis.

scholarly journals Phaseolin type and heat treatment influence the biochemistry of protein digestion in the rat intestine

2008 ◽  
Vol 99 (3) ◽  
pp. 531-539 ◽  
Author(s):  
Carlos A. Montoya ◽  
Pascal Leterme ◽  
Stephen Beebe ◽  
Wolfgang B. Souffrant ◽  
Daniel Mollé ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Western Blotting ◽  
Sole Source ◽  
Protein Band ◽  
Molecular Weights ◽  
Endogenous Protein ◽  
Anionic Trypsin ◽  
Endogenous Proteins ◽  
Protein Components

The study aimed to investigate thein vivodigestion ofPhaseolus vulgarisphaseolin types differing in their subunit pattern composition. Diets contained either casein as the sole source of protein or a mixture (1:1) of casein and pure Sanilac (S), Tendergreen (T) or Inca (I) phaseolin either unheated or heated. Rats were fed for 11 d with the experimental diets. Their ileal content and mucosa were collected and prepared for electrophoresis, Western blotting, densitometry and MS. Differences in digestion among native phaseolin types were observed for intact phaseolin at molecular weights (MW) of 47–50·5 kDa and for an undigested fragment at MW of 19–21·5 kDa in ileal digesta. In both cases, the concentration of these protein bands was lower for I phaseolin than for S or T phaseolin (P < 0·05). In the mucosa, the concentration of a protein band at MW of 20·5–21·5 kDa was lower for S phaseolin as compared to T or I phaseolin (P < 0·001). The presence of phaseolin subunits and their fragments was confirmed by Western blotting. MS analysis revealed the presence of undigested α and β subunit fragments from phaseolin and endogenous proteins (anionic trypsin I and pancreatic α-amylase) in ileal digesta. Thermal treatment improved digestion (P < 0·01), acting on both dietary and endogenous protein components. In conclusion, this study provides evidence for differences in intestinal digestion among phaseolin types, S phaseolin being more resistant and I phaseolin more susceptible. These differences were affected by the origin of the phaseolin subunit precursor. Heat treatment enhanced phaseolin digestion.

scholarly journals Synthesis of proteins from [35S]methionine by guinea pig megakaryocytes in vivo and time course of appearance of newly synthesized proteins in platelets

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 887-891 ◽  
Author(s):  
BP Schick
Keyword(s):  
Protein Synthesis ◽  
Guinea Pigs ◽  
Time Course ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Sds Page ◽  
Relationship Of

The relationship of protein synthesis to megakaryocyte maturation has been studied in guinea pigs in vivo. Guinea pigs were injected with a single dose of [35S]methionine. Megakaryocytes and platelets were isolated daily for 4 days, and proteins from both cells were isolated by DEAE-Sephacel chromatography and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. All proteins in megakaryocytes corresponding to stained bands on the SDS- PAGE gels were radiolabeled at 3 hours after injection. The greatest loss of radioactivity from the megakaryocytes occurred between 1 and 3 days after injection. Only trace labeling of platelet proteins was seen at 3 hours, representing almost entirely three bands at molecular weights 47,000, 52,000, and 66,000. At 24 hours only about 13% of the maximal labeling was present, but not all proteins were labeled. The maximal labeling was at 3 days. The pattern of labeling of platelets at 3 days was identical to that of megakaryocytes at 3 hours. The protein pattern of nonmegakaryocytic marrow cells was different from that of the platelets and megakaryocytes. Data presented here suggest that most protein synthesis in megakaryocytes is completed at least 24 hours before release of the platelets to the circulation, and suggest some specificity in the proteins that are synthesized at the terminal stages of maturation.

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 Synthesis of proteins from [35S]methionine by guinea pig megakaryocytes in vivo and time course of appearance of newly synthesized proteins in platelets

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 887-891 ◽  
Author(s):  
BP Schick
Keyword(s):  
Protein Synthesis ◽  
Guinea Pigs ◽  
Time Course ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Sds Page ◽  
Relationship Of

Abstract The relationship of protein synthesis to megakaryocyte maturation has been studied in guinea pigs in vivo. Guinea pigs were injected with a single dose of [35S]methionine. Megakaryocytes and platelets were isolated daily for 4 days, and proteins from both cells were isolated by DEAE-Sephacel chromatography and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. All proteins in megakaryocytes corresponding to stained bands on the SDS- PAGE gels were radiolabeled at 3 hours after injection. The greatest loss of radioactivity from the megakaryocytes occurred between 1 and 3 days after injection. Only trace labeling of platelet proteins was seen at 3 hours, representing almost entirely three bands at molecular weights 47,000, 52,000, and 66,000. At 24 hours only about 13% of the maximal labeling was present, but not all proteins were labeled. The maximal labeling was at 3 days. The pattern of labeling of platelets at 3 days was identical to that of megakaryocytes at 3 hours. The protein pattern of nonmegakaryocytic marrow cells was different from that of the platelets and megakaryocytes. Data presented here suggest that most protein synthesis in megakaryocytes is completed at least 24 hours before release of the platelets to the circulation, and suggest some specificity in the proteins that are synthesized at the terminal stages of maturation.

scholarly journals Biosynthesis of collagen and other matrix proteins by articular cartilage in experimental osteoarthrosis

1980 ◽  
Vol 188 (3) ◽  
pp. 823-837 ◽  
Author(s):  
D R Eyre ◽  
C A McDevitt ◽  
M E Billingham ◽  
H Muir
Keyword(s):  
Articular Cartilage ◽  
Bond Cleavage ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dry Weight ◽  
Type Ii Collagen ◽  
Protein Band ◽  
The Matrix

Osteoarthrosis was induced in one knee joint of dogs by an established surgical procedure. Changes in the articular cartilage in the biosynthesis of collagen and other proteins were sought by radiochemical labelling in vivo, with the following findings. (1) Collagen synthesis was stimulated in all cartilage surfaces of the experimental joints at 2, 8 and 24 weeks after surgery. Systemic labelling with [3H]proline showed that over 10 times more collagen was being deposited per dry weight of experimental cartilage compared with control cartilage in the unoperated knee. (2) Type-II collagen was the radiolabelled product in all samples of experimental cartilage ranging in quality from undamaged to overtly fibrillated, and was the only collagen detected chemically in the matrix of osteoarthrotic cartilage from either dog or human joints. (3) Hydroxylysine glycosylation was examined in the newly synthesized cartilage collagen by labelling dog joints in vivo with [3H]lysine. In experimental knees the new collagen was less glycosylated than in controls. However, no difference in glycosylation of the total collagen in the tissues was observed by chemical analysis. (4) Over half the protein-bound tritium was extracted by 4 M-guanidinium chloride from control cartilage labelled with [3H]proline, compared with one-quarter or less from experimental cartilage. Two-thirds of the extracted tritium separated in the upper fraction on density-gradient centrifugation in CsCl under associative conditions. Much of this ran with a single protein band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions. The identity of this protein was unknown, although it resembled serum albumin in mobility afte disulphide-bond cleavage.

scholarly journals Incorrect Molecular Weights due to inaccurate Prestained Protein Molecular Weight Markers that are used for Gel Electrophoresis and Western Blotting

2020 ◽  
Author(s):  
Rômulo Leão Silva Neris ◽  
Ajuni Kaur ◽  
Aldrin V. Gomes
Keyword(s):  
Molecular Weight ◽  
Molecular Mass ◽  
Western Blotting ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Molecular Weights ◽  
Molecular Masses ◽  
Protein Molecular Weight

ABSTRACTThe most widely used Western blotting protein standards are prestained proteins of known molecular mass (kDa). They are also utilized for sodium dodecyl sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) to determine the molecular mass of proteins separated by electrophoresis. The objective of this study was to assess the reliability of different commercially available protein standards in predicting accurate protein molecular weights. We performed this experiment by running Criterion TGX gels with five prestained protein standards (Thermo Fisher SeeBlue Plus 2, Bio-Rad Precision Plus Protein Dual-color, Thermo Fisher Spectra Multi-color, Novex-Sharp Pre-stained, and Invitrogen iBright Pre-Stained). To evaluate their accuracy, we utilized highly purified Bovine Serum Albumin (BSA, 66.44 kDa) and Cytochrome C (Cyto C, 11.62 kDa). We also made use of the dimers of BSA (132.88 kDa) and Cyt C (23.24 kDa) that are present on SDS-PAGE gels. Our results suggest that three of the standards were less accurate at higher molecular masses with the iBright marker having the highest error in determining the expected 132.88 kDa molecular weight. The SeeBlue Plus 2 was accurate at identifying the 132.88 kDa molecular weight protein band but was less reliable for the three other lower molecular weight proteins. These findings have significant implications for the determination of protein masses because researchers rely on these standards to evaluate the molecular masses of their protein(s). We suggest that at least two different protein standards should be initially used in electrophoresis gels and for Western blotting in order to get accurate protein molecular weight results.

scholarly journals The structure and subunit composition of the particulate NADH-ubiquinone reductase of bovine heart mitochondria

1976 ◽  
Vol 154 (2) ◽  
pp. 295-305 ◽  
Author(s):  
C. I Ragan
Keyword(s):  
Molecular Weight ◽  
Complex I ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Heart Mitochondria ◽  
Molar Ratio ◽  
Polypeptide Composition ◽  
Molecular Weights ◽  
Bovine Heart

Preparations of NADH-ubiquinone reductase from bovine heart mitochondria (Complex I) were shown to contain at least 16 polypeptides by gel electrophoresis in the presence of sodium dodecyl sulphate. 2. High-molecular-weight soluble NADH dehydrogenase prepared from Triton X-100 extracts of submitochondrial particles [Baugh & King (1972) Biochem. Biophys. Res. Commun. 49, 1165-1173] was similar to Complex I in its polypeptide composition. 3. Solubilization of Complex I by phospholipase A treatment and subsequent sucrose-density-gradient centrifugation did not alter the polypeptide composition. 4. Lysophosphatidylcholine treatment of Complex I caused some selective solubilization of a polypeptide of mol.wt. 33000 previosuly postulated to be the transmembrane component of Complex I in the mitochondrial membrane [Ragan (1975) in Energy Transducing Membranes: Structure, Function and Reconstitution (Bennun, Bacila & Najjar, eds.), Junk, The Hague, in the press]. 5. Chaotropic resolution of Complex I caused solubilization of polypeptides of molecular weights 75000, 53000, 29000, 26000 and 15500 and traces of others in the 10000-20000-mol.wt.range. 6. The major components of the iron-protein fraction from chaotropic resolution had molecular weights of 75000, 53000 and 29000, whereas the flavoprotein contained polypeptides of molecular weights 53000 and 26000 in a 1:1 molar ratio. 7. Iodination of Complex I by lactoperoxidase indicated that the water-soluble polypeptides released by chaotropic resolution, in particular those of the flavoprotein fraction, were largely buried in the intact Complex. 8. The polypeptides of molecular weights 75000, 53000, 42000, 39000, 33000, 29000 and 26000 were present in 1:2:1:1:1:1:1 molar proportions. The two subunits of molecular weight 53000 are probably non-identical.

scholarly journals Purification of two hexosaminidases from human kidney

1977 ◽  
Vol 163 (1) ◽  
pp. 133-140 ◽  
Author(s):  
D V Marinkovic ◽  
J N Marinkovic
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Human Kidney ◽  
Protein Band ◽  
Iodoacetic Acid ◽  
Homogeneous State ◽  
Molecular Weights ◽  
Hexosaminidase A

Hexosaminidase forms A and B were isolated from human kidney in a homogeneous state as demonstrated by electrophoretic and enzymic criteria. The enzymes were stable for at least 18 months when stored at -20 degrees C in 0.025 M-phosphate buffer, pH 6.5. The molecular weights of forms A and B were estimated by gel filtration to be 111 000 +/- 1500 and 114 000 +/- 1600 respectively. The molecular weights of hexosamidase A and B subunits were determined by using polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Hexosaminidase A dissociated into one subunit with mol.wt. 68 000. Hexosaminidase B dissociated into three subunits with mol. wts. 100 000, 68 000 and 37000 respectively, and one protein band of mol.wt. 140 000. After treatment of hexosaminidases A and B with iodoacetic acid, the molecular weights of the carboxymethylated polypeptide subunits were also estimated. Carboxymethylated hexosaminidase A dissociated into one major subunit of mol.wt. 18 000 and two other protein bands of mol.wts. 65 000 and 100 000. Carboxymethylated hexosaminidase B dissociated into one major subunit for mol.wt. 19 000 and an additional band of mol.wt. 37 000. The Km of the enzymes for the synthetic substrate p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside was 0.8 mM. Both enzymes were inhibited or activated by various metal ions. Double pH optima for the enzymes were found at pH 4.5 and 4.8.

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