Purification of infectious pancreatic necrosis (IPN) virus and comparison of polypeptide composition of different isolates

1978 ◽  
Vol 24 (1) ◽  
pp. 19-27 ◽  
Author(s):  
N. Chang ◽  
R. D. MacDonald ◽  
T. Yamamoto
Keyword(s):  
Gel Electrophoresis ◽  
Pancreatic Necrosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Particle Diameter ◽  
Virus Infectivity ◽  
Electron Microscopic ◽  
Infectious Pancreatic Necrosis ◽  
Virus Isolates

Infectious pancreatic necrosis (IPN) virus was partially purified by freon extraction of infected CHSE-214 cells and concentrated by polyethylene glycol (PEG) precipitation of virus from the medium. Both methods resulted in virus concentrates that could be further purified by two CsCl gradient centrifugations with little loss of infectivity. A recovery of 80 to 100% of the virus infectivity was obtained and over 100-fold concentration of viral infectivity was achieved by these methods. This purification was used to compare 10 isolates of IPN virus with regard to their physiochemical properties by electron microscopy, buoyant density in CsCl, and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified virions. Electron-microscopic observations showed that the virus isolates were identical in that they were isometric, hexagonal in profile, and had a particle diameter of 71 nm. The buoyant densities of the virus isolates in CsCl were found to be 1.33 g/ml. SDS-gel electrophoresis of the virus isolates revealed the presence of three polypeptides of molecular weight 50, 30, and 27 × 103 daltons designated as VP50, VP30, and VP27, respectively.

Plant NAD-Dependent Glutamate Dehydrogenase. Purification, Molecular Properties and Metal Ion Activation of the Enzymes from Lemna minor and Pisum sativum

1980 ◽  
Vol 35 (3-4) ◽  
pp. 213-221 ◽  
Author(s):  
Heinz-Walter Scheid ◽  
Adelheid Ehmke ◽  
Thomas Hartmann
Keyword(s):  
Amino Acid ◽  
Pisum Sativum ◽  
Glutamate Dehydrogenase ◽  
Gel Electrophoresis ◽  
Metal Ion ◽  
Lemna Minor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Electron Microscopic

Abstract Glutamate dehydrogenase (ʟ-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) has been purified to homogeneity from Lemna minor and seeds of Pisum sativum. As established by polyacrylamide gel electrophoresis the Pisum-enzyme constitutes a multiple pattern of seven char­ge isoenzymes whereas the Lemna enzyme shows one single protein band. Molecular weights of 230 000 were calculated for both enzymes by sedimentation equilibrium measurements (Pisum-enzyme) and comparative gel filtration (Lemna-enzyme). Sodium dodecyl sulfate gel electrophoresis and electron microscopic observations revealed that both enzymes are composed of four identical subunits (molecular weight 58 500) arranged in a tetraedric structure. The amino acid compositions of both enzymes are similar to those of various hexameric glutamate dehydrogenases. The N-terminal amino acid of the Pisum-enzyme is alanine. Both enzymes require Ca2+ for maximal catalytic activity. For the Lemna-enzyme the K0.5 values for Ca2+ are 22 µᴍ (NADH-dependent reaction) and 4 µᴍ (NAD+ -dependent reaction), respectively. Ca2+ which to some extent can be replaced by Zn2+ does not affect the enzyme aggregation but seems to govern a reversible equilibrium between catalytically active and inactive enzyme forms.

Electrophoretic and electron microscopic examination of the adductor and diductor muscles of an articulate brachiopod, Terebratalia transversa

1982 ◽  
Vol 60 (4) ◽  
pp. 550-559 ◽  
Author(s):  
William P. Eshleman ◽  
Jerrel L. Wilkens ◽  
Michael J. Cavey
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Light Chains ◽  
Electron Microscopic ◽  
Electrophoretic Patterns ◽  
Transmission Electron ◽  
Adductor Muscles ◽  
Regulation Of Contraction

The proteins of the striated adductor muscles, smooth adductor muscles, and diductor muscles of the articulate brachiopod Terebratalia transversa have been examined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Electrophoretic patterns indicate the presence of paramyosin in all of these valve muscles. Tentative identification has also been made of the proteins responsible for actin and for myosin regulation of contraction (troponin–tropomyosin and myosin light chains, respectively). The myofilaments of the striated adductor cells, smooth adductor cells, and diductor cells have been characterized by transmission electron microscopy. The smooth adductor cells and the diductor cells exhibit very thick myofilaments which are fusiform in shape, exceptionally long, and axially banded. Morphological features of these thick myofilaments are consistent with those of paramyosin filaments found in other muscles and myoepithelia. Although the striated adductor cells contain paramyosin, it is not manifest in the thick myofilaments.

scholarly journals A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage

1981 ◽  
Vol 197 (2) ◽  
pp. 355-366 ◽  
Author(s):  
D Heinegård ◽  
M Paulsson ◽  
S Inerot ◽  
C Carlström
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Chondroitin Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Partial Specific Volume ◽  
Gel Chromatography ◽  
Guanidinium Chloride ◽  
Cscl Density Gradient

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40-1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.

scholarly journals Microvilli of the human term placenta Isolation and subfractionation by centrifugation in sucrose density gradients

1981 ◽  
Vol 196 (1) ◽  
pp. 121-132 ◽  
Author(s):  
P Truman ◽  
J S Wakefield ◽  
H C Ford
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Density Gradients ◽  
Electron Microscopic ◽  
Microscopic Appearance ◽  
Human Term Placenta ◽  
Morphology And Morphometry ◽  
Microscopic Morphology

Human placental microvilli were isolated and separated into two fractions by centrifugation in sucrose density gradients. Electron-microscopic morphology and morphometry, the distribution of enzymic activities and the results of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of proteins were used to assess the purity of the final preparations and to define their properties. The combined evidence strongly suggested that the preparations contained negligible material that was not plasma membrane. The two fractions of microvilli differed in buoyant density, protein composition, enzyme specific activities and microscopic appearance. Some of these differences were explained by the absence of internal structure in the microvilli of the lighter fraction.

scholarly journals Isolation and protein composition of gap junctions from rabbit hearts

1982 ◽  
Vol 205 (1) ◽  
pp. 189-194 ◽  
Author(s):  
C K Manjunath ◽  
G E Goings ◽  
E Page
Keyword(s):  
Gap Junctions ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Polyacrylamide Gel ◽  
Major Protein ◽  
Electron Microscopic ◽  
Gap Junctional

We have modified a method for isolating gap-junctional membrane from mouse hearts [Kensler & Goodenough (1980) J. Cell Biol. 86, 755-764] to isolate gap junctions of comparable purity from rabbit hearts more rapidly, with better yield, and without resort to non-ionic detergents. Purification was monitored by electron microscopy of thin-sectioned membrane pellets and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gap junctions were obtained as vesicles whose mean surface area approximated that of junctions in intact myocardial cells. About 10-20% of the vesicles were ferritin-impermeable. Approx. 125 micrograms of membrane protein was obtained per 8 g of rabbit heart. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of purified gap junctions showed five major protein bands of mol.wts. 46 000, 44 000, 33 000, 30 000 and 28 500 that co-purified with the junctions. This protein composition was nearly identical with that published for gap junctions of mouse hearts, and differed markedly from the protein composition of gap junctions from non-excitable cells (lens and liver). The constancy of junctional protein composition between hearts of two different species and its non-identity with that from liver and lens suggest that, although gap-junctional structure in mammalian tissues seems to be remarkably similar by electron-microscopic techniques, junctional-channel protein composition actually varies from tissue to tissue and may be adapted to the permeability requirements of the tissue.

Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
Author(s):  
Nobuhiko Yoshida ◽  
Shingi Imaoka ◽  
Hajime Hirata ◽  
Michio Matsuda ◽  
Shinji Asakura
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tetraacetic Acid ◽  
Fibrin Monomer ◽  
Sds Page ◽  
Thrombotic Tendency ◽  
Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

1982 ◽  
Vol 47 (01) ◽  
pp. 014-018 ◽  
Author(s):  
H Sumi ◽  
N Toki ◽  
S Takasugi ◽  
S Maehara ◽  
M Maruyama ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Chromatography ◽  
Urinary Trypsin Inhibitor ◽  
Plasmin Activity ◽  
Molecular Weights ◽  
Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

Identifikasi Daging Tikus Pada Produk Baso Dengan Metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2018 ◽  
Vol 26 (2) ◽  
pp. 058
Author(s):  
Anna P. Roswiem ◽  
Triayu Septiani
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Sds Page

<em>Bahan<strong> </strong>baku untuk membuat baso adalah daging hewan, pada umumnya dari daging sapi, ayam, ikan dan babi. Di beberapa daerah di Indonesia terjadi kasus baso tikus. Tujuan penelitian ini adalah menguji ada tidaknya kandungan daging tikus pada produk baso yang dijual di pasar Cempaka Putih-Kecamatan Kramat Jakarta Pusat dan di pedagang baso atau mie baso di sekitar kampus Universitas YARSI Jakarta. Daging adalah protein salah satu metode untuk mengidentifikasi protein adalah metode Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE).<strong> </strong>Hasil penelitian menunjukkan bahwa dari 6 sampel baso terindikasi ada 2 sampel baso dengan nomor 1 dan 5 yang dibuat dari campuran daging sapi dan tikus; ada 1 sampel baso dengan nomor 6 yang terbuat dari daging tikus; dan 2 sampel baso dengan nomor 2 dan 3 yang terbuat dari campuran sapi  dan babi, dan hanya 1 sampel baso dengan nomor sampel 4 yang benar-benar terbuat dari daging sapi.</em>

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