Comparison of the Effect of Various Methods of Dissociation on the Protein Composition of Rat Liver Ribosomal Subunits

1975 ◽  
Vol 53 (9) ◽  
pp. 935-942 ◽  
Author(s):  
Nabil Hanna ◽  
Claude Godin
Keyword(s):  
Rat Liver ◽  
Ribosomal Proteins ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Composition ◽  
Small Subunit ◽  
Protein Patterns ◽  
Major Loss ◽  
Centrifugation Technique ◽  
High Concentrations

Rat liver ribosomes were dissociated into subunits using EDTA, sodium pyrophosphate, high concentrations of KCl, as well as by incubation with puromycin in presence of 0.5 M KCl. The subunits obtained were analyzed using the density gradient centrifugation technique and their ribosomal proteins were separated by means of two-dimensional polyacrylamide gel electrophoresis. The ribosomal protein patterns of the two subunits isolated using each of the dissociating method were compared to the protein patterns of monosomes prepared by puromycin treatment alone. Our results revealed that the use of chelating agents to dissociate the ribosomes resulted in the loss of some ribosomal proteins from the small subunit. On the other hand, the use of KCl in high concentrations to dissociate the ribsosomes did not appear to cause any major loss of proteins from the ribosomes except for some acidic proteins.

scholarly journals Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 155 (1) ◽  
pp. 107-115 ◽  
Author(s):  
T Noguchi ◽  
E Okuno ◽  
Y Minatogawa ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Aminotransferase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

Preliminary biochemical characterization of ‘veil’ structure purified fromTheileria sergenti-, T. buffeli- andT. orientalis-infected bovine erythrocytes

Parasitology ◽  
1992 ◽  
Vol 104 (2) ◽  
pp. 207-213 ◽  
Author(s):  
C. Sugimoto ◽  
S. Kawazu ◽  
M. Sato ◽  
T. Kamio ◽  
K. Fujisaki
Keyword(s):  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Microscopical Examination ◽  
Protein Patterns ◽  
Basic Proteins ◽  
Percoll Density Gradient Centrifugation ◽  
The Veil ◽  
Electron Microscopical ◽  
Bovine Erythrocytes

SUMMARYA structure called ‘veil’ inTheileria sergenti-, T. buffeli- orT. orientalis-infected bovine erythrocytes was purified from erythrocyte lysates by Percoll density-gradient centrifugation. On electron microscopical examination, the veils consisted of electron-dense material showing a periodicity of striations and were not surrounded by membranes. Analyses of veil proteins by one- and two-dimensional polyacrylamide gel electrophoresis revealed that the veils contain haemoglobin and several other basic proteins of molecular weight ranging from 15·5 to 46 kDa. By comparing the protein patterns of the veil with those of purified piroplasms and uninfected bovine erythrocytes, these basic proteins appeared to be of parasite origin. It would appear likely that the veils formed by intra-erythrocytic precipitation of haemoglobin and proteins excreted by the parasites. Differences in veil constituents were found betweenT. sergenti, T. buffeliandT. orientalis.

Relationships among species of Verticillium: protein composition of spores and mycelium

1971 ◽  
Vol 49 (8) ◽  
pp. 1293-1297 ◽  
Author(s):  
Gilles Pelletier ◽  
Robert Hall
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Morphological Differentiation ◽  
Protein Composition ◽  
Soluble Proteins ◽  
Polyacrylamide Gel ◽  
Close Similarity ◽  
Protein Patterns ◽  
Electrophoresis Of Proteins

Buffer-soluble proteins extracted from six morphologically different isolates of Verticillium were separated by polyacrylamide gel-electrophoresis. Protein patterns from the six isolates were different from one another whether extracts were prepared from conidia, from young colonies composed of mycelium and conidia, or from 6-day-old mycelium. However, the nature of the patterns, and therefore the degree of differences among species patterns, was influenced by the types of cells from which the extracts were prepared.Patterns of proteins from V. tricorpus, V. nigrescens, and an isolate of uncertain identity (isolate 2) which produced chlamydospores and dark mycelium were clearly different from one another whether extracts were prepared from conidia or mycelium. In contrast, conidia of V. albo-atrum, of V. dahliae, and of an isolate which did not produce pigmented structures produced very similar patterns which differed by only a few protein bands. This close similarity of patterns supports the view that V. albo-atrum and V. dahliae are genetically closely related.The protein composition of conidia differed from that of mycelium. In V. albo-atrum, spore extracts contained at least three proteins not detected in mycelium extracts. Differences between spores and mycelium were even greater in V. nigrescens and isolate 2. Analysis of V. dahliae showed differences between spores, 3-day-old mycelium, and 6-day-old mycelium.Our results support the view that gel-electrophoresis of proteins is useful as a taxonomic tool provided attention is given to the degree of morphological differentiation of the materials to be compared.

scholarly journals Simplification of ribosomes in bacteria with tiny genomes

2019 ◽  
Author(s):  
Daria D. Nikolaeva ◽  
Mikhail S. Gelfand ◽  
Sofya K. Garushyants
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Protein Biosynthesis ◽  
Large Subunit ◽  
Protein Composition ◽  
Small Subunit ◽  
Genome Reduction ◽  
Bacterial Genomes ◽  
Shine Dalgarno Sequence

AbstractThe ribosome is an essential cellular machine performing protein biosynthesis. Its structure and composition are highly conserved in all species. However, some bacteria have been reported to have an incomplete set of ribosomal proteins. We have analyzed ribosomal protein composition in 214 small bacterial genomes (< 1 Mb) and found that although the ribosome composition is fairly stable, some ribosomal proteins may be absent, especially in bacteria with dramatically reduced genomes. The protein composition of the large subunit is less conserved than that of the small subunit. We have identified the set of frequently lost ribosomal proteins and demonstrated that they tend to be situated on the ribosome surface and have fewer contacts to other ribosome components. Moreover, some proteins are lost in an evolutionary correlated manner. The reduction of rRNA is also common in bacteria with tiny genomes with deletions mostly occurring in free loops. Finally, the loss of the anti-Shine-Dalgarno sequence is associated with the genome reduction, the number of lost ribosomal proteins, and with the loss of bL9 and TF.

scholarly journals Purification of a protein having pore forming activity from the rat liver mitochondrial outer membrane

1982 ◽  
Vol 208 (1) ◽  
pp. 77-82 ◽  
Author(s):  
M Lindén ◽  
P Gellerfors ◽  
B D Nelson
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mitochondrial Outer Membrane ◽  
Rat Liver Mitochondria ◽  
Charge Heterogeneity

A protein with pore-forming activity has been isolated from the outer membrane of rat liver mitochondria. The purification involves sucrose gradient centrifugation, differential centrifugation in the presence of Triton X-100, and DEAE-Sepharose and CM-Sepharose chromatography. The yield of the purified protein was approx. 2% of the total outer membrane proteins. The protein, when inserted into soya bean phospholipid vesicles, increases the [3H]sucrose permeability of the vesicles but had no effect on the permeability of high-molecular-weight [14C]dextran (Mr 70 000). The protein is very active, since as little as 3-4 micrograms of protein per mg of phospholipid is required for the complete release of [3H]sucrose from the vesicles. Sucrose diffusion channels could not be reconstituted with other membrane proteins such as rat liver cytochrome oxidase or cytochrome b5. Purified pore protein revealed a single band of apparent Mr 30000 when resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This polypeptide could be further resolved by isoelectric focusing into a major (pI7.9) and two relatively minor (pI7.6 and 7.2) components. Proteolytic mapping with V8 proteinase from Staphylococcus aureus suggests that these probably represent a single component showing charge heterogeneity. The reason for the charge heterogeneity is not known. The amino acid composition of the protein revealed 47.8% polar amino acids with a relatively high lysine content.

Analysis of two-dimensional protein patterns from developmental stages of the potato cyst-nematode, Globodera rostochiensis

Parasitology ◽  
1992 ◽  
Vol 105 (3) ◽  
pp. 461-474 ◽  
Author(s):  
J. M. De Boer ◽  
H. A. Overmars ◽  
J. Bakker ◽  
F. J. Gommers
Keyword(s):  
Developmental Stages ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Composition ◽  
Globodera Rostochiensis ◽  
Cyst Nematode ◽  
Potato Cyst Nematode ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Silver Stain ◽  
Molecular Weights

SUMMARYTwo-dimensional polyacrylamide gel electrophoresis was used to examine the differences in total protein composition between two motile stages and two sedentary stages of the potato cyst-nematode, Globodera rostochiensis. Using a sensitive silver stain, 542 reproducible protein spots were distinguished. A list of these spots is presented, showing their apparent molecular weights, estimated isoelectric points, and occurrences in the different developmental stages. When the protein patterns were compared, 401 spots were found to change their presence or size in one or more of the four developmental stages. It is therefore estimated that during the post-embryonic development of G. rostochiensis, 74% of the polypeptides undergo modulation of their synthesis, or are affected by protein degradation or modification. In the motile stages several abundant proteins were present, which disappeared or decreased in concentration in the sedentary stages. Some of these proteins are presumably muscle proteins, and their modulation may illustrate the degeneration of body-wall musculature in the sedentary stages. It is concluded that the potato cyst-nematode has a very dynamic protein metabolism.

scholarly journals Simplification of Ribosomes in Bacteria with Tiny Genomes

2020 ◽  
Vol 38 (1) ◽  
pp. 58-66 ◽  
Author(s):  
Daria D Nikolaeva ◽  
Mikhail S Gelfand ◽  
Sofya K Garushyants
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Protein Biosynthesis ◽  
Large Subunit ◽  
Protein Composition ◽  
Small Subunit ◽  
Bacterial Genomes ◽  
Shine Dalgarno Sequence

Abstract The ribosome is an essential cellular machine performing protein biosynthesis. Its structure and composition are highly conserved in all species. However, some bacteria have been reported to have an incomplete set of ribosomal proteins. We have analyzed ribosomal protein composition in 214 small bacterial genomes (&lt;1 Mb) and found that although the ribosome composition is fairly stable, some ribosomal proteins may be absent, especially in bacteria with dramatically reduced genomes. The protein composition of the large subunit is less conserved than that of the small subunit. We have identified the set of frequently lost ribosomal proteins and demonstrated that they tend to be positioned on the ribosome surface and have fewer contacts to other ribosome components. Moreover, some proteins are lost in an evolutionary correlated manner. The reduction of ribosomal RNA is also common, with deletions mostly occurring in free loops. Finally, the loss of the anti-Shine–Dalgarno sequence is associated with the loss of a higher number of ribosomal proteins.

scholarly journals The composition of rat liver microsomes. The structural proteins of rat liver microsomes

1969 ◽  
Vol 114 (1) ◽  
pp. 41-48 ◽  
Author(s):  
K. A. Ward ◽  
J K Pollak
Keyword(s):  
Rat Liver ◽  
Ribosomal Proteins ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Liver Microsomes ◽  
Electrophoretic Pattern ◽  
Structural Proteins ◽  
Rat Liver Microsomes ◽  
Structural Protein ◽  
Electrophoretic Patterns

1. The structural-protein component of microsomal membranes was isolated by three separate methods. Analysis by polyacrylamide-gel electrophoresis indicated that the microsomal structural component is made up of a heterogeneous group of proteins. These proteins were further characterized by their phospholipid-binding capacity. The electrophoretic patterns of microsomal structural proteins were found to differ significantly from those of mitochondrial structural proteins. 2. The reticulosomal fraction was also characterized by electrophoresis with reference to total microsomal proteins, microsomal structural proteins and ribosomal proteins. The reticulosomes gave an electrophoretic pattern significantly different from those of the other three preparations examined. It is suggested that reticulosomes consist largely of enzymic proteins of the endoplasmic reticulum.

scholarly journals Enzymic iodination of eukaryotic ribosomal subunits. Characterization and analysis by two-dimensional gel electrophoresis

1975 ◽  
Vol 152 (2) ◽  
pp. 373-378 ◽  
Author(s):  
David P. Leader
Keyword(s):  
Rat Liver ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Large Subunit ◽  
Small Subunit ◽  
Suitable Method ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Ribosomal Subunits

1. Conditions are described for the enzymic iodination of ribosomal subunits from rat liver. The reaction is relatively insensitive to broad changes in the concentration of KCl, allowing subunits to be studied under conditions which minimize their dimerization. 2. Mixtures of extracted ribosomal proteins were iodinated with 125I, the proteins separated by two-dimensional gel electrophoresis and the radioactivity in each protein was determined. Thus 19 out of 23 of the proteins of the small subunit and 25 out of 33 of the proteins of the large subunit were labelled. Iodination should therefore be a suitable method for studying the topography of the ribosomal proteins of rat liver. 3. When the intact 40S subunit (rather than the extracted mixture of proteins) was iodinated, 18 of the 19 proteins were still labelled. However five of these were labelled less strongly than before. When the intact 60S subunit was iodinated, 17 of the 25 proteins were still labelled, although six of these were labelled less strongly. 4. These results show that in rat liver most of the ribosomal proteins of both subunits are at least partially at the surface of the particles. They are also consistent with the idea that the proportion of the ribosomal proteins in the interior of the particle may be greater for the 60S subunit than for the 40S subunit.

Sign in / Sign up

Export Citation Format

Share Document