Proteins from different classes of liver nuclei in normal and thioacetamide-treated rats

1. In normal rats the amounts of each of the main types of nuclear protein, i.e. soluble proteins, histones, non-histone chromosomal proteins and residual proteins, vary within the different classes of rat liver nuclei fractionated by zonal centrifugation. 2. Heterogeneity is observed in the non-histone chromosomal proteins prepared from different classes of liver nuclei. These differences were observed by analysis of the proteins both by sodium dodecyl sulphate–polyacrylamide-gel electrophoresis and electrofocusing electrophoresis. They are most evident between the non-histone chromosomal proteins obtained from stromal and parenchymal nuclei. However, some differences are also found for the parenchymal nuclei, between the diploid parenchymal and the tetraploid parenchymal, and between them and the nuclei involved in the synthesis of DNA respectively. 3. Drastic alterations in the nuclear proteins are found after the administration of thioacetamide. The changes observed are complex and not uniform. They vary with the age of the animal and the type of nucleus. In general an increase in the soluble proteins and non-histone chromosomal proteins and a decrease in the residual proteins is observed. There is a decrease in the specific radioactivity of soluble and residual proteins. 4. Electrophoretic analysis of the non-histone chromosomal proteins showed that specific changes occurred after administration of thioacetamide, which are different in adolescent and young adult rats.

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The characterization of the non-histone chromosomal proteins of the main classes of nuclei from rat brain fractionated by zonal centrifugation

Biochemical Journal ◽

10.1042/bj1770331 ◽

1979 ◽

Vol 177 (1) ◽

pp. 331-346 ◽

Cited By ~ 6

Author(s):

Sonia G. Tsitilou ◽

David Cox ◽

Anthony P. Mathias ◽

David Ridge

Keyword(s):

Rat Brain ◽

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Brain Cells ◽

Adult Rats ◽

Chromosomal Proteins ◽

Neuronal Nuclei ◽

Infant Rats

1. Non-histone chromosomal proteins were isolated from the cell nuclei of whole rat brain and nuclei from different types of brain cells. 2. Brain nuclei were fractionated by zonal centrifugation into five zones deriving from five main categories of brain cells. These are the neuronals, astrocytes I, astrocytes II, oligodendrocytes I and oligodendrocytes II. 3. The non-histone chromosomal proteins were analysed by (a) sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, (b) electrofocusing electrophoresis and (c) two-dimensional electrophoresis. The results of this analysis showed a limited specific pattern of non-histone chromosomal proteins from the different classes of nuclei. Differences were found to exist between the proteins from neuronal and glial nuclei. In particular one polypeptide band with mol.wt. 10000 and pI8.5 was found to be present in the non-histone protein fractions of neuronal nuclei, and absent from the corresponding fractions of nearly all the other classes of nuclei. 4. Two other classes of nuclear proteins, buffered-saline-soluble and 0.35m-NaCl-soluble, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis along with the non-histone chromosomal. The similarities and differences among these groups of proteins are discussed. 5. The patterns of non-histone chromosomal proteins during development were investigated by studying them in two age groups of animals: in infant rats (10 days old) and adult rats. The polypeptide that was found to be specific for the proteins of neuronal nuclei of adult rats is present in all the classes of nuclei of infant rats.

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Labelling of Proteins by Isolated Rat Liver Nuclei

Canadian Journal of Biochemistry ◽

10.1139/o72-026 ◽

1972 ◽

Vol 50 (2) ◽

pp. 190-199 ◽

Cited By ~ 14

Author(s):

K. M. Anderson ◽

M. Slavik ◽

O. P. Elebute

Keyword(s):

Protein Synthesis ◽

Rat Liver ◽

Nuclear Protein ◽

Sucrose Solution ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Rat Liver Nuclei ◽

Teleological Argument ◽

Liver Nuclei ◽

Triton X

Rat liver nuclei, isolated in hypertonic sucrose solution and washed with Triton X-100, incorporate radioactive amino acids into hot trichloroacetic acid insoluble materials.Optical and biochemical evidence of nuclear purity is presented. The temperature-dependent incorporation continued for 20–30 min, and was proportional to the concentrations of both nuclear protein between 0.5–1.5 mg/ml, and radioactive amino acid. The radioactive product was degraded by pronase, and a number of inhibitors reduced incorporation, but only if present at [Formula: see text]. Proteins extracted from labelled nuclei and microsomes and examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis at pH 7.2 exhibited different patterns of radioactivity. This provides further support for the concept of protein synthesis intrinsic to rat liver nuclei.A teleological argument for the function of nuclear protein synthesis is discussed.

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Soleus fiber force and maximal shortening velocity after non-weight bearing with intermittent activity

Journal of Applied Physiology ◽

10.1152/jappl.1996.80.3.981 ◽

1996 ◽

Vol 80 (3) ◽

pp. 981-987 ◽

Cited By ~ 34

Author(s):

J. J. Widrick ◽

J. J. Bangart ◽

M. Karhanek ◽

R. H. Fitts

Keyword(s):

Isometric Force ◽

Weight Bearing ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cross Sectional Area ◽

Type I ◽

Sectional Area ◽

Shortening Velocity ◽

Adult Rats ◽

Cross Sectional

This study examined the effectiveness of intermittent weight bearing (IWB) as a countermeasure to non-weight-bearing (NWB)-induced alterations in soleus type I fiber force (in mN), tension (Po; force per fiber cross-sectional area in kN/m-2), and maximal unloaded shortening velocity (Vo, in fiber lengths/s). Adult rats were assigned to one of the following groups: normal weight bearing (WB), 14 days of hindlimb NWB (NWB group), and 14 days of hindlimb NWB with IWB treatments (IWB group). The IWB treatment consisted of four 10-min periods of standing WB each day. Single, chemically permeabilized soleus fiber segments were mounted between a force transducer and position motor and were studied at maximal Ca2+ activation, after which type I fiber myosin heavy-chain composition was confirmed by sodium dodecyl sufate-polyacrylamide gel electrophoresis. NWB resulted in a loss in relative soleus mass (-45%), with type I fibers displaying reductions in diameter (-28%) and peak isometric force (-55%) and an increase in Vo (+33%). In addition, NWB induced a 16% reduction in type I fiber Po, a 41% reduction in type I fiber peak elastic modulus [Eo, defined as (delta force/delta length) x (fiber length/fiber cross-sectional area] and a significant increase in the Po/Eo ratio. In contrast to NWB, IWB reduced the loss of relative soleus mass (by 22%) and attenuated alterations in type I fiber diameter (by 36%), peak force (by 29%), and Vo (by 48%) but had no significant effect on Po, Eo, or Po/Eo. These results indicate that a modest restoration of WB activity during 14 days of NWB is sufficient to attenuate type I fiber atrophy and to partially restore type I peak isometric force and Vo to WB levels. However, the NWB-induced reductions in Po and Eo, which we hypothesize to be due to a decline in the number and stiffness of cross bridges, respectively, are considerably less responsive to this countermeasure treatment.

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An analysis of discoidin I binding sites in Dictyostelium discoideum (NC4)

Biochemical Journal ◽

10.1042/bj2000083 ◽

1981 ◽

Vol 200 (1) ◽

pp. 83-91 ◽

Cited By ~ 7

Author(s):

I C Madley ◽

B D Hames

Keyword(s):

Binding Sites ◽

Bacterial Contamination ◽

Wild Type Strain ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Specific Radioactivity ◽

Wild Type ◽

Stage Of Development ◽

Affinity Constants ◽

Radioactivity Measurements

Vegetative wild-type (strain NC4) D. discoideum cells and cells at the 10h stage of development (aggregation) were harvested in the presence of 0.5 M-galactose to remove any endogenous discoidin I already bound to the cell surface, and fixed with glutaraldehyde. Affinity-purified 125I-labelled discoidin I bound to these fixed cells in a specific manner, greater than or equal to 95% of binding being inhibited by 0.5 M-galactose. Binding of 125I-labelled discoidin I was essentially complete in 90 min at 22 degrees C. Based on specific radioactivity measurements, vegetative (0h) D. discoideum (NC4) cells bind approx. 8.4 x 10(5) discoidin I tetramers/cell and aggregated (10h) cells bind 5.1 x 10(5) discoidin I tetramers/cell, each exhibiting apparent positive co-operativity of binding with highest limiting affinity constants (Ka) of approx. 1 x 10(7) and 2 x 10(7) M-1, respectively. Klebsiella aerogenes, the food source used for growth of D. discoideum NC4 amoebae, also binds 125I-labelled discoidin I and this is greater than 99% inhibited by 0.5 M-galactose. However, at the levels of bacterial contamination present, greater than 97% of 125I-labelled discoidin I binding to D. discoideum cell preparations was to the cells themselves. Confirmation of the number of discoidin I tetramers bound per D. discoideum cell was obtained by elution of bound 125I-labelled discoidin I followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and then quantification by scanning of stained discoidin I bands.

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Electrophoretic Analysis of Polypeptides of Plasma Membranes from Bovine Pituitary Gland

Canadian Journal of Biochemistry ◽

10.1139/o74-089 ◽

1974 ◽

Vol 52 (7) ◽

pp. 620-630

Author(s):

André Lemay ◽

Fernand Labrie

Keyword(s):

Plasma Membranes ◽

Polyacrylamide Gel Electrophoresis ◽

Broad Band ◽

Sodium Dodecyl ◽

Electrophoretic Pattern ◽

Apparent Molecular Weight ◽

Electrophoretic Analysis ◽

Polyacrylamide Gel ◽

Protein Components ◽

Membrane Components

Purified plasma membranes from bovine hypophyseal tissue have been fractionated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under various conditions of pH and acrylamide concentrations. The best separation of protein components is achieved at a concentration of 7.5% acrylamide and at pH 7.1. Under these conditions, the electrophoretic pattern consistently shows 36 protein bands ranging in molecular weights from 250 000 to 15 000. Only one broad band, having an apparent molecular weight of 150 000, stains for glycoproteins by the period acid – Schiff technique. After electrophoresis on a two-dimensional polyacrylamide gel system using disc gels containing urea and Triton X-100 in the first dimension and SDS in the second dimension, approximately 45 different protein components can be identified. Less than 12% of the membrane proteins are solubilized by washing the membranes with 1 M KCl or NH4Cl. Denaturating agents like urea and lithium 3,4-diiodosalycilate solubilize 55–60% of membrane components. Adenohypophyseal plasma membranes show an eleetrophoretic pattern completely different from that obtained with membranes isolated from the intermediate or posterior pituitary lobes.

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Purification and properties of a new glutathione-dependent thiol:disulphide oxidoreductase from rat liver

Biochemical Journal ◽

10.1042/bj2070133 ◽

1982 ◽

Vol 207 (1) ◽

pp. 133-138 ◽

Cited By ~ 20

Author(s):

M G Battelli ◽

E Lorenzoni

Keyword(s):

Rat Liver ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Xanthine Dehydrogenase ◽

Enzymic Activity ◽

Oxidized Glutathione ◽

Adult Rats ◽

Purification And Properties ◽

Rat Liver Cytosol

A new GSSG-dependent thiol:disulphide oxidoreductase was extensively purified from rat liver cytosol. The enzymic protein shows molecular weight 40 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and 43 000 as determined by thin-layer gel filtration on Bio-Gel P-100. The pI is 8.1. This enzyme converts rat liver xanthine dehydrogenase into an oxidase, in the presence of oxidized glutathione. Other disulphide compounds are either inactive or far less active than oxidized glutathione in the enzymic oxidation of rat liver xanthine dehydrogenase. The enzyme also catalyses the reduction of the disulphide bond of ricin and acts as a thioltransferase and as a GSH:insulin transhydrogenase. The enzymic activity was measured in various organs of newborn and adult rats.

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Isolation and Analysis of Non-Histone Chromatin Proteins from Rat-Liver Nuclei by Three Different Methods

Canadian Journal of Biochemistry ◽

10.1139/o74-159 ◽

1974 ◽

Vol 52 (12) ◽

pp. 1143-1153 ◽

Cited By ~ 31

Author(s):

D. Suria ◽

C. C. Liew

Keyword(s):

Rat Liver ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Soluble Proteins ◽

Polyacrylamide Gel ◽

Pulse Labelling ◽

Rat Liver Nuclei ◽

Liver Nuclei ◽

Chromatin Proteins

Non-histone chromatin proteins were isolated from rat-liver nuclei by three different methods, and defined as (I) phenol-soluble proteins, (II) SDS-soluble proteins and (III) proteins not adsorbed by cation-exchange chromatography. About 62–70% of chromatin proteins were recovered from the total nuclear proteins. The yield of non-histone chromatin proteins varied from 17 to 26% of chromatin proteins, depending on the method used. The amino-acid composition of these proteins showed that they are acidic in nature. Their phosphorus content was found to be 0.9, 1.1, and 1.4%, respectively, according to method I, II, or III. In-vivo pulse-labelling experiments indicated that chromatin proteins were highly labelled with 3H-acetate and 32P-phosphoric acid. In particular, the specific activities of 32P incorporation were higher in all non-histone chromatin proteins isolated as compared with histones. One-dimensional SDS–polyacrylamide gel electrophoresis showed that at least 26 similar fractions can be detected in the samples prepared by these three methods.The similarity of some of the proteins obtained from methods I and III was further confirmed by fractionation of the non-histone chromatin proteins in an isoelectro-focusing system followed by a second-dimensional SDS–polyacrylamide gel electrophoresis. It was found that more than 100 components could be identified. However, some minor variations of the non-histone chromatin proteins were detected by this system. The differences in proteins isolated by these methods are mainly quantitative rather than qualitative. The methods examined are not specific for the fractionation of a certain class of non-histone chromatin proteins.

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Characterization of human platelet glycoprotein antigens giving rise to individual immunoprecipitates in crossed-immunoelectrophoresis

Blood ◽

10.1182/blood.v58.6.1190.1190 ◽

1981 ◽

Vol 58 (6) ◽

pp. 1190-1197 ◽

Cited By ~ 43

Author(s):

TJ Kunicki ◽

AT Nurden ◽

D Pidard ◽

NR Russell ◽

JP Caen

Keyword(s):

Sodium Dodecyl ◽

Electrophoretic Analysis ◽

Human Platelets ◽

Soluble Proteins ◽

Platelet Glycoprotein ◽

Membrane Glycoprotein ◽

Membrane Glycoproteins ◽

Reference Pattern ◽

Crossed Immunoelectrophoresis ◽

Normal Individuals

Abstract Washed human platelets were labeled with 125I by the lactoperoxidase- catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.

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Synthesis and turnover of plasma-membrane proteins and glycoproteins in a neuroblastoma cell line

Biochemical Journal ◽

10.1042/bj1540057 ◽

1976 ◽

Vol 154 (1) ◽

pp. 57-64 ◽

Cited By ~ 20

Author(s):

R A Mathews ◽

T C Johnson ◽

J E Hudson

Keyword(s):

Steady State ◽

Membrane Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Surface Membrane ◽

Neuroblastoma Cell ◽

Sodium Dodecyl ◽

Neuroblastoma Cells ◽

Specific Radioactivity ◽

Neuroblastoma Cell Line ◽

Total Membrane

A kinetic analysis of the appearance of 14C-labelled proteins in the surface membranes isolated from exponentially growing neuroblastoma cells (N2a) showed that the total membrane proteins reached a steady-state specific radioactivity in 18-20 h. However, examination of individual protein bands resolved by sodium dodecyl sulphate-urea-polyacrylamide-gel electrophoresis illustrated that differences in the kinetics of specific surface-membrane proteins could be detected. Although most of the protein bands reached a steady-state specific radioactivity at a time similar to that for total membrane proteins, at least two bands (mol. wt. 180000 and 130000) attained the steady-state within 8-10 h. It was shown by the use of dual-labelling techniques that these two protein bands turned over in the surface membranes of neuroblastoma N2a cells at least 180 and 150% faster than the total membrane protein. These two proteins were glycosylated and located on the outer surface of the cells, since they were labelled with radioactive carbohydrates and readily removed by treatment of the intact neuroblastoma cell with proteinases.

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Quantitative aspects of mucus glycoprotein biosynthesis in rat gastric mucosa

Biochemical Journal ◽

10.1042/bj2280227 ◽

1985 ◽

Vol 228 (1) ◽

pp. 227-232 ◽

Cited By ~ 10

Author(s):

T Jentjens ◽

G J Strous

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Electrophoretic Analysis ◽

Rat Stomach ◽

Free System ◽

Mucus Glycoprotein ◽

Wide Range ◽

Rat Gastric Mucosa ◽

Polypeptide Backbone ◽

Mucus Glycoproteins

The synthesis of the polypeptide backbone of mucus glycoproteins in rat stomach was studied. CsCl centrifugation of the homogenate of [3H]serine pulse-chase labelled stomach or mucosal scrapings showed that [3H]serine was mainly incorporated into molecules having a density identical to that of proteins and that only 8-12% was incorporated into macromolecules with the density of mucus glycoproteins. [3H]-Galactose, however, was almost exclusively incorporated into macromolecules with a density identical to that of mucus glycoproteins. Electrophoretic analysis of the CsCl fraction containing the mucus glycoprotein revealed that 78% of the [3H]serine-labelled macromolecules had an electrophoretic behaviour identical to that of mucus glycoproteins. Thus, only a small portion (about 6-10%) of incorporated [3H]serine was present in the backbone of the mucus glycoprotein. Translation in a wheat germ cell-free system of total RNA derived from both whole stomach and superficial mucosal scrapings, using either [35S]methionine or [35S]cysteine as radioactive amino acid, yielded a wide range of proteins. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, one major translation product of whole stomach RNA had an apparent Mr (43000) identical to that of rat pepsinogen. As this polypeptide could not be found amongst the translation products of RNA from scrapings it probably was pepsinogen. The present data provide strong evidence that the backbone polypeptide of mucus glycoproteins only accounts for a small part of the proteins synthesized by mucus-producing cells.

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