scholarly journals Immunoglobin M biosynthesis. Intracellular accumulation of 7s subunits

1969 ◽  
Vol 115 (2) ◽  
pp. 163-169 ◽  
Author(s):  
R. M. E. Parkhouse ◽  
Brigitte A. Askonas
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Antiserum ◽  
Weight Ratio ◽  
Sucrose Density Gradient ◽  
Immunoglobulin M ◽  
Light Chains ◽  
Sucrose Density Gradient Centrifugation ◽  
System Cell

Immunoglobulin M biosynthesis was studied with mouse plasma cell tumour MOPC 104E as a model system. Cell suspensions prepared from solid tumours were incubated in vitro with tritiated leucine; the radioactivity incorporated into intracellular and secreted proteins was analysed by polyacrylamide-gel electrophoresis, sucrose-density-gradient centrifugation and precipitation with rabbit antiserum specific for the macroglobulin. The tumour was found to secrete immunoglobulin M and light chains in a 1:2 weight ratio, with lag periods of 20–30min. Within the cells there was a 7s component precipitable with specific antiserum to the macroglobulin that was shown to consist of heavy and light chains. This 7s subunit of the macroglobulin appeared to accumulate in the intracellular environment, so that even after long periods of incubation (3hr.) no more than trace amounts of fully assembled 19s molecules could be detected in cell lysates. Polymerization of the subunits into the pentamer therefore appears to take place shortly before, or simultaneously with, secretion of the molecules.

scholarly journals Immunoglobulin M biosynthesis. Production of intermediates and excess of light chain in mouse myeloma MOPC 104E

1971 ◽  
Vol 123 (4) ◽  
pp. 635-641 ◽  
Author(s):  
R. M. E. Parkhouse
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Immunoglobulin M ◽  
Sucrose Density Gradient Centrifugation ◽  
Chain Synthesis

Immunoglobulin M (IgM) biosynthesis was studied with mouse plasma-cell tumour MOPC 104E as a model system. Cell suspensions prepared from solid tumours were incubated in vitro with [3H]leucine; the radioactivity incorporated into intracellular and secreted proteins was analysed by sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The tumour secretes IgM and light chains. ‘Pulse–chase’ experiments indicated average secretion times of 1.5h for light chain and 2.5h for IgM. The order of disulphide-bond assembly within the cell was shown to be heavy chain+light chain → heavy chain–light chain intermediate → IgMs. The 7S subunit (IgMs) was polymerized into IgM just before or at the time of secretion. Measurements of heavy-chain/light-chain radioactivity ratios in intracellular HL and IgMs and secreted IgM demonstrated the existence of a light-chain pool participating in IgM biosynthesis. The size of the light-chain pool, together with analysis of clones isolated in vivo, suggested that the tumour contains cells in which light-chain synthesis is in excess of heavy-chain production.

scholarly journals Assembly of immunoglobulin M. Blocked thiol groups of intracellular 7S subunits

1971 ◽  
Vol 123 (4) ◽  
pp. 629-634 ◽  
Author(s):  
Brigitte A. Askonas ◽  
R. M. E. Parkhouse
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Reducing Agent ◽  
Immunoglobulin M ◽  
Cysteine Residues ◽  
Sucrose Density Gradient Centrifugation ◽  
Spontaneous Polymerization ◽  
Low Protein ◽  
Mouse Myeloma

We have shown previously that immunoglobulin M (IgM) is present within IgM-forming cells mainly in its 7S subunit form (IgMs), whereas only fully assembled IgM pentamers are secreted. There is no spontaneous polymerization of intracellular IgMs in cell lysates, suggesting that the 7S subunits had blocked cysteine residues. This suggestion was explored and confirmed in the present paper. Radioactive IgM (secreted) and IgMs (intracellular) were prepared by sucrose-density-gradient centrifugation after incubation of cells of the IgM-producing mouse myeloma MOPC 104E with [3H]leucine. We investigated the susceptibility to reduction of fully assembled mouse IgM and its reconstitution from subunits by analysis by polyacrylamide-gel electrophoresis under dissociating conditions. With increasing concentrations of dithioerythritol, interchain disulphide bonds were cleaved in the following order: inter-IgMs subunit, intra-IgMs subunit H-H, intra-IgMs subunit H-L. Removal of the reducing agent from IgM-reduction mixtures by filtration through Sephadex G-25 caused partial reconstitution of IgM at low protein concentrations (5–100μg/ml) and total reconstitution at higher protein concentrations (300μg/ml or more). Isolated radioactive intracellular IgMs showed no tendency to polymerize unless first treated with a reducing agent; under optimum conditions removal of the reducing agent caused 70% of the subunits to be assembled into IgM. Similar assembly occurred when IgMs was isolated from cells that had been lysed in the presence of an irreversible alkylating reagent (iodoacetamide). The intracellular IgMs cysteine residues responsible for inter-IgMs linkage therefore appear to be reversibly blocked within the cells. Assembly into IgM is thus controlled by removal of this block during secretion.

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

scholarly journals Thermolability of 28S ribosomal ribonucleic acid from the liver of Crotalus durissus terrificus (Ophidia, Reptilia)

1973 ◽  
Vol 135 (1) ◽  
pp. 73-79 ◽  
Author(s):  
J. F. Giorgini ◽  
F. L. De Lucca
Keyword(s):  
Molecular Weight ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Dimethyl Sulphoxide ◽  
Low Molecular Weight ◽  
28S Rrna ◽  
Sucrose Density Gradient Centrifugation ◽  
Crotalus Durissus Terrificus ◽  
Crotalus Durissus

Instability of 28S rRNA of Crotalus durissus terrificus liver was observed during hotphenol extraction: purified 28S rRNA is converted into an 18S RNA component by heat treatment. It was also found that ‘6S’ and ‘8S’ low-molecular-weight RNA species were released during the thermal conversion. This conversion and the release of the low-molecular-weight species were also induced by 8m-urea and 80% (v/v) dimethyl sulphoxide at 0°C. Evidence is presented that this phenomenon is an irreversible process and results from the rupture of hydrogen bonds. The 18S RNA product was shown to be homogeneous by polyacrylamide-gel electrophoresis and by sucrose-density-gradient centrifugation. The base composition of the 18S RNA products obtained by heat, urea or dimethyl sulphoxide treatments was similar. The C+G content of the 18S RNA product was different from that of the native 18S rRNA, but similar to that of 28S rRNA.

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

scholarly journals Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA

2007 ◽  
Vol 405 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Jørgen de Jonge ◽  
Johanna M. Leenhouts ◽  
Marijke Holtrop ◽  
Pieter Schoen ◽  
Peter Scherrer ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Gradient Centrifugation ◽  
Cationic Lipid ◽  
Sucrose Density Gradient ◽  
Target Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Viral Membrane ◽  
Viral Envelopes

Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA–virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA–virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA–virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA–virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.

scholarly journals Isolation of 10-nm filaments from astrocytes in the mouse optic nerve.

1981 ◽  
Vol 88 (1) ◽  
pp. 245-250 ◽  
Author(s):  
S Tsukita ◽  
H Ishikawa ◽  
M Kurokawa
Keyword(s):  
Optic Nerve ◽  
Peptide Mapping ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Sucrose Density Gradient Centrifugation ◽  
Optic Nerves ◽  
10 Nm Filaments

Astroglial filaments approximately 10 nm in diameter were isolated from degenerated mouse optic nerves by Triton X-100 and DNase I treatments followed by sucrose density gradient centrifugation. 2-4 wk after bilateral enucleation, optic nerves contained virtually a single population of 10-nm filaments (astroglial filaments), free from neurofilaments. In negative-staining and thin-section electron microscopy, the isolated filaments were seen as nonbranching linear structures with smooth contour, and were morphologically identical to those in situ. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the isolated filaments to be composed of two major polypeptides with molecular weights of 45,000 and 55,000, present in an approximate molar ratio of 1:1. These findings, together with the results of one-dimensional peptide mapping and solubility study, indicate that the astroglial filaments in the mouse optic nerve are primarily composed of these two polypeptides.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

Synapsin-1 is found in a microtubule-associated complex of proteins isolated from bovine brain

1990 ◽  
Vol 68 (11) ◽  
pp. 1256-1261 ◽  
Author(s):  
Karen P. Farrell ◽  
Robert A. B. Keates
Keyword(s):  
Peptide Mapping ◽  
Gradient Centrifugation ◽  
Staining Intensity ◽  
Sucrose Density Gradient ◽  
Bovine Brain ◽  
Sucrose Density Gradient Centrifugation ◽  
Usual Procedure ◽  
Brain Spectrin ◽  
Synapsin 1

We have prepared microtubules from brain tissue by stabilizing the cellular microtubules in 6.7 M glycerol buffer, instead of the usual procedure which extracts the solubilized protein and then reassembles microtubules in vitro at some later time. There are substantial differences in the microtubule associated proteins obtained by the two methods, and brain spectrin is a major component of the stabilized microtubules. We have now modified the buffer used for the isolation of stabilized microtubules to minimize their tendency to aggregate. When the stabilized microtubules were further purified by sucrose density gradient centrifugation, we were able to distinguish previously unidentified polypeptides at 49, 74 (doublet), and 100 kilodaltons (doublet). These bands maintained staining intensity in the same proportion to tubulin as in the original homogenate, whereas background proteins were diminished in staining intensity. We now report the identification of the 74-kilodalton doublet polypeptides as synapsin-1 by peptide mapping. Synapsin-1 is a protein known to bind to brain spectrin and also to microtubules, and may thus serve as a linker between these cytoskeletal components.Key words: microtubule-associated protein, synapsin, spectrin, tubulin, cytoskeleton.

Sign in / Sign up

Export Citation Format

Share Document