scholarly journals Membrane-bound ribosomes of myeloma cells. V. Subcellular distribution of immunoglobulin mRNA molecules.

1981 ◽  
Vol 88 (1) ◽  
pp. 37-41 ◽  
Author(s):  
B Mechler
Keyword(s):  
Endoplasmic Reticulum ◽  
Subcellular Distribution ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Class ◽  
Template Activity ◽  
Myeloma Cells ◽  
Reticulocyte Lysate ◽  
Membrane Bound ◽  
Mouse Myeloma

The subcellular distribution of the most abundant mRNA sequences, particularly those of the immunoglobulin heavy (Ig H) and light (IG L) chain mRNA sequences, of MOPC 21 (P3K) mouse myeloma cells has been examined by translating the mRNA of various subcellular fractions in a messenger-dependent reticulocyte lysate (MDL) and by identifying Ig products with the use of a specific antiserum. Analyses of the distribution of the mRNA template activity and the translation products by SDS polyacrylamide gel electrophoresis reveal that approximately 85% of the mRNA present in the free ribosomal fraction is incorporated into polysomes and that the remainder is present as mRNP particles. On the endoplasmic reticulum (ER) the mRNA is found entirely in polysomes. In general, the size class of free (F) and membrane-bound (MB) polysomes corresponds to the size of their translation products. Thus, mRNAs coding Ig H (5.0 x 10(5) daltons in size) and Ig L (2.5 x 10(5) daltons in size) are incorporated into polysomes formed of 12 and 6 ribosomes, respectively. About 10% of the Ig mRNAs are not bound to membranes. A third of these are associated with mRNPs and the remainder incorporated into F polysomes of the same size as the Ig-synthesizing MB polysomes.

scholarly journals Membrane-bound ribosomes of myeloma cells. IV. mRNA complexity of free and membrane-bound polysomes.

1981 ◽  
Vol 88 (1) ◽  
pp. 29-36 ◽  
Author(s):  
B Mechler ◽  
T H Rabbitts
Keyword(s):  
Cross Hybridization ◽  
Myeloma Cells ◽  
Mrna Species ◽  
Sequence Complexity ◽  
Polysomal Mrna ◽  
Reticulocyte Lysate ◽  
Cdna Hybridization ◽  
Membrane Bound ◽  
Mouse Myeloma

We have analyzed the sequence complexity, frequency distribution, and template activity of free (F) and membrane-bound (MB) polysomal mRNA populations of MOPC 21 (P3K) mouse myeloma cells. Using the technique of mRNA-cDNA hybridization, we find that F poly(A)+ RNA, which represent 60% of total polysomal mRNA, consists of approximately 8,000 different mRNA sequences distributed in three abundance classes, while MB poly(A)+ RNA (20% of total polysomal mRNA) includes only 230 mRNA species and almost completely lacks very infrequent mRNA species. Cross-hybridization indicates that MB mRNA sequences are also present in F mRNA, but in reduced concentrations. Translation of F and MB RNA fractions in a messenger-dependent reticulocyte lysate indicates that essentially all MB RNA contains poly(A), whereas 25% of F mRNA lacks poly(A). Furthermore, the use of a cDNA highly specific for the immunoglobulin light (Ig L) chain mRNA allows the determination of the subcellular content of this message. Ig L mRNA, representing approximately 5% of total polysomal poly(A)+ RNA, is one of the most abundant MB mRNAs. 90% of Ig L mRNA is found in MB polysomes and 10% in F polysomes.

scholarly journals Transfer of carbohydrates on to nascent glycoprotein of vesicular stomatitis virus

1979 ◽  
Vol 181 (2) ◽  
pp. 295-300 ◽  
Author(s):  
J Kruppa
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Viral Envelope ◽  
Pulse Labelling ◽  
Viral Envelope Protein ◽  
Viral Glycoprotein ◽  
Membrane Bound ◽  
Vesicular Stomatitis

I studied the glycosylation in vivo of a viral envelope protein, the glycoprotein of vesicular stomatitis virus (VSV), by pulse labelling of virus-infected HeLa cells with 3H-labelled monosaccharides (mannose, glucosamine). Radioactivity was incorporated into the fraction of membrane-bound polyribosomes, although metabolic conversion of [3H]-mannose into amino acids was negligible. Dissociation of bound polyribosomes revealed that the radioactively co-purified with the peptidyl-tRNA. The nascent peptides were released by alkaline hydrolysis, immunoprecipitated and analysed by polyacrylamide-gel electrophoresis. It is apparent from the size distribution of the [3H]mannose-labelled nascent chains that attachment of carbohydrate starts when approximately half of the amino acid sequence of the viral glycoprotein has been synthesized.

scholarly journals Membrane proteins of chromaffin granules. Dopamine β-hydroxylase, a major constituent

1972 ◽  
Vol 129 (1) ◽  
pp. 187-195 ◽  
Author(s):  
Heide Hörtnagl ◽  
H. Winkler ◽  
H. Lochs
Keyword(s):  
Membrane Proteins ◽  
Gel Electrophoresis ◽  
Cetylpyridinium Chloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Major Constituent ◽  
Chromaffin Granules ◽  
Lower Mobility ◽  
Membrane Bound ◽  
Immunological Cross Reaction

1. Soluble lysates and membranes were prepared from chromaffin granules isolated from bovine adrenal medulla. The detergent N-cetylpyridinium chloride was used for solubilizing the membrane proteins, including the membrane-bound dopamine (2,4-dihydroxyphenethylamine) β-hydroxylase. The solubilized proteins were fractionated by Sephadex chromatography in the presence of N-cetylpyridinium chloride. The major component of the membrane proteins, i.e. chromomembrin A, was identified as the enzyme dopamine β-hydroxylase. 2. The addition of N-cetylpyridinium chloride to the soluble lysate caused precipitation of up to 96% of the proteins, but only a small proportion of the dopamine β-hydroxylase activity was precipitated. The only protein demonstrable in the supernatant by polyacrylamide-gel electrophoresis was the protein that has a lower mobility than chromogranin A in disc gel electrophoresis. This component has been identified previously as dopamine β-hydroxylase. Thus, this method provides an extremely simple isolation procedure for dopamine β-hydroxylase. 3. A comparison of the membrane-bound and soluble dopamine β-hydroxylases revealed the identity of these two preparations. Both were activated by N-cetylpyridinium chloride, they migrated identically in polyacrylamide-gel electrophoresis, their amino acid composition was very similar and an immunological cross-reaction could be demonstrated.

scholarly journals Biosynthesis of rat liver pI-6.1 esterase, a carboxylesterase of the cisternal space of the endoplasmic reticulum

1987 ◽  
Vol 248 (2) ◽  
pp. 545-550 ◽  
Author(s):  
M Robbi ◽  
H Beaufay
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Antiserum ◽  
Secretory Proteins ◽  
Structural Element ◽  
Oligosaccharide Moiety ◽  
Reticulocyte Lysate ◽  
Dog Pancreas ◽  
Processed Products

Biosynthesis of the rat liver microsomal esterase with pI 6.1 was investigated in cell-free systems and in cultured hepatocytes, by using a rabbit antiserum. Protein synthesis directed by total rat liver RNA in wheatgerm extract or reticulocyte lysate generated a single immunoprecipitable product, also found with the RNA extracted from bound, but not from free, polysomes. When dog pancreas microsomal fractions were included, reticulocyte lysates gave two processed products, a prominent one slightly larger, and another slightly smaller, than the precursor, both resistant to exogenous proteinases and, hence, segregated within vesicles. The processing was co-translational; it consisted of the removal of a peptide fragment and, for the large component, the addition of a single oligosaccharide chain. Indeed, this component bound to concanavalin A-Sepharose and gave the small one (approximately 2000 Mr loss) by cleavage with endo-beta-N-acetylglucosaminidase H (endo-H). A single labelled peptide was precipitated from hepatocytes incubated with [35S]methionine. Its apparent Mr was decreased by approximately 2000 after treatment with endo-H; it was then identical with that of an unglycosylated form produced in hepatocytes poisoned with tunicamycin. Even in that case, immunoreactive peptides were not detected in the culture medium. Whether synthesized in reticulocyte lysate or in hepatocytes, the glycosylated forms migrated in SDS/polyacrylamide-gel electrophoresis as the purified enzyme labelled with [3H]di-isopropyl fluorophosphate. Thus, although pI-6.1 esterase is not secreted, its biosynthesis is, as yet, indistinguishable from that of secretory proteins. Its oligosaccharide moiety is apparently not the structural element that retains it in the endoplasmic reticulum.

scholarly journals Role of membrane-bound and free polyribosomes in the synthesis of cytochrome c in rat liver

1974 ◽  
Vol 140 (2) ◽  
pp. 157-167 ◽  
Author(s):  
Néstor F. González-Cadavid ◽  
Carmen Sáez De Córdova
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Cell Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Membrane Bound

The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [14C]leucine and δ-amino[14C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.

scholarly journals Secretion, cleavage and binding of complement component C3 by the human monocytic cell line U937

1989 ◽  
Vol 261 (2) ◽  
pp. 407-413 ◽  
Author(s):  
C M Maison ◽  
C L Villiers ◽  
M G Colomb
Keyword(s):  
Gel Electrophoresis ◽  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complement Component ◽  
Polyacrylamide Gel ◽  
Classical Pathway ◽  
U937 Cells ◽  
Complement Component C3 ◽  
Thioester Bond ◽  
Membrane Bound

Secretion of complement component C3 by U937 cells was studied. Preliminary evidence for a cell-associated proteolytic activity specific for C3 is given, as well as for a covalent-like binding of C3 fragments to the cell membranes. Secretion of C3, in the presence of 10 ng of phorbol 12-myristate 13-acetate/ml, is 120-140 ng/10(6) cells per 24 h on the third day after addition of the activator. As shown by SDS/polyacrylamide-gel electrophoresis, the intracellular pro-C3 (200 kDa) and the extracellular secreted C3 (alpha-chain 110 kDa and beta-chain 75 kDa) are identical with the forms of C3 previously characterized from human serum. Incubation of U937 cells in the presence of exogenous radiolabelled C3 shows that membrane-bound proteinase(s), not related to the classical-pathway or the alternative-pathway C3 convertases, is (are) able to cleave C3; this cleavage leads to the binding of the resulting C3 fragments to the cell membrane through reaction of membrane acceptors with the carbonyl group of C3 revealed after disruption of the intramolecular thioester bond. The proteolysis appears to be fairly specific to C3, as C4, which also possesses an intramolecular thioester bond, is not cleaved and does not bind to the cells. p-Nitrophenyl p'-guanidinobenzoate (1 mM) and di-isopropyl phosphorofluoridate (2 mM) are potent inhibitors of the proteolysis, whereas soya-bean trypsin inhibitor (1 mM), leupeptin (0.1 mg/ml) and 1,10-phenanthroline (1 mM) were ineffective. Immunological characterization of the cell-bound C3 fragments with monoclonal antibodies shows an evolution of the proteolysis of the fragments from iC3b to C3dg epitopes. Extraction of membrane-bound fragments by detergent, followed by SDS/polyacrylamide-gel electrophoresis, shows two fragments, of 43 kDa and 46 kDa, with C3dg-like characteristics.

scholarly journals Membrane-bound ribosomes of myeloma cells. VI. Initiation of immunoglobulin mRNA translation occurs on free ribosomes.

1981 ◽  
Vol 88 (1) ◽  
pp. 42-50 ◽  
Author(s):  
B Mechler
Keyword(s):  
Polypeptide Chain ◽  
Mrna Translation ◽  
Normal Growth ◽  
Kinetic Studies ◽  
Myeloma Cells ◽  
Nascent Polypeptide ◽  
Membrane Attachment ◽  
Membrane Bound ◽  
Terminal Amino ◽  
Mouse Myeloma

Immunoglobulin heavy (Ig H) and light (Ig L) chain mRNA molecules have been released from the endoplasmic reticulum (ER) membranes as free (F) mRNP particles when MOPC 21 (P3K) mouse myeloma cells are exposed to a hypertonic initiation block (HIB). The subsequent fate of these mRNA sequences has been examined when the cells are returned to normal growth medium. Upon return to isotonicity, all previously translated mRNA molecules reassociate with ribosomes and form functional polysomes. Ig H mRNA is found incorporated first into F polysomes and then into membrane-bound (MB) polysomes. Kinetic studies indicate that the time of passage of Ig H mRNA in F polysomes is approximately 30 s, during which a nascent polypeptide chain of approximately 80 amino acids would have been completed. When the rate of polypeptide elongation is depressed with emetine during the recovery from HIB, both Ig H and L mRNA molecules accumulate in small F polysomes. These results indicate that the formation of Ig-synthesizing polysomes proceeds in the sequence: mRNA leads to F polysomes leads to MB polysomes. With the additional observation that during HIB recovery puromycin completely prevents the reassociation of Ig mRNA with the ER, these findings support a model of MB polysome formation in which the specificity of membrane attachment is determined by the nature of the N-terminal amino acid sequence of the nascent polypeptide chain.

An Improved Method for the Preparation of the Four Ribonucleic Acids of Cowpea Chlorotic Mottle Virus

1981 ◽  
Vol 36 (1-2) ◽  
pp. 157-163
Author(s):  
P. E. Dickerson ◽  
M. C. Malomi ◽  
J. R. O. Dawson ◽  
B. A. M. Morris-Krsinich ◽  
A. R. Trim
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mottle Virus ◽  
Cowpea Chlorotic Mottle Virus ◽  
Ribonucleic Acids ◽  
Highly Active ◽  
Chlorotic Mottle Virus ◽  
Chlorotic Mottle ◽  
Reticulocyte Lysate ◽  
Whole Plants

Abstract The four components of the genome of cowpea chlorotic mottle virus have been prepared in a highly active and highly purified state by a method based on their resolution by polyacrylamide gel electrophoresis. Activity and purity have been confirmed by gel electrophoresis under denaturing and non-denaturing conditions, infectivity tests on whole plants, translation in an mRNA dependent rabbit reticulocyte lysate and fingerprinting of T1 ribonuclease digests after labelling with 32P at the 5′ ends of the oligonucleotide products. The quality of the RNAs obtained was superior to those previously obtained by zonal centrifugation but only comparatively small batches of whole RNA can be handled with ease.

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