scholarly journals CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS

1970 ◽  
Vol 45 (1) ◽  
pp. 146-157 ◽  
Author(s):  
D. D. Sabatini ◽  
G. Blobel
Keyword(s):  
Amino Acid ◽  
Messenger Rna ◽  
Close Association ◽  
Membrane Integrity ◽  
Sucrose Density Gradient ◽  
Electron Microscopic ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Cell Fractions

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.

scholarly journals Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

1977 ◽  
Vol 74 (2) ◽  
pp. 414-427 ◽  
Author(s):  
J Kruppa ◽  
DD Sabatini
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Messenger Rna ◽  
High Salt ◽  
Salt Medium ◽  
Ribosomal Subunits ◽  
Microsomal Membranes ◽  
Low Ionic Strength

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.

Proteins synthesized by rabbit reticulocyte membrane-bound ribosomes

1982 ◽  
Vol 60 (5) ◽  
pp. 580-585 ◽  
Author(s):  
Réal Lemieux ◽  
Claude Godin
Keyword(s):  
Protein Synthesis ◽  
Ionic Strength ◽  
Messenger Rna ◽  
Rna Degradation ◽  
Main Protein ◽  
Membrane Bound ◽  
Phenol Treatment

Rabbit reticulocyte membrane-bound ribosomes liberated by deoxycholate treatment contain degraded forms of ribosomal and messenger RNA. This degradation occurs after the liberation of the ribosomes from the membranes by the detergent because intact ribosomal and messenger RNA can be extracted from washed membranes by phenol treatment. Increasing the ionic strength of the detergent buffer prevents this RNA degradation and allows the recovery of membrane-bound ribosomes capable of protein synthesis. Comparison of the proteins synthesized in vitro by the polyribosomes shows that the main protein produced by both free and membrane-bound ribosomes is globin. However, the two types of polyribosomes could be distinguished by the nonglobin proteins they produce.

scholarly journals CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS

1970 ◽  
Vol 45 (1) ◽  
pp. 130-145 ◽  
Author(s):  
G. Blobel ◽  
D. D. Sabatini
Keyword(s):  
Messenger Rna ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Subunit Structure ◽  
Amino Acid Residues ◽  
Nascent Polypeptide ◽  
Nascent Chain ◽  
Cell Fractions ◽  
Carboxy Terminal

Free ribosomes containing nascent polypeptide chains labeled in vitro were submitted to proteolysis at 0° by a mixture of trypsin and chymotrypsin. Sucrose gradient analysis showed that polysome patterns are retained even after 24 hr of proteolysis in the cold, while messenger RNA-free ribosomes (generated progressively during in vitro incorporation) are, within 2 hr, completely dissociated into subunits by trypsin. Although ribosomes and subunits are not extensively degraded into smaller fragments during low temperature proteolysis, changes in the acrylamide gel electrophoresis pattern showed that most ribosomal proteins are accessible to and are partially degraded by the proteases. Ribosome-bound nascent polypeptides are partially resistant to proteolysis at 0°, although they are totally digested at 37° or when the ribosomal subunit structure is disrupted by other means. Radioactivity incorporated into nascent chains during incubation times shorter than 3 min was mostly resistant to digestion at 0°. A larger fraction of the initial radioactivity became degraded in ribosomes which incorporated for longer times. In these ribosomes, the amount of radioactivity which was resistant to proteolysis was constant and independent of the initial value, which reflects the labeled length of the nascent chains. These results suggest that the growing end of the nascent polypeptide is resistant to digestion and is protected from proteolytic attack by the ribosomal structure. A pulse and chase experiment confirmed this suggestion, showing that the protected segment is located at the carboxy-terminal end of the nascent chain. The protected segment was contained in the large ribosomal subunit and had a length of ∼39 amino acid residues, as estimated by chromatography on Sephadex G-50.

scholarly journals Identification of storage-protein messenger RNA of the fleshfly Sarcophaga peregrina

1982 ◽  
Vol 203 (3) ◽  
pp. 571-575 ◽  
Author(s):  
T Tahara ◽  
Y Maeda ◽  
A Kuroiwa ◽  
K Ueno ◽  
M Obinata ◽  
...  
Keyword(s):  
Density Gradient ◽  
Storage Protein ◽  
Messenger Rna ◽  
Fat Body ◽  
Density Gradient Centrifugation ◽  
Signal Sequence ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation

Storage-protein mRNA was found to be abundant in poly(A)-containing RNA extracted from the fat-body of third-instar larvae of Sarcophaga peregrina (fleshfly). This RNA sedimented at the position of 19S on sucrose-density-gradient centrifugation and the product of its translation in vitro was 75K protein (protein of mol.wt. 75 000), which was precipitated specifically with antibody against storage protein. This product was suggested to contain a signal sequence that is missing in mature storage protein. The poly(A)-containing RNA was also found to contain much of another mRNA coding for 25K protein (protein of mol.wt. 25 000), but the function of this protein is unknown.

scholarly journals Human carboxypeptidase E. Isolation and characterization of the cDNA, sequence conservation, expression and processing in vitro

1990 ◽  
Vol 267 (2) ◽  
pp. 517-525 ◽  
Author(s):  
E Manser ◽  
D Fernandez ◽  
L Loo ◽  
P Y Goh ◽  
C Monfries ◽  
...  
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Sequence Similarity ◽  
Coding Region ◽  
Sequence Comparisons ◽  
Brain Membrane ◽  
Carboxypeptidase E ◽  
Isolation And Characterization ◽  
Microsomal Membranes

Carboxypeptidase E (CPE), which cleaves C-terminal amino acid residues and is involved in neuropeptide processing, is itself subject to intracellular processing. Human CPE cDNA was isolated and sequence comparisons were made with those of a previously isolated brain cDNA (M1622) encoding rat CPE and of other human carboxypeptidases (M and N). Human (2.5 kb) and rat (2.1 kb) CPE cDNAs approximated to the size of their respective mRNAs; additional sequences were located in putative 5′ and 3′ untranslated regions of human CPE mRNA. There is 79% sequence similarity between human and rat CPE cDNAs, with greater similarity (89%) over the coding region and short sections of the non-coding sequence. The predicted 476-amino acid-residue sequences of human and rat preproCPEs are highly conserved (96% identity), with lower degree of similarity of the N-terminal signal peptide (76%). Human CPE showed 51% and 43% sequence similarity to human CPN and CPM respectively, with discrete regions of divergence dispersed between the highly conserved mechanistically implicated regions. Antiserum generated from a fusion protein, synthesized in Escherichia coli from constructs of the human cDNA, recognized an approx. 50 kDa membrane protein and a smaller soluble protein in rat and human brain preparations, corresponding to the two forms of native CPE. Human CPE mRNA transcripts directed the synthesis in reticulocyte lysate of a 54 kDa translation product, which in the presence of dog pancreas microsomal membranes was co-translationally processed with cleavage, insertion into membranes and glycosylation. Three processed forms were generated, the largest (56 kDa) and smallest (52 kDa) being equally glycosylated. The membrane association of the processed translation products and of native brain membrane CPE, detected immunologically, was resistant to moderate alkali but not pH 11.5 extraction. These results are consistent with secondary-structure predictions that CPE is a peripheral membrane protein. The dissimilar regions of human carboxypeptidases may provide information on sequences responsible for their different cellular disposition.

scholarly journals Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses

1982 ◽  
Vol 92 (2) ◽  
pp. 269-276 ◽  
Author(s):  
AH Bunt ◽  
JC Saari
Keyword(s):  
Intravitreal Injection ◽  
Time Course ◽  
Apparent Molecular Weight ◽  
Sucrose Density Gradient ◽  
Neutral Sugar ◽  
Electron Microscopic ◽  
Outer Segments ◽  
Photoreceptor Membranes ◽  
Electron Microscopic Radioautography

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.

scholarly journals The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

2008 ◽  
Vol 19 (9) ◽  
pp. 3944-3955 ◽  
Author(s):  
Li Wang ◽  
Mary A. Bittner ◽  
Daniel Axelrod ◽  
Ronald W. Holz
Keyword(s):  
Plasma Membrane ◽  
Chromaffin Cells ◽  
Close Association ◽  
Living Cells ◽  
Full Length ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Functional Implications ◽  
Membrane Bound

We investigated the functional and structural implications of SNAP25 having two SNARE motifs (SN1 and SN2). A membrane-bound, intramolecular FRET probe was constructed to report on the folding of N-terminal SN1 and C-terminal SN2 in living cells. Membrane-bound constructs containing either or both SNARE motifs were also singly labeled with donor or acceptor fluorophores. Interaction of probes with other SNAREs was monitored by the formation of SDS-resistant complexes and by changes in FRET measured in vitro using spectroscopy and in the plasma membrane of living cells using TIRF microscopy. The probes formed the predicted SDS-resistant SNARE complexes. FRET measurements revealed that syntaxin induced a close association of the N-termini of SN1 and SN2. This association required that the SNARE motifs reside in the same molecule. Unexpectedly, the syntaxin-induced FRET was prevented by VAMP. Both full-length SNAP25 constructs and the combination of its separated, membrane-bound constituent chains supported secretion in permeabilized chromaffin cells that had been allowed to rundown. However, only full-length SNAP25 constructs enabled robust secretion from intact cells or permeabilized cells before rundown. The experiments suggest that the bidentate structure permits specific conformations in complexes with syntaxin and VAMP and facilitates the function of SN1 and SN2 in exocytosis.

scholarly journals The effect of whole-body X-irradiation of guinea pigs on liver ribosomes

1968 ◽  
Vol 109 (4) ◽  
pp. 495-505 ◽  
Author(s):  
E. J. Hidvégi ◽  
J. Holland ◽  
Elisabeth Bölöni ◽  
P. Lónai ◽  
F. Antoni ◽  
...  
Keyword(s):  
Amino Acid ◽  
Guinea Pigs ◽  
Sucrose Density Gradient ◽  
Whole Body ◽  
Radiation Doses ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
X Irradiation ◽  
Liver Ribosomes

1. The size distribution of aggregates of liver ribosomes and their protein-synthesizing ability in vitro were studied shortly after X-irradiation of guinea pigs. 2. Sucrose-density-gradient analysis of the mitochondrial supernatant after treatment with deoxycholate revealed a gradual increase in the number of polysomes, reaching a maximum between 9 and 15 hr. after irradiation. At that period the amount of ribonucleoprotein particles reached a level 25–30% above the control. This finding was confirmed by analytical-ultracentrifugal analysis and electron microscopy. Experiments were conducted to exclude the possibility that the enrichment of polysomes in the irradiated animals had occurred during the isolation procedure. 3. The protein-synthesizing ability of total ribosomal particles was measured in vitro. This showed an increase in amino acid incorporation parallel to the progressive enrichment of polysomes. At radiation doses of up to 1000r. the protein-synthesizing capacity was dependent on the radiation dose: the higher the dose the higher the amino acid incorporation, reaching 40–60% above the control at the period of maximal polysome enrichment. Amino acid incorporation remained at this level after radiation doses of between 1000 and 3000r. The enhanced protein-synthesizing activity was due solely to the increase in the proportion of polysomes, since irradiation was without effect on the activity of single ribosomes. 4. The results of the experiments are discussed in the light of our knowledge of the effect of radiation on protein synthesis.

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