scholarly journals Two fractions of rough endoplasmic reticulum from rat liver. II. Cytoplasmic messenger RNA's which code for albumin and mitochondrial proteins are distributed differently between the two fractions.

1977 ◽  
Vol 72 (3) ◽  
pp. 726-743 ◽  
Author(s):  
G C Shore ◽  
J R Tata
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Polyacrylamide Gels ◽  
Free System ◽  
Wheat Embryo ◽  
Cell Free System ◽  
Polypeptide Synthesis ◽  
Membrane Bound ◽  
Albumin Mrna

Subcellular fractions were obtained from rat liver homogenates under conditions which prevented degradation of polysomes (pH 8.5 and high ionic strength). Rough endoplasmic reticulum (RER) was recovered in high yields from a low-speed nuclear pellet (rapidly sedimenting endoplasmic reticulum, RSER) and from a postmitochondrial supernate (rough microsomes). The polysomal RNA content of these two fractions was very similar. When polyA+-RNA's were translated inthe mRNA-dependent wheat embryo cell-free system, both fractions yielded polypeptide products which had similar electrophoretic patterns on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Activities of messenger RNA's which code for albumin and for polypeptides destined for transport to the inner membrane and matrix of mitochondria (i.e. 'mitoplasts') were assayed by translating in the more active rabbit reticulocyte cell-free system followed by immunoprecipitation of radioactive products and coelectrophoresis with immunoprecipitated marker proteins on SDS-polyacrylamide gels. These tests indicated that albumin mRNA is about equally distributed between the two fractions of RER, or slightly enriched in the RSER fraction when activity is expressed as a percentage of total polypeptide synthesis. Activities of cytoplasmic mRNA's which code for at least some mitoplast proteins could be detected in both fractions, but all were enriched in the rough microsome fraction, not the RSER (two- to threefold when corrected for differences in total polypeptide synthesis in the lysate). Comparisons of mRNA's from free vs. membrane-bound polysomes indicated that most of the albumin mRNA activity (86-91%) and mitoplast protein mRNA activities (75%) were present in the bound fraction. Assuming that RSER and rough microsomes do not derive exclusively from different cells types, the evidence suggests that, compared to albumin and most other membrane-bound mRNA's, cytoplasmic mRNA's coding for mitoplast proteins may be preferentially segregated or compartmentalized within the cell on the microsomal class of RER.

scholarly journals Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver

1991 ◽  
Vol 266 (7) ◽  
pp. 4322-4328 ◽  
Author(s):  
P Moreau ◽  
M Rodriguez ◽  
C Cassagne ◽  
D M Morré ◽  
D J Morré
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Electrophoretic protein blots as aids in choosing fixatives for immunocytochemistry.

1988 ◽  
Vol 36 (4) ◽  
pp. 441-446 ◽  
Author(s):  
J Paiement ◽  
L Roy
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Liver Protein ◽  
Polyacrylamide Gels ◽  
Immunogold Technique ◽  
Immunocytochemical Studies

We used electrophoretic protein blots prepared from polyacrylamide gels to test the effect of different fixatives on the antigenicity of rough endoplasmic reticulum (RER) peptides from rat liver. Protein blots were prepared by the procedure of Towbin et al. (Proc Natl Acad Sci USA 76:4350, 1979), treated with different fixatives, rinsed to inactivate non-specific reactive sites, and then reacted with rabbit polyclonal anti-rat liver RER antibodies, followed by peroxidase-conjugated anti-rabbit antibodies. On the basis of differences in immunostaining densities as determined by densitometry, we found that RER peptides displayed differential sensitivities to various fixatives. Anti-rat liver RER antibodies and the immunogold technique were applied to methacrylate sections of in vitro fixed rat liver rough microsomes. Specific labeling was observed over the microsomes and was shown by quantitation to vary in a similar manner to the immunostaining of specific peptides in protein blots following different fixations. We conclude that protein blots may serve as useful tools for screening the effects of different fixatives on cell antigenicity, and therefore may be helpful in immunocytochemical studies.

Effect of polyamines on polypeptide synthesis in rat liver cell-free system

1973 ◽  
Vol 299 (2) ◽  
pp. 325-330 ◽  
Author(s):  
Kazuei Igarashi ◽  
Kimiko Hikami ◽  
Kumiko Sugawara ◽  
Seiyu Hirose
Keyword(s):  
Rat Liver ◽  
Liver Cell ◽  
Free System ◽  
Cell Free System ◽  
Polypeptide Synthesis

scholarly journals Temperature- and acceptor-specificity of cell-free vesicular transfer from transitional endoplasmic reticulum to the cis Golgi apparatus

1992 ◽  
Vol 288 (3) ◽  
pp. 969-976 ◽  
Author(s):  
S Dunkle ◽  
T Reust ◽  
D D Nowack ◽  
L Waits ◽  
M Paulik ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Free System ◽  
Cell Free System ◽  
Transitional Endoplasmic Reticulum ◽  
Acceptor Specificity ◽  
Membrane Compartment ◽  
A Cell

The temperature dependence and specificity of transfer of membrane constituents from donor transitional endoplasmic reticulum to the cis Golgi apparatus were investigated using a cell-free system from rat liver. The radiolabelled transitional endoplasmic reticulum donors were prepared from slices of rat liver prelabelled with [14C]leucine. The acceptor Golgi apparatus elements were unlabelled and immobilized on nitrocellulose. When Golgi apparatus stacks were separated by preparative free-flow electrophoresis into subfractions enriched in cisternae derived from the cis, medial and trans portions of the stack respectively, efficient specific transfer was observed only to cis elements. Trans elements were devoid of specific acceptor capacity. Similarly, when transfer was determined as a function of temperature, a transition was observed in transfer activity between 12 degrees C and 18 degrees C similar to that seen in vivo for formation of the so-called 16 degrees C cis Golgi-located membrane compartment. Transfer at temperatures below 16 degrees C and transfer to trans Golgi apparatus compartments at temperatures either above or below 16 degrees C was similar and unspecific. The unspecific transfer at low temperature was pH independent, whereas specific transfer was greatest at the physiological pH of 7, and was reduced to 10% and 18% of that occurring at pH 8 and pH 5.5 respectively. These findings show that the cell-free system derived from rat liver exhibits a high degree of fidelity to transfer in vivo, an efficiency approaching that observed in vivo, and a nearly absolute acceptor specificity for cis Golgi apparatus. The acceptor-, temperature- and pH-specificity of the cell-free transfer, as well as the saturation kinetics exhibited with respect to acceptor Golgi apparatus, support the concept of transition-vesicle-specific docking sites of finite number associated with cis Golgi apparatus cisternae.

Isolation of globin mRNA from spleen of anemic rabbits and its translation in wheat embryo cell-free system

1978 ◽  
Vol 43 (4) ◽  
pp. 1184-1189
Author(s):  
Ota Fuchs ◽  
Jitka Borová ◽  
Přemysl Poňka ◽  
Jan Neuwirt
Keyword(s):  
Embryo Cell ◽  
Globin Mrna ◽  
Free System ◽  
Wheat Embryo ◽  
Cell Free System
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