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Ultrastructure and polysome content of the microsomal subfractions of mouse plasmacytoma cells
Abstract:
The endoplasmic reticulum (ER) of MPC-11 cells released as vesicles upon cell disruption by nitrogen cavitation was separated from the bulk of mitochondria, lysosomes and plasma membranes by a low speed centrifugation. The ER membranes were fractionated on discontinuous sucrose gradients into heavy rough (HR), light rough (LR) and smooth (S) membranes. The morphology of subcellular fractions was studied by electron microscopy and the ER membranes were shown to be virtually free of contaminating organelles. The S fraction was easily distinguishable because of the lack or ribosomes but there were no apparent morphological differences between the HR and LR fractions. Of total activity in the microsomal subfractions, 70% of the UDPase and 67% of the 5'-nucleotidase activity was associated with the S fraction. Polysomes were present in the HR, LR and nuclear-associated ER fractions but not in the S fraction. The HR and LR fractions did not appear to be contaminated to any great extent with free polysomes. RNA/protein and RNA/phospholipid ratios of the HR fraction were higher than those of the LR fraction, indicating a greater density of ribosomes in the former fraction. These ratios were much lower in the S fraction reflecting the low ribosome content.
Insights
Researchers isolated endoplasmic reticulum (ER) membranes from MPC-11 cells, separating rough and smooth fractions. The smooth ER fraction showed high UDPase and 5'-nucleotidase activity, indicating distinct functional roles.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The endoplasmic reticulum (ER) is a crucial organelle involved in protein and lipid synthesis.
- Fractionating ER membranes is essential for understanding the specific functions of different ER subdomains.
Purpose of the Study:
- To isolate and characterize different fractions of endoplasmic reticulum (ER) membranes from MPC-11 cells.
- To investigate the distribution of enzymatic activities and ribosomes within these ER fractions.
Main Methods:
- MPC-11 cells were disrupted using nitrogen cavitation.
- Endoplasmic reticulum (ER) vesicles were separated by low-speed centrifugation.
- ER membranes were fractionated into heavy rough (HR), light rough (LR), and smooth (S) fractions using discontinuous sucrose gradients.
- Morphological analysis was performed using electron microscopy.
- Enzymatic activities (UDPhase, 5 -nucleotidase) and RNA/protein/phospholipid ratios were measured.
Main Results:
- ER membranes were successfully isolated with minimal contamination from other organelles.
- The smooth (S) ER fraction exhibited high UDPase (70%) and 5 -nucleotidase (67%) activity.
- Rough ER fractions (HR and LR) contained polysomes, with higher ribosome density in the HR fraction indicated by RNA/protein and RNA/phospholipid ratios.
- No significant morphological differences were observed between HR and LR fractions.
Conclusions:
- The study successfully fractionated ER membranes, distinguishing smooth ER from rough ER.
- Smooth ER is enriched in specific enzymatic activities like UDPase and 5 -nucleotidase.
- Ribosome distribution differs between heavy rough and light rough ER fractions, suggesting functional specialization.