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Microsomal components in relation to amino acid incorporation by preparations from the developing rat brain
Abstract:
1. After incorporation of [(14)C]valine in vitro, cerebral microsomes were separated into membrane-bound and free ribosomes by sucrose-density-gradient centrifugation. 2. In preparations from both 4-day-old and adult rats, free and bound ribosomes incorporated [(14)C]valine. Free ribosomes could be found as polysomes, which were highly active. 3. Microsomes labelled with [(14)C]valine in vitro were fractionated after deoxycholate treatment into a preliminary sediment, sedimented at 105000g (5min.), and ribonucleoprotein particles, sedimented at 150000g (70min.), to determine the role of membrane-bound ribosomes. In the adult the ribonucleoprotein particles retained most of the radioactivity, whereas in the young the preliminary sediment was as highly labelled as the ribonucleoprotein particles. 4. The labelled preliminary sediment from young preparations was both ribonuclease- and deoxycholate-resistant, and the nature of this material is discussed in terms of a possible structural component of microsomal membranes.
Insights
Free and membrane-bound ribosomes in rat brain microsomes incorporate radiolabeled valine. In young rats, a unique, resistant microsomal component binds this label, suggesting a structural role.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Ribosomes are crucial for protein synthesis, existing freely in the cytoplasm or bound to the endoplasmic reticulum.
- Membrane-bound ribosomes are involved in synthesizing proteins destined for secretion or insertion into membranes.
- Understanding the differential roles and properties of free versus membrane-bound ribosomes is key to comprehending cellular protein production.
Purpose of the Study:
- To investigate the incorporation of [(14)C]valine by free and membrane-bound ribosomes in rat cerebral microsomes.
- To characterize the distribution of radioactivity in different subcellular fractions, comparing young and adult rats.
- To elucidate the potential role of membrane-bound ribosomes and associated structures in protein synthesis.
Main Methods:
- In vitro incorporation of [(14)C]valine into rat cerebral microsomes.
- Separation of free and membrane-bound ribosomes using sucrose-density-gradient centrifugation.
- Fractionation of labeled microsomes with deoxycholate and differential ultracentrifugation (105,000g and 150,000g).
- Treatment with ribonuclease and deoxycholate to assess the nature of labeled components.
Main Results:
- Both free and membrane-bound ribosomes from young and adult rats incorporated [(14)C]valine.
- Free ribosomes were observed as highly active polysomes.
- In adult rats, radioactivity was predominantly in ribonucleoprotein particles after fractionation.
- In young rats, the preliminary sediment (105,000g) showed radioactivity comparable to ribonucleoprotein particles.
- The preliminary sediment from young rats was resistant to ribonuclease and deoxycholate.
Conclusions:
- Both free and membrane-bound ribosomes actively synthesize proteins in the rat brain.
- Age-dependent differences exist in the distribution of newly synthesized proteins associated with membrane-bound ribosomes.
- A ribonuclease- and deoxycholate-resistant component in young rat brain microsomes may represent a structural element of the microsomal membrane involved in protein synthesis or targeting.