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CMP-N-acetylneuraminic acid: isolation from and penetration into mouse liver microsomes
Abstract:
We present three lines of evidence for the existence of an intramicrosomal pool of CMP-NeuNAc in liver slices incubated with 3H-NeuNAc: first, enrichment in the microsomal fraction over the cell homogenate of CMP-3H-NeuNAc relative to CMP-14C-NeuNAc, the latter added as marker for sugar nucleotide trapped between and adsorbed onto vesicles; second, removal from microsomes by isotonic sucrose washing of only half of the CMP-3H-NeuNAc synthesized in vivo under conditions which remove all the CMP-14C-NeuNAc; and third, complete loss of both sugar nucleotides upon treatment of microsomes by hypotonic shock or with low concentrations of detergents. Incubation of CMP-NeuNAc with microsomes in vitro showed that the intact sugar nucleotide was penetrating the vesicles. When the sugar nucleotide is labeled in the sugar moiety, microsomal vesicles accumulate soluble radiolabeled products. This accumulation was saturable and dependent on time, temperature and concentration of sugar nucleotide, and was also inhibited by substrate analogues and by pretreatment of microsomes with proteases. Together, these results provide strong evidence for the existence of a specific transport system for CMP-NeuNAc in microsomes.
Insights
Researchers found evidence for an internal pool of CMP-NeuNAc within liver microsomes. This suggests a specific transport system facilitates CMP-NeuNAc entry into these cellular vesicles.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Transport
Background:
- Microsomes are key cellular organelles involved in various metabolic processes.
- Cytidine monophosphate N-acetylneuraminic acid (CMP-NeuNAc) is a crucial precursor for sialic acid biosynthesis.
- Understanding the compartmentalization and transport of CMP-NeuNAc is vital for elucidating sialylation pathways.
Purpose of the Study:
- To investigate the existence and characteristics of an intramicrosomal pool of CMP-NeuNAc.
- To determine if a specific transport mechanism mediates CMP-NeuNAc uptake into liver microsomes.
Main Methods:
- Incubation of liver slices with radiolabeled NeuNAc (3H-NeuNAc).
- Fractionation of cell homogenates to isolate microsomes.
- Differential washing and hypotonic shock treatments to assess CMP-NeuNAc localization.
- In vitro incubation of isolated microsomes with CMP-NeuNAc and analysis of radiolabeled product accumulation.
Main Results:
- Enrichment of CMP-3H-NeuNAc in microsomes compared to cell homogenates.
- Partial removal of synthesized CMP-3H-NeuNAc by sucrose washing, unlike a marker compound (CMP-14C-NeuNAc).
- Evidence of intact CMP-NeuNAc penetration into vesicles and saturable, temperature-dependent accumulation of radiolabeled products, inhibited by analogues and proteases.
Conclusions:
- Strong evidence supports the presence of an intramicrosomal pool of CMP-NeuNAc.
- A specific transport system for CMP-NeuNAc into liver microsomes is indicated.
- These findings advance the understanding of sialic acid precursor transport and metabolism.
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