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The submicrosomal distribution of dolichyl phosphate and dolichyl phosphate phosphatase in rat liver
Abstract:
Rat liver microsomes were isolated and fractionated into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER), and the purity of these preparations was determined. The dolichyl phosphate (Dol-P) content of whole microsomes and of each of the submicrosomal fractions was estimated using high pressure liquid chromatography. Dol-P accounts for 4 and 40% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in whole liver and in purified microsomes, respectively. Concentrations equal to 58, 77, and 108 ng of Dol-P/mg of protein were found in Golgi, SER, and RER, respectively. These values represent 3, 36, and 54% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in each of these same fractions, respectively. Increases in the Dol-P content of rat liver were observed as early as 12 h after turpentine-induced inflammation and increased 2-fold over 36 h. In this system, Dol-P accounts for no more than 50% of the sum of all phosphorylated and pyrophosphorylated dolichol intermediates present. The specific activity for dolichyl phosphate phosphatase was highest by more than a factor of 2 in Golgi membrane. Specific activities obtained for SER and RER were 42 and 11% of those present in Golgi. The major requirement for Dol-P is thought to be for the saccharide and oligosaccharide transferase reactions which are presumed to take place in RER. The discovery of significant quantities of Dol-P in Golgi and SER is consistent with a possible role of Dol-P in the transport of sugars required for glycoprotein synthesis and processing from a cytosolic to luminal orientation.
Insights
Dolichyl phosphate (Dol-P) is crucial for glycoprotein synthesis, with higher concentrations found in rat liver endoplasmic reticulum and Golgi. Its levels increase during inflammation, suggesting a role in cellular stress responses.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Dolichyl phosphate (Dol-P) is a key lipid-linked oligosaccharide precursor essential for N-linked glycoprotein synthesis.
- Understanding Dol-P distribution and metabolism within cellular compartments is vital for elucidating its role in protein glycosylation.
Purpose of the Study:
- To quantify dolichyl phosphate (Dol-P) content in different rat liver subcellular fractions (Golgi, SER, RER).
- To investigate the changes in Dol-P levels during turpentine-induced inflammation.
- To determine the distribution and specific activity of dolichyl phosphate phosphatase (Dol-Pase) in these fractions.
Main Methods:
- Isolation and fractionation of rat liver microsomes into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER).
- Quantification of dolichyl phosphate (Dol-P) using high-performance liquid chromatography (HPLC).
- Measurement of dolichyl phosphate phosphatase (Dol-Pase) specific activity in subcellular fractions.
Main Results:
- Dolichyl phosphate (Dol-P) concentrations were highest in RER (108 ng/mg protein), followed by SER (77 ng/mg) and Golgi (58 ng/mg).
- Dol-P constituted a significant portion of total dolichol derivatives in SER (36%) and RER (54%), and also in Golgi (3%).
- Dol-P levels in rat liver increased by twofold within 36 hours of turpentine-induced inflammation.
- Dolichyl phosphate phosphatase (Dol-Pase) activity was highest in Golgi membranes, significantly exceeding that in SER and RER.
Conclusions:
- The distribution of Dol-P in Golgi and SER suggests a role beyond the RER-localized glycoprotein synthesis, potentially in sugar transport for glycoprotein processing.
- Increased Dol-P levels during inflammation indicate its involvement in cellular stress responses.
- Differential Dol-Pase activity across fractions highlights compartment-specific regulation of dolichol metabolism.