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Characterization of protein kinase activity associated with rat liver polysomal messenger ribonucleoprotein particles
Abstract:
Poly(adenylic acid)-containing rat liver polysomal messenger ribonucleoprotein particles (pmRNP) were isolated and found to contain protein kinase activity. The association of the enzyme(s) with the particles was confirmed by experiments showing that the protein kinase activity comigrated with the pmRNP on metrizamide gradients and bound to oligo-(dT)-cellulose columns only under conditions where the pmRNP bound. The following properties were determined for the pmRNP-associated kinase(s). Casein and phosvitin were preferred substrates over histone and protamine. The optimal MgCl2 and KCl concentrations were found to be 12.5 and 50 mM, respectively. MnCl2 and CaCl2 could not replace MgCl2 and were inhibitory at low concentrations. The optimum pH range was 7.7--9.0, and the enzyme activity was cAMP independent. A molecular weight of 55 000--60 000 was determined for the kinase(s) by sucrose gradient analysis. The enzyme(s) was capable of phosphorylating proteins endogenous to the pmRNP. Membrane-bound pmRNP contained much less kinase activity than free pmRNP while pmRNP from hepatoma 7777 contained an elevated level of the enzyme(s). The relationship between the protein kinase activity and one of the pmRNP proteins of molecular weight 66 000 is discussed.
Insights
Rat liver polysomal messenger ribonucleoprotein particles (pmRNP) contain protein kinase activity. This enzyme phosphorylates endogenous pmRNP proteins and shows altered levels in specific conditions.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Messenger ribonucleoprotein particles (mRNPs) are crucial for gene expression.
- Protein kinase activity associated with mRNPs suggests regulatory roles in translation.
Purpose of the Study:
- To isolate and characterize protein kinase activity associated with poly(adenylic acid)-containing rat liver polysomal mRNPs (pmRNPs).
- To investigate the properties and potential substrates of the pmRNP-associated kinase(s).
Main Methods:
- Isolation of pmRNPs using metrizamide gradients and oligo-(dT)-cellulose chromatography.
- Enzyme assays to determine substrate preference, optimal conditions (MgCl2, KCl, pH), and molecular weight.
- Analysis of kinase activity in membrane-bound vs. free pmRNPs and in hepatoma cells.
Main Results:
- Protein kinase activity was confirmed to be associated with pmRNPs.
- The kinase preferred casein and phosvitin as substrates, with optimal MgCl2 at 12.5 mM and KCl at 50 mM.
- Optimal pH was 7.7-9.0, activity was cAMP-independent, and molecular weight was 55,000-60,000.
- The kinase phosphorylated endogenous pmRNP proteins, with higher activity in free pmRNPs and elevated levels in hepatoma 7777 pmRNPs.
Conclusions:
- Rat liver pmRNPs possess associated protein kinase activity with specific biochemical properties.
- This kinase may play a role in regulating gene expression by phosphorylating endogenous pmRNP proteins.
- Alterations in kinase activity in different cellular compartments and in hepatoma suggest its involvement in disease states.