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Identification of protein kinase C and its potential substrate in Entamoeba histolytica
F De Meester1, D Mirelman, T Stolarsky
1MacAuthur Center for Molecular Biology of Parasitic Diseases, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
1. Protein kinase C (PKC) activity has been identified in various strains of the human parasite, Entamoeba histolytica. 2. An amoebic protein of mol. wt 78,000 was recognized by polyclonal antibodies raised against the 82,000 mol. wt rat brain protein kinase C. 3. A partially purified PKC preparation from E. histolytica phosphorylated histone I in the presence of calcium, phospholipids and diacylglycerol, and specifically bound tritiated phorbol ester at an apparent KD of 9 nM. 4. A relocalization of the amoebic PKC activity from the cytosol to the membrane fraction was observed when trophozoites were actively phagocytising bacteria. Under these conditions, a labelled phosphoprotein of mol. wt 68,000 was identified. 5. Similar to what was found during macrophage activation, a myristoylated mol. wt 68,000 protein was detected in amoebae grown in the absence of bacteria, but not in amoebae which were active in phagocytosis.
Insights
Protein kinase C (PKC) activity was found in Entamoeba histolytica. This enzyme
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Protein kinase C (PKC) plays crucial roles in cellular signaling pathways.
- Entamoeba histolytica is a significant human parasite responsible for amoebiasis.
- Understanding signaling mechanisms in E. histolytica is vital for developing therapeutic strategies.
Purpose of the Study:
- To investigate the presence and characteristics of Protein Kinase C (PKC) in Entamoeba histolytica.
- To explore the function and localization of amoebic PKC during phagocytosis.
Main Methods:
- Immunological detection using antibodies against rat brain PKC.
- Biochemical assays for kinase activity using histone I phosphorylation.
- Ligand binding assays with tritiated phorbol ester.
- Subcellular fractionation to determine PKC localization.
Main Results:
- Amoebic PKC (78,000 mol. wt) was recognized by antibodies against rat brain PKC.
- Partially purified E. histolytica PKC phosphorylated histone I and bound phorbol ester (KD = 9 nM).
- PKC translocated from cytosol to membrane during bacterial phagocytosis, coinciding with the appearance of a 68,000 mol. wt phosphoprotein.
Conclusions:
- Entamoeba histolytica possesses functional Protein Kinase C (PKC) activity.
- Amoebic PKC is involved in the cellular response to phagocytosis, with a relocalization and potential substrate phosphorylation observed.
- Further research into amoebic PKC could reveal novel drug targets for treating amoebiasis.