Related Experiment Videos
Magnesium-dependent stimulation of protein synthesis by the insulin mimic, pervanadate
D M Barnes1, D B Sykes, J J Smith
1Laboratory of Cellular and Molecular Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Abstract:
The insulin mimic, peroxide of vanadate (pervanadate), stimulated 35S-methionine incorporation into Xenopus oocyte protein in a Mg(2+)-dependent manner. Reducing the extracellular Mg2+ concentration from 1.0 to 0.1 mM decreased the pervanadate-stimulated component of incorporation by 35%; with 0.01 mM Mg2+ or lower, the pervanadate-stimulated component was abolished. In addition, reducing extracellular Mg2+ to 0.01 mM inhibited about 50% of the insulin-stimulated component of methionine incorporation. Mg2+ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn2+ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg2+. Reducing extracellular Ca2+ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 microliter droplet of buffer containing the Mg2+ indicator dye, mag-fura-2, and pervanadate, apparent droplet Mg2+ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg2+. In contrast, apparent extracellular Mg2+ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg2+, but not Ca2+, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg2+, and this change in membrane transport may be an early event in signalling the increase in translation.
Insights
Peroxide of vanadate (pervanadate), an insulin mimic, stimulates protein synthesis in Xenopus oocytes by increasing magnesium uptake. This process is dependent on extracellular magnesium ions, not calcium.
Area of Science:
- Cellular Biology
- Biochemistry
- Molecular Signaling
Background:
- Insulin and its mimics are known to regulate protein synthesis.
- The role of extracellular ions in mediating these signaling pathways is not fully understood.
Purpose of the Study:
- To investigate the role of extracellular magnesium (Mg2+) and calcium (Ca2+) in pervanadate- and insulin-stimulated protein synthesis in Xenopus oocytes.
- To determine if pervanadate or insulin induce changes in extracellular Mg2+ concentration.
Main Methods:
- Xenopus oocytes were used to study 35S-methionine incorporation into proteins.
- Extracellular Mg2+ and Ca2+ concentrations were manipulated.
- A Mg2+ indicator dye (mag-fura-2) was used to measure changes in extracellular Mg2+.
Main Results:
- Pervanadate-stimulated protein synthesis was dependent on extracellular Mg2+, with reduced Mg2+ significantly decreasing incorporation.
- Insulin-stimulated protein synthesis was partially dependent on extracellular Mg2+.
- Pervanadate and insulin induced a net uptake of extracellular Mg2+ into the oocytes.
- Extracellular Ca2+ did not affect protein synthesis stimulated by pervanadate or insulin.
Conclusions:
- Extracellular Mg2+, but not Ca2+, is crucial for pervanadate-stimulated protein synthesis in Xenopus oocytes.
- Insulin-stimulated protein synthesis also shows a partial dependence on extracellular Mg2+.
- Pervanadate-induced Mg2+ uptake may be an early signaling event in the stimulation of translation.