Evidence that intracellular magnesium is present in cells at a regulatory concentration for protein synthesis.

Abstract

When extracellular magnesium is reduced by a factor of 50 (from 1.0 to 0.02 mM), the total intracellular magnesium of a spontaneously transformed clone of 3T3 cells decreases by 30-50%. Protein synthesis rates in these cells were measured as the intracellular magnesium decreased. Protein synthesis rates and magnesium content were found to decrease in parallel with each other. At 3 hr, a decrease to 84% ofcontrol values of magnesium content was accompanied by a decrease to 85% of control values of leucine incorporation rates. A larger inhibition had occurred by 12 hr, when the magnesium had decreased to 67% and leucine incorporation rates had decreased to 57%. When magnesium was restored to maiumdeprived cells, both magnesinm content and leucine incorporation increased about 2-fold by 1 hr. In the experiments reported here, initial small changes in magnesium content are associated with changes in protein synthesis rates. This strongly suggests that magnesium is present at a regulatory rather than excess concentration for protein synthesis. The results are consistent with a role for intracellular magnedum in the regulation of protein synthesis and support the hypothesis that magnesium has a central role in the regulation of metabolism and growth.Protein synthesis rates in in vitro systems are known to be affected by magnesium (1). However, there has been little work devoted to characterizing the effects of magnesium on protein synthesis in intact cells. Perhaps the main reason is that magnesium is difficult to vary within cells-increases or decreases by a factor of 10 of extracellular magnesium have no effect on the total intracellular magnesium contents of the cell (2).In an earlier paper, intracellular magnesium of 3T3 cells was varied by lowering the extracellular calcium concentration to 0.02 mM and then varying extracellular magnesium from 1 to 20 mM and to even higher inhibitory magnesium concentrations (3 ity, 51.6 Ci/mmol; 1 Ci = 37 GBq) was used at an activity of 2 ,uCi/ml in 2 ml of medium for the 60-mm dishes.[3H]Thymidine (original specific activity, 20 Ci/mmol) was used at an activity of 1 puCi/ml in 2 ml of medium. The length of the labeling period is given in the figure legends. All labeling media were made using medium of the same composition as the growth medium of the cells at the time of the label. At the end of the labeling period, cultures were washed three times with ice-cold Tris-buffered saline, extracted with ice-cold 5% trichloroacetic acid for 10 min, washed twice with trichloroacetic acid, and then solubilized in 2 ml of 0.2 M NaOH. Samples of the NaOH were taken for scintillation counting and protein determination. Duplicate protein determinations were run.Ion Measurements. The method of Sanui and Rubin (8) was used. Briefly, cultures were first washed three times with C02-free 0.25 M sucrose (pH 7). This was followed by one wash with carbonated 0.25 M sucrose (pH 4), whose purpose was to displace externally bound cations by protons. The cells were scraped into distilled deioni...

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