Mechanism of inhibition of polypeptide chain initiation in calcium-depleted Ehrlich ascites tumor cells.
Protein synthesis in Ehrlich ascites tumor cells is inhibited when cellular calcium is depleted by the addition of EGTA to the growth medium. This inhibition is at the level of polypeptide chain initiation as evidenced by a disaggregation of polyribosomes accompanied by a significant elevation in 80-S monomers. To identify direct effects of calcium on the protein synthesis apparatus we have developed a calcium-dependent, cell-free protein-synthesizing system from the Ehrlich cells by using 1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a recently developed chelator with a high (greater than 10(5)) selectivity for calcium (pKa = 6.97) over magnesium (pKa = 1.77). BAPTA inhibits protein synthesis by 70% at 1 mM and 90% at 2 mM. This effect was reversed by calcium but not by other cations tested. The levels of 43-S complexes (i.e., 40-S subunits containing bound methionyl-tRNAf.eIF-2.GTP) were significantly lower in the calcium-deprived incubations, indicating either inhibition of the rate of formation or decreased stability of 43-S complexes. Analysis of 43-S complexes on CsCl gradients showed that in BAPTA-treated lysates, 40-S subunits containing eIF-3, completely disappeared and the residual methionyl-tRNA-containing complexes were bound to 40-S subunits lacking eIF-3. Our results demonstrate a direct involvement of Ca2+ in protein synthesis and we have localized the effect of calcium deprivation to decreased binding of eIF-2 and eIF-3 to 40-S subunits.
Kumar
R V
RV
Cancer Center, University of Rochester, New York 14642.
Wolfman
A
A
Panniers
R
R
Henshaw
E C
EC
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002286:Carcinoma, Ehrlich Tumor; D002474:Cell-Free System; D002614:Chelating Agents; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D066298:In Vitro Techniques; D051379:Mice; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects; D010448:Peptide Initiation Factors / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D011506:Proteins / Q000502:physiology; D012270:Ribosomes / Q000378:metabolism
D002614:Chelating Agents; D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D011506:Proteins; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium