Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP.
Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.
Dholakia
J N
JN
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216.
Xu
Z
Z
Hille
M B
MB
Wahba
A J
AJ
D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D015852:Eukaryotic Initiation Factor-2 / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006153:Guanosine Diphosphate / Q000378:metabolism*; D007700:Kinetics; D010063:Ovum / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012617:Sea Urchins / Q000196:embryology / Q000378:metabolism*
D015852:Eukaryotic Initiation Factor-2; D006153:Guanosine Diphosphate