Alain Viel

Size exclusion chromatography and universal calibration of gel columns

We have tested the proposal (M. Potschka (1987) Anal. Biochem. 162, 47-64) that the elution position of macromolecules by gel chromatography is better correlated with the viscosity-based Stokes radius (R eta) than with the Stokes radius (RS) calculated from the frictional coefficient. By the use of different gel matrices (agarose, Sephadex, TSK silica gel columns) we found that the elution of dextran fractions and reduced proteins, denatured with guanidinium hydrochloride, agreed with their R eta, using water-soluble, globular proteins for gel calibration. However, the elution of large sodium dodecyl sulfate-protein complexes and other elongated macromolecules (fibrinogen, tropomyosin) was systematically retarded while polyethylene glycol and polyethylene glycol detergents eluted earlier than water-soluble, globular protein as a function of R eta. The same was the case for bacteriorhodopsin, solubilized by C12E8 or Triton X-100. It is concluded that for steric reasons size exclusion chromatography is more sensitive than hydrodynamic measurements to the detailed conformation of macromolecules (rods and random coils) and that for this reason gels with inert pores cannot be universally calibrated for all kinds of macromolecules.

Purification of two thermostable components of messenger ribonucleoprotein particles (mRNPs) from Xenopus laevis oocytes, belonging to a novel class of RNA‐binding proteins

We have purified and partially sequenced two proteins from Xenopus laevis previtellogenic oocytes, belonging to messenger ribonucleoprotein particles (mRNPs). The purification procedure rests on the thermostability of these proteins, which remain soluble after heating the cell extracts at 80°C. The thermostable proteins can be identified with two of the most abundant components (mRNP3 and mRNP4) of the mRNPs, described by Darnbrough and Ford (1981) [Eur. J. Biochem. 118, 415–424]. mRNP3 and mRNP4 are homologous to each other, but to no other protein of known sequence. The abundance and semi‐periodic distribution of proline residues in mRNF3 and mRNF4 sequences suggest that these RNA‐binding proteins adopt an unusual type of conformation

Elongation factor 1α (EF-1α) is concentrated in the Balbiani body and accumulates coordinately with the ribosomes during oogenesis of Xenopus laevis☆

Abstract In Xenopus laevis oocytes two distinct systems catalyze the mRNA-dependent binding of aminoacyl tRNA to the A site of ribosomes. These systems are elongation factor 1α (EF-1α) and the 42S nucleoprotein particle. This particle is also implicated in the long-term storage of 5S RNA and aminoacyl tRNA during early oogenesis. We report here that the ribosomes and the storage particles are distributed uniformly in the cytoplasm of previtellogenic (stage I) oocytes. In contrast, EF-1α is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body. When the Balbiani body disperses in early vitellogenic oocytes (stage II), EF-1α becomes evenly distributed in the cytoplasm. The main phase of EF-1α accumulation follows the disappearance of the 42S particles (stage II), but coincides with the main phase of ribosome accumulation (stages III and IV).

Thesaurin a, the major protein of Xenopus laevis previtellogenic oocytes, present in the 42 S particles, is homologous to elongation factor EF-1α

We have purified in SDS X. laevis thesaurin a (M r 50 000) which is part of the 42 S storage particles. Its N‐terminal amino acid is blocked and several peptides obtained by V8 protease treatment were purified and sequenced. As expected from one of the functional roles of the 42 S particles (tRNA binding, protection against deacylation and exchange with the ribosome), the amino acid sequence of thesaurin a was found to be closely related to that of the elongation factor EF‐1α. We suggest that all three proteins involved in 5 S RNA and tRNA storage in previtellogenic oocytes, TFIIIA, thesaurin a and thesaurin b, have a dual function: storage and a role in transcription or in protein synthesis.

Ise EF−1α associated with tubulin in X.laevis oocytes

In Xenopus laevis oocytes EF−1α is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiany body. When the mitochondrial mass disperses in early vitellogenic oocytes EF−1α becomes evenely distributed in the cytoplasm. The local concentration of EF‐1α in oocytes probably results from the association of EF‐1α and EF‐1β with tubulin. Three observations are in favor of this interpretation. First, a large fraction of EF−1α in X.laevis oocytes occurs as a complex with EF−1βγ. Second, purified EF‐1γ has a strong affinity for tubulin. Third, tubulin is extremely concentrated in the mitochondrial mass and disperses exactly at the same stage of oogenesis as EF−1α. A similar association has been recently postulated between tubulin and an EF‐1α‐like protein located in the centrosphere of sea urchin eggs.