Possibility of the transformation of eEF-2 (100 kDa) to eEF-2 (65 kDa) in the peptide elongation process in vitro.

Two active eEF-2 polypeptides of approximately 100 and 65 kDa were copurified from rat liver cells and separated. The fate of eEF-2 (100 kDa) during its binding to ribosomes and in the translocation step of the peptide elongation process was investigated. It was shown that eEF-2 (100 kDa) did not change its form during the process of binding to the ribosomes. In the postribosomal supernatant, obtained from the postincubation mixture of the elongation process, only eEF-2 (65 kDa) was found. These results suggest that the form of eEF-2 (100 kDa), when bound to the ribosome during the elongation process, is transformed to eEF-2 (65 kDa).

Effect of the components extracted from the needles of Taxus baccata on protein biosynthesis in a cell-free rat liver system.

Various species of Taxus contain taxanes that promote polymerization and stabilization of microtubules. They have been reported as antineoplastic compounds with highly effective chemiotherapeutic application. A decrease in incorporation of the radiolabelled precursors into DNA, RNA and proteins in vivo has been reported too. The preliminary results have shown that also the other compounds present in the aqueous extract from Taxus baccata needles, participate in the inhibition of the protein biosynthesis. The binding site of eEF-2 on the ribosome seems to be the target of this inhibition process.

Three novel type I collagen mutations in osteogenesis imperfecta type IV probands are associated with discrepancies between electrophoretic migration of osteoblast and fibroblast collagen.

In three cases of type IV osteogenesis imperfecta (OI), we identified unique point mutations in type I collagen alpha1(I) cDNA. In two cases, the appearance of dimers indicated the presence of cysteine substitutions in the alpha1(I) protein chain. Cyanogen bromide digestion localized these cross-links to CB8 and 3, respectively. In the third case, the overmodification pattern of the CNBr peptides was compatible with a substitution in the aa 123-402 region of either type I collagen chain. We identified a unique point mutation in each proband, which resulted in substitutions for glycine residues in a 300-aa region of the alpha1(I) helix, specifically, Gly to Ala at codon 220 (GGT-->GCT), Gly to Cys at codon 349 (GGT-->TGT) and Gly to Cys at codon 523 (GGT-->TGT). We compared each proband's fibroblast and osteoblast collagen directly, as well as with fibroblast and osteoblast controls. For all cases, the OI osteoblast collagen was more electrophoretically delayed than OI fibroblast collagen. In the patient with G349C, OI fibroblast and osteoblast collagen synthesized in the presence of alpha,alpha'-dipyridyl co-migrated on gels, demonstrating that the electrophoretic discrepancy resulted from differences in post-translational modification. Melting temperature curves for stability of the collagen helix yielded an identical Tm for control fibroblast and osteoblast collagen (41.2 degrees C). By contrast, for collagen with the gly349-->cys substitution, the Tm of the fibroblast collagen was 1 degree C lower than the Tm of the osteoblast collagen. These data indicate that the metabolism of mutant collagen might be cell-specific and has significant implications for understanding the phenotype/genotype correlations and the pathophysiology of OI.

Multiformity of elongation factor eEF-2 isolated from rat liver cells.

Two fractions of eEF-2 (M(r) approx. 100,000 and M(r) approx. 65,000) were isolated from post-ribosomal supernatant of the rat liver cells. Only eEF-2, with mol. weight of about 100,000 Da, can be phosphorylated, but only eEF-2, with mol. weight of about 65,000 Da, was isolated from the active polyribosomes. The existence of two eEF-2 forms with different properties in the rat liver cells is striking and uncovers new aspects for the cellular function of this protein.

Purification and characterization of the protein kinase eEF-2 isolated from rat liver cells.

The elongation factor 2 (eEF-2) protein kinase was isolated from rat liver cells, purified and partly characterized. It was found that the enzyme exists in an inactive form in the homogenate of rat liver. The active fraction of kinase eEF-2 was obtained after removal of the inhibitory substance by hydroxyapatite column chromatography. The purified enzyme is an electrophoretically homogeneous protein with relative molecular mass of approximately 90,000 and isoelectric point, pI = 5.9. The enzyme specifically phosphorylates the elongation factor eEF-2 in the presence of calmodulin and Ca2+.

Purification and properties of the heterogeneous subunits of elongation factor EF-1 from Guerin epithelioma cells.

Elongation factor EF-1 from Guerin epithelioma was separated into two subunit forms EF-1A and EF-1B by chromatography in the presence of 25% glycerol, successively on CM-Sephadex and DEAE-Sephadex. It was shown that EF-1A is a thermolabile, single polypeptide which catalyses the binding of aminoacyl-tRNA to ribosomes, similarly as eukaryotic EF-1 alpha or prokaryotic EF-Tu. EF-1B was characterized as a complex composed of at least two polypeptides. One of them is EF-1A, the other EF-1C, which stimulates EF-1A activity and protects this elongation factor from thermal inactivation.

The effect of phosphorylation of the EF-2 isolated from rat liver cells on protein biosynthesis in vitro.

The activity of EF-2 was distinctly decreased after phosphorylation catalysed by a partly purified calmodulin and Ca2+ dependent protein kinase III. At the same time 32P from [gamma-32P]ATP was incorporated into EF-2 molecule. After dephosphorylation of EF-2 catalysed by alkaline phosphatase the activity of this factor was increased. This suggests that the phosphorylation-dephosphorylation of EF-2 is the regulatory process in the elongation step of the translation. Preliminary purification of the kinase III from rat liver resulted in 8-fold purified enzyme with a recovery of 60%.