Altered sensitivity of eukaryotic elongation factor 2 for trypsin after phosphorylation and ribosomal binding

We have used variations in the trypsin sensitivity of eukaryotic protein synthesis elongation factor 2 (eEF-2) to probe for structural alterations induced by phosphorylation, ribosomal binding, or guanosine nucleotides. We could not detect any nucleotide-related effect on the tryptic cleavage rate of Arg66. However, eEF-2 was protected from trypsin after ribosomal binding. Also, phosphorylation of eEF-2 led to a protection of Arg66. This indicates that phosphorylation leads to a structural rearrangement that could explain the reduced affinity of the phosphorylated factor for ribosomes (Carlberg, U., Nilsson, A., and Nyg?rd, O. (1990) Eur. J. Biochem. 191, 639-645). Cleavage of Arg66 led to a complete loss of the ability of the factor to be phosphorylated. Furthermore, ribosome-bound eEF-2 was found to be inaccessible for phosphorylation. Based on these findings and previously published data, we suggest that the region around the sites of phosphorylation and trypsin cleavage is vitally important for the factor function and ribosomal binding.     

The influence of neuropeptide Y and norepinephrine on ovulation in the rat ovary.

Neuropeptide Y (NPY) was measured in tissue extracts from ovaries of rats treated with pregnant mare serum gonadotropin (PMSG). The extracted NPY-immunoreactive material was identical to synthetic human NPY with regard to size and hydrophobicity as evaluated by gel filtration and high performance liquid chromatography. The concentration of NPY was related to the estrous cycle and a maximum was observed in relation to the endogenous luteinizing hormone (LH) peak. NPY immunoreactivity was demonstrated by immunohistochemistry to be localized within nerve fibers supplying blood vessels and follicles. The increase in the NPY content could not be related to accumulation around specific ovarian structures. Employing an in vitro set-up, NPY (10(-7) M) was unable to induce ovulation and did not increase the ovulation rate in LH-stimulated ovaries. The combination of NPY (10(-7) M) and NE (10(-7) M) did not significantly increase the number of ovulations compared to that induced by NE (10(-7) M) alone. In conclusion, NPY content in the ovary is related to the estrous cycle, but NPY does not seem to have any direct effect on the ovulatory process.     

Genetic evidence that the putative receptor binding domain of apolipoprotein B (residues 3130 to 3630) is not the only region of the protein involved in interaction with the low density lipoprotein receptor

We have searched for sequence differences in the region of the apolipoprotein B (apo B) gene encoding amino acids 3130-3630 in eight individuals with reduced affinity of low density lipoprotein (LDL) for the normal LDL-receptor. All individuals were hypercholesterolaemic and were selected either on the basis of reduced fractional catabolic rate (FCR) of autologous LDL or substantially reduced binding of their LDL to normal LDL-receptors determined by an in vitro cell growth assay using the U937 macrophage-like cell line. Segments of the apo B gene were amplified by the polymerase chain reaction. Using a combination of cloning and sequencing the amplified fragment, together with chemical cleavage mismatch analysis, no sequence differences were identified in this region of the gene. We therefore conclude that variation outside the region of the apo B gene that codes for amino acids 3130-3630 must be responsible for the reduced LDL clearance in these patients.