No abstract

We report that residues Lys-16 and Asp-l19 play critical roles in the guanine nudeotide binding and, consequently, the biological function of the Ha-ras-encoded protein (Ha). Substitution of an asparagine residue for Lys-16 reduces the affinity of Ha for GDP and GTP by a factor of 100 but does not alter the specificity of nucleotide binding. The replacement of Asp-119 with an alanine residue reduces the affinity of Ha for GDP and GTP by a factor of 20 and reduces the relative affinity of Ha for GDP over IDP from 200-500 to 10. Based on these observations, a structural model for the GDP/GTP-binding site of Ha is proposed. By microinjecting purified proteins into NIH 3T3 cells, we observed that the ability of [Ala'19] Comparisons of amino acid sequences suggested two peptide regions of Ha-ras protein (Ha) p21 that might be involved in nucleotide interactions. Ha p21 sequence 5-20 is homologous to sequences present in a large series of nucleotide-binding proteins, with the sequence Gly-Lys that occurs at positions 15-16 being invariant (14). Structural studies of adenylate kinase have implicated the involvement of this region with binding to the phosphate groups of the nucleotide (15). The sequence Asn-Lys-Xaa-Asp that occurs at positions 116-119 is less universal but occurs in the known sequences for G proteins [e.g., EF-Tu, EF-G, and a-transducin (16, 17)].To examine the role of these residues in Ha-ras protein function, we investigated the biochemical properties of Ha proteins having amino acid substitutions at positions 16, 117, and 119. The biological properties of these proteins were determined in both mammalian and yeast cells. MATERIALS AND METHODSProtein Biochemistry. Oligonucleotide mutagenesis (11) of the genes for Ha p21 and [Val12,Thr59Ha was used to create ras variants, with all mutations confirmed by nucleotide sequencing. Mutant proteins were expressed in Escherichia coli strain HB101 and purified by chromatography on DEAESephacel and Sephadex G-75 in buffer A (50 mM sodium Hepes, pH 7.5/1 mM sodium EDTA/1 mM sodium EGTA/ 1 mM dithiothreitol/1 mM phenylmethylsulfonyl fluoride/0.01% n-octyl glucoside), as described (11). For [Ala'19]Ha, 5 mM MgCl2 and 10 ,M GDP were included in buffer A through the DEAE-Sephacel chromatography (4°C), and then purification was continued in buffer A by highperformance liquid chromatography (LKB) on a column of Superose 12 (Pharmacia) at 22°C, followed by immediate chilling. Protein determinations and other assays were as described (11).In trypsin-digestion experiments, pure Ha proteins (2-4 ,ug) were incubated at 4°C in 10 ,u of 50 mM sodium Hepes, pH 7.5/10 mM MgCl2/1 mM dithiothreitol with nucleotides (Sigma) for 15 min prior to the addition of 1 ug of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma). Reactions were terminated by the addition of 3 ,ug of soybean trypsin inhibitor (Sigma).Equilibrium-dialysis measurements of nucleotide binding utilized 4-mm dialysis tubing with a 12-to 14-kDa cutoff (Spectra/Por 2, Spectrum Medic...

We examined the effects of treatment with rHuTNF on food consumption and body weight in C3H/HeJ mice. rHuTNF was administered intraperitoneally either by injections of 3, 12, or 24 micrograms twice a day or by implantation of osmotic pumps that released 0.75, 3, or 12 micrograms per day. Dose-dependent reductions in both food intake and weight were induced by rHuTNF. However, in spite of continued exposure to rHuTNF, the mice developed a resistance to rHuTNF and resumed their pretreatment food intake and weight. Non-immunological factors may play a role in the development of this tolerance, since it developed rapidly and faded within 2 wk of cessation of exposure to rHuTNF.