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Radioaetive leueine was ineorporated by N eurospora crassa mitoehondria in vivo in the presence of cyeloheximide. When the membrane protein of these mitochondria was ehromatographieally separated on oleyl polymethaerylie aeid resin, & nurober of fraetions were obtained whieh differ with respeet to their eontents of radioaetivity and eytoehromes. The highest speeifie radioaetivity was found in the fraction eontaining eytoehrome aa3• This fraetion proved to be a pure and enzymatically aetive cytoehrome oxidase. Its ratio of absorbanee at 280 nm (ox)/ 443 nm (red.) was 2.1. By means of sodium dodeeylsulfate gel-electrophoresis, this enzymewas separated into five polypeptides with molecular weights of 30000, 20000, 13000, 10000, and 8000. Only the polypeptide with the molecular weight 20000 displayed a high specific radioaetivity.
Radioactive amino acids were incorporated in vivo into N eurospora crassa cells, and the mitochondrial ribosomes were isolated. The incorporation of radioactivity into the proteins of these ribosomes was inhibited by cycloheximide, but not by chloramphenicol. It is therefore concluded that these proteins are synthesized on the cycloheximide sensitive and chloramphenicol insensitive cytoplasmic ribosomes.
Interleukin 4 (IL-4) exerts a decisive role in the coord.ination of proteelive immune responses against parasites, particularly helminths. A disregulation of ll.r4 function is possibly involved in the genesis of allergic disease states. The search for important amino acid residues in human ll.r4 by mutational analysis of charged invariant amino acid positions identified two distinct functional sites in the 4-helix-bundle protein. Site 1 was marked by amino acid substitutions of the glutamic acid at position 9 in helix A and arginine at position 88 in helix C. Exchanges at both positions led to IL-4 variants deficient in binding to the extracellular domain of the ll.r4 receptor (IL-4ReJ. In parallel, up to 1000-fold increased concentrations of this type of variant were required to induce T -cell proliferation and B-eeil CD23 expression. Site 2 was marked by amino acid exchanges in helix D at positions 121, 124 and 125 (arginine, tyrosine and serine respectively in the wild-type).ß.A variants affected at site 2 exhibited partial agonist activity during T -cell proliferation; however, they still bound with high affinity to IL-4Rex. [The generation of an IL-4 antagonist by replacing tyrosine 124 with aspartic acid has been described before by Kruse et al. (1992) (EMBO }., 11, 3237-3244)]. These findings indicate that IL-4 functions by bind.ing IL-4Rex via site 1 which is constituted by residues on helices A and C. They further suggest that the association of a second, still undetined receptor protein with site 2 in helix D activates the receptor system and generates a transmembrane signal.
Cell kinetic studies of T cells stimulated with the interleukin 2 (11-2), D-4, or both lymphokines were performed with conventional [3H] thymidine incorporation and with the bivariate BrdU/Hoechst technique. 11-2 and 11-4 are able to drive phytohemagglutininactivated T cells through more than one cell cycle. Neither synergistic nor inhibitory efl'ect on T -cell proliferationwas seen for the stimulation with both 11-2 and 11-4 as compared with the effect ofll-2 alone. The quantitative data ofthe cell cycle distribution ofphytohemagglutininactivated T cells suggestthat the population ofll-4-responsive cells is at least an overlapping population, if not a real subset of the ·population of the 11-2-responsive cells.
Mutant proteins (muteins) of human lnterleukin-4 (llA) were constructed by means of in vitro mutagenesis. The muteins were expressed in E. co/1, submitted to a renaturation and purification protocol and analysed for biological activity. Exchange of the cysteines at either position 46 or 99 which form one of the three disulfide bridges resulted. in a nearly co•mplete loss · of biological actiyity and an unstable protein. The exchange of tyrosine 124 also inactivated the protein, while a mutation of tyrosine 56 left some residual activity. Exchange of the other four cysteines or of · the single tryptophane had smaller etTects.
Receptor binding properties of four-helix-bundle growth factors deduced from electrostatic analysis
(1994)
Hormones of the hematopoietin class mediate signal transduction by binding to specific transmembrane receptors. Structural data show that the human growth hormone (hGH) forms a complex with a homodimeric receptor and that hGH is a member of a class of hematopoietins possessing an antiparallel 4-a-helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormonereceptor recognition. In order to examine the specificity of hormone-receptor complexation, an analysis was made of the electrostatic potentials of hGH, interleukin-2 (IL-2), interleukin-4 (IL-4), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the hGH and IL-4 receptors. The binding surfaces of hGH and its receptor, and of IL-4 and its receptor, show complementary electrostatic potentials. The potentials of the hGH and its receptor display approximately 2-fold rotational symmetry because the receptor subunits are identical. In contrast, the potentials of GM-CSF and IL-2 Iack such symmetry, consistent with their known high affinity for hetero-oligomeric receptors. Analysis of the electrostatic potentials supports a recently proposed hetero-oligomeric model for a high-affinity IL-4 receptor and suggests a possible new receptor binding mode for G-CSF; it also provides valuable information for guiding structural and mutagenesis studies of signal-transducing proteins and their receptors.
The effects of the interlenkins IL-7 and IL-9 on cell cycle progression were investigated by conventional [3H]thymidine incorporation and by the bivariate BrdU/Hoechst technique. 8oth IL· 7 and IL-9 drive phytohemagglutinin-activated T cells through more than one cell cycle, but IL-7 wasmorepotent on cell cycle progression than IL-9. Neither synergistic nor inhibitory effects were seen between various combinations of the lymphokines IL-7, IL-9 and IL-4 compared to each lymphokine alone. When T cells are activated with phytohemagglutinin for 3 days, all or most IL-4 responsive cells respond to IL-7 as weil, whereas only a part of IL-7 responders are IL-4 responders. In contrast, when T cells are activated with phytohemagglutinin for 7 days, the quantitative data of the cell cycle distribution soggest that the population of IL-7 responders is at least an overlapping, if not a real subset of the population of the IL-4 responders.