Stefan Klauser

Activation of system L heterodimeric amino acid exchangers by intracellular substrates

System L‐type transport of large neutral amino acids is mediated by ubiquitous LAT1‐4F2hc and epithelial LAT2‐4F2hc. These heterodimers are thought to function as obligatory exchangers, but only influx properties have been studied in some detail up until now. Here we measured their intracellular substrate selectivity, affinity and exchange stoichiometry using the Xenopus oocyte expression system. Quantification of amino acid influx and efflux by HPLC demonstrated an obligatory amino acid exchange with 1:1 stoichiometry. Strong, differential trans‐stimulations of amino acid influx by injected amino acids showed that the intracellular substrate availability limits the transport rate and that the efflux selectivity range resembles that of influx. Compared with high extracellular apparent affinities, LAT1‐ and LAT2‐4F2hc displayed much lower intracellular apparent affinities (apparent Km in the millimolar range). Thus, the two system L amino acid transporters that are implicated in cell growth (LAT1‐4F2hc) and transcellular transport (LAT2‐4F2hc) are obligatory exchangers with relatively symmetrical substrate selectivities but strongly asymmetrical substrate affinities such that the intracellular amino acid concentration controls their activity.

Sex-peptide activates juvenile hormone biosynthesis in the Drosophila melanogaster corpus allatum

Mating elicits two well-defined reactions in sexually matured females of many insects: reduction of receptivity and increased oviposition. These post-mating responses have been shown to be induced by factors synthesized in the reproductive tract of the adult male and transferred in the seminal fluid to the female during copulation. One of these factors, named sex-peptide (SP), has been identified in Drosophila melanogaster. Using an in vitro radiochemical assay, we show that synthetic sex-peptide considerably activates juvenile hormone III-bisepoxide (JHB3) synthesis in corpus allatum (CA) excised from Days 3 and 4 post-eclosion virgin females. Base levels are significantly lower at emergence (Day 0) than on subsequent days, and only weak stimulation is obtained on Day 1, while none is obtained on Day 2, where maximal basal synthesis occurs. The CA of mated females cannot be stimulated further for at least 7 days, but regain responsiveness by Day 10 after mating. Synthesis of JHB3 stimulated by SP in vitro persists for at least 4 h after removal of the peptide. Development of responsiveness of the CA to SP in vitro is compared with development of the post-mating reactions of sex-peptide injected virgin females. Our results suggest that the CA is a direct target for SP in vivo and that sexual maturity is established separately for the two post-mating reactions.

Identification and isolation of a bactericidal domain in chicken egg white lysozyme

Chicken egg white lysozyme exhibits antimicrobial activity against both Gram‐positive and Gram‐negative bacteria. Fractionation of clostripain‐digested lysozyme yielded a pentadecapeptide with antimicrobial activity but without muramidase activity. The peptide was isolated and its sequence found to be I‐V‐S‐D‐G‐N‐G‐M‐N‐A‐W‐V‐A‐W‐R (amino acids 98–112 of chicken egg white lysozyme). A synthesized peptide of identical sequence had the same bactericidal activity as the natural peptide. Replacement of Trp 108 with tyrosine significantly reduced the antibacterial capacity of the peptide. By replacement of Trp 111 with tyrosine the antibacterial activity was lost. Replacement of Asn 106 with the positively charged arginine strongly increased the antibacterial capacity of I‐V‐S‐D‐G‐N‐G‐M‐N‐A‐W‐V‐A‐W‐R. The peptide I‐V‐S‐D‐G‐N‐G‐M consisting of the eight amino acids of the N‐terminal side had no bactericidal properties, whereas the peptide N‐A‐W‐V‐A‐W‐R of the C‐terminal side retained some bactericidal activity. Replacement of asparagine 106 by arginine (R‐A‐W‐V‐A‐W‐R) increased the bactericidal activity considerably. The D enantiomer of R‐A‐W‐V‐A‐W‐R was as active as the L form against five of the tested bacteria, but substantially less active against Serratia marcescens, Micrococcus luteus,Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lentus. For these bacterial species some stereospecific complementarity between receptor structures of the bacteria and the peptide can be assumed.

Comparison of the high-performance liquid chromatography of peptides and proteins on 100- and 300-Å reversed-phase supports

Abstract The chromatographic separations of peptides and proteins on commercially available 100- and 300-A pore size reversed-phase columns have been compared using various buffer systems. The larger-pore-size packing exhibits a slightly more hydrophilic character while maintaining flow and back-pressure characteristics typical of 10-μm supports. In addition to equal or improved resolving capabilities for smaller amino acid derivatives and peptides, this column material is notably superior to the 100-A for the chromatography of proteins with molecular weights exceeding ca . 15,000.

The macrophage response to bacteria: Flow of L-arginine through the nitric oxide and urea pathways and induction of tumoricidal activity

The consequences of the interaction of heat-killed bacteria and lipopolysaccharide (LPS) with a pure population of bone marrow-derived mononuclear phagocytes (BMMø) were investigated, utilizing changes in the flow of L-arginine and expression of tumoricidal activity as parameters of macrophage (mø) function. Gram negative bacteria and LPS proved potent in inducing the flow of L-arginine through the nitric oxide and the urea pathways but were mostly poor in eliciting tumoricidal activity. Gram positive bacteria affected the metabolism of L-arginine only little but were often efficient in triggering tumoricidal activity. The findings show that the mø response to bacteria, which may determine the outcome of their interaction with the host, may differ considerably depending on the type of bacteria.

Induction of nitric oxide synthase is a necessary precondition for expression of tumor necrosis factor-independent tumoricidal activity by activated macrophages

Various bacteria and bacterial products induce in pure, lymphocyte-free bone marrow-derived mononuclear phagocytes (BMMø) the generation of tumor necrosis factor, nitric oxide (NO) synthase, NO and nitrite (NO2-), the flow of L-arginine to citrulline, and tumoricidal activity. The flow of L-arginine to citrulline and formation of NO/NO2- on the one hand and expression of tumoricidal activity were not always closely related; however, these parameters were suppressed in a dose-dependent manner by the flavoprotein inhibitor, diphenyleneiodonium (DPI) and the L-arginine analogue, NG-monomethyl-L-arginine (NMMA). The findings support the concept of a central role of the NO synthase pathway in the generation of tumor necrosis factor-independent tumoricidal activity by activated macrophages but the exact conditions which enable the transfer of the lytic principle from the effector to the target cell remain to be elucidated.

Identification of an aprotinin antiviral domain

Digestion of the proteinase inhibitor aprotinin, by clostripain, a cysteine proteinase, yielded five oligopeptide fragments. Two fragments exhibited both antiviral and antibacterial activities, two fragments only antiviral activity, and one fragment showed no antimicrobial activity. One of the former oligopeptides showed antiviral activity against human herpes simplex virus type 1 and bovine parainfluenza virus type 3. It consisted of the hexapeptide Y‐F‐Y‐N‐A‐K corresponding to amino acids 21–26 of intact aprotinin. An identical synthetic peptide had the same antiviral spectrum as the natural hexapeptide, exhibited no antibacterial activity, and was also devoid of trypsin inhibiting activity. Intact aprotinin, in contrast, is ineffective against human herpes simplex virus 1 and bovine parainfluenza virus 3 but possesses antibacterial properties against several bacterial species [(1992) J. Appl. Bact. 72, 180–187].

Structural and biological studies on native bovine Cu,Zn-metallothionein-3

Following the discovery of the neuronal growth inhibitory factor, classified as metallothionein3 (MT-3), by Uchida et. al. 1991 [1] we have focussed our research on the structure-function analysis of this protein. MT-3 is a central nervous system (CNS) specific metallothionein-like protein, which has been linked to Alzheimer’s disease (AD) [1]. The amino acid sequence of human MT-3 (68 amino acids) exhibits 70% sequence identity to mammalian metallothioneins (MT-1 and MT-2) (61 amino acids), including the preserved array of 20 Cys residues. In MT-1 and MT-2 these 20 Cys residues are involved in the formation of two metal-thiolate clusters, i.e. MeII 3(Cys)9 and MeII 4(Cys)11 [2]. Relative to mammalian MT-1 and MT-2 sequences, MT-3 contains two conserved inserts, a single Thr in the N-terminal and a Glu-rich hexapeptide in the C-terminal region (see Fig. 1). Despite their high sequence identity only MT-3 exhibits a growth inhibitory activity in neuronal cell culture studies, indicating that the — so far unknown — structure of MT-3 must differ from that of the other MTs. Furthermore, it has been demonstrated that the N-terminal metal binding domain MT-3(1–32) is sufficient for the growth inhibitory activity [3, 6]. In contrast to MTs, which usually contain 7 Zn(II) ions, native MT-3 isolated from bovine brain possesses an unusual metal ion composition, i.e. 4-5 Cu(I) and 2-2.5 Zn(II) per polypeptide chain, suggesting the presence of different cluster structures in this protein [4].