An amphipathic model peptide, KLALKLALKALKAAKLA-NH2, and its complete double D-amino acid replacement set was used to analyze the process of peptide binding at lipid vesicles of different surface charge and to determine the structure of the lipid-bound peptides using CD spectroscopy. The relationship between peptide helicity, model membrane permeability, and biological activity has been studied by dye release from liposomes and investigation of antibacterial and hemolytic activity. The accumulation of cationic KLAL peptides at and the membrane-disturbing effect on bilayers of high negative surface charge were found to be dominated by charge interactions. Independent of any structural propensity, the cationic peptide side chains bind to the anionic phosphatidylglycerol moieties. The charge interactions hold the peptides at the bilayer surface, where they may disturb preferentially lipid headgroup organization by formation of peptide-lipid clusters. In contrast, KLAL peptide interaction with bilayers of low negative surface charge is highly dependent on peptide helicity. With decreasing amounts of anionic phosphatidylglycerol in the bilayer the membrane-disturbing effect of KLAL and other helical analogs substantially increases despite drastically reduced binding affinity. Less helical peptides exhibit reduced bilayer-disturbing activity, showing that the hydrophobic helix domain is decisive for binding at and inducing permeability in membranes of low negative surface charge. It is suggested that hydrophobic interactions drive the penetration of the amphipathic peptide structure into the inner membrane region, thus disturbing the arrangement of the lipid acyl chains and causing local disruption. On the basis of the proposed model for membrane disturbance, interactions modulating antibacterial and hemolytic activity are discussed.

Substance P and other polycationic peptides are thought to stimulate mast cell degranulation via direct activation of G proteins. We investigated the ability of extracellularly applied substance P to translocate into mast cells and the ability of intracellularly applied substance P to stimulate degranulation. In addition, we studied by reverse transcription–-PCR whether substance P-specific receptors are present in the mast cell membrane. To study translocation, a biologically active and enzymatically stable fluorescent analogue of substance P was synthesized. A rapid, substance P receptor- and energy-independent uptake of this peptide into pertussis toxin-treated and -untreated mast cells was demonstrated using confocal laser scanning microscopy. The peptide was shown to localize preferentially on or inside the mast cell granules using electron microscopic autoradiography with 125I-labeled all-D substance P and 3H-labeled substance P. Cell membrane capacitance measurements using the patch-clamp technique demonstrated that intracellularly applied substance P induced calcium transients and activated mast cell exocytosis with a time delay that depended on peptide concentration (delay of 100–500 s at concentrations of substance P from 50 to 5 μM). Degranulation in response to intracellularly applied substance P was inhibited by GDPβS and pertussis toxin, suggesting that substance P acts via G protein activation. These results support the recently proposed model of a receptor-independent mechanism of peptide-induced mast cell degranulation, which assumes a direct interaction of peptides with G protein α subunits subsequent to their translocation across the plasma membrane.

The kon gene of Escherichia coli encodes the ATP-dependent serine protease La and belongs to the family of c32-dependent heat shock genes. In this paper, we report the cloning and characterization of the Ion gene from the gram-positive bacterium Bacillus subtilis. The nucleotide sequence of the Ion locus, which is localized upstream of the heDL4XCDBL operon, was determined. The Ion gene codes for an 87-kDa protein consisting of 774 amino acid residues. A comparison of the deduced amino acid sequence with previously described ion gene products from E. coli, Bacillus brevis, and Myxococcus xanthus revealed strong homologies among all known bacterial Lon proteins. Like the E. coli ion gene, the B. subtilis Ion gene is induced by heat shock. Furthermore, the amount of Ion-specific mRNA is increased after salt, ethanol, and oxidative stress as well as after treatment with puromycin. The potential promoter region does not show similarities to promoters recognized by o+32 of E. coli but contains sequences which resemble promoters recognized by the vegetative RNA polymerase EoA of B. subtilis. A second gene designated orJX is suggested to be transcribed together with ion and encodes a protein with 195 amino acid residues and a calculated molecular weight of 22,000.ATP-dependent proteases are involved in the regulation of the level of a number of proteins with short half-lives, such as SulA and RcsA of Escherichia coli (25,26). In addition to biologically active proteins, many damaged and abnormal proteins resulting from misfolding, premature termination, or denaturation are subjected to ATP-dependent proteolytic degradation. During various stresses, increasing amounts of misfolded and damaged proteins may accumulate. Hence, the ATP-dependent proteases are also important during stress. In E. coli, two forms of energy-dependent proteolytic systems, the Lon (La) (11,14) and Clp (32,36) proteases, have been fairly well characterized. Lon (12,22,46) and ClpP (37) belong to the family of heat shock proteins. The ATP-dependent serine protease La is encoded by the ion gene of E. coli. Mutations in ion result in a pleiotropic phenotype of E. coli cells, with an increased sensitivity to UV light, mucoidy, filamentous growth, and defects in the lysogenicity of some bacteriophages and in the degradation of regulatory or abnormal proteins (26).Although extracellular proteases have been a matter of extensive investigation in various bacilli because of their industrial application, the knowledge of the structure and function of ATP-dependent proteases in Bacillus species is still very limited. stresses. According to their regulation, the heat shock proteins of B. subtifis can be arranged into at least two groups (29). Gene products of the groESL and the dnaK operons represent members of the first group. The heat induction of these proteins requires the vegetative sigma factor orA of B. subtifis (9) and a conserved palindromic structure (CIRCE) just downstream of the start point of transcription (50,67,70). The alternative sigma factor...

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The tetrahydroisoquinoline (TIQ) salsolinol (SAL), a condensation product of dopamine and pyruvate or acetaldehyde, is one of the neuropharmacologically active alkaloids in mammals. Previous HPLC studies have shown that the R-enantiomer of SAL is largely predominant, or is the only enantiomer in the urine of healthy subjects, whereas the S-enantiomer was found predominant in the urine of alcoholics. An enzymatic pathway for SAL formation that is influenced by chronic alcohol intake was proposed. However, our analyses showed that the SAL detectable in human urine and plasma is racemic, at least in healthy subjects. No change of the enantiomeric distribution was observed after an acute alcohol ingestion (1 g alcohol/kg body weight). Using a new method for the resolution of the SAL enantiomers and gas chromatography mass spectrometry analysis, the SAL enantiomers were quantified in the urine and plasma of 24 subjects before and after the intake of alcohol. Special dietary conditions were observed to avoid interferences by the SAL of the foodstuff. Although the distribution of SAL enantiomers was not changed after alcohol intake, the total urinary SAL output and the plasma concentration of SAL were significantly influenced in different ways. Only five subjects showed a significant increase both in plasma SAL concentration and in the total urinary SAL output, whereas 19 subjects showed decreased or unchanged SAL levels after alcohol administration. Data also show that only the subjects with low baseline levels (mean of 0.148 ng SAL/ml plasma) tend to increase SAL levels after ethanol ingestion, which may imply some genetic basis for the response.