Synthetic magainin analogues with improved antimicrobial activity

Based on modifications to enhance the alpha-helical structure of the broad spectrum antibiotic magainin 2, a series of analogues have been synthesized which display an increase up to two orders of magnitude in antimicrobial activity and, in the most favorable case, no appreciable increase in hemolytic activity over magainin 1 at the concentrations tested.     

Primary sequence mapping of human apolipoprotein B-100 epitopes. Comparisons of trypsin accessibility and immunoreactivity and implication for apoB conformation

Differential trypsin-accessibility and monoclonal antibodies (Mabs) to human apolipoprotein (apo) B-100 are both important tools for probing apoB structure and conformation on low-density lipoproteins (LDL). In this study, we have mapped greater than 80% of the C-terminal region (720 residues) of LDL apoB-100 using trypsin digestion. Our results extend our previous data [Yang et al. (1986) Nature (Lond.) 323, 738-742] confirming that the C-terminal region of about 420 residues of apoB-100 is largely inaccessible to trypsin, whereas the part just preceding this region has interspersed trypsin-accessible and inaccessible peptides. We have determined the amino acid sequence of specific apoB-100 peptides containing epitopes recognized by four separate Mabs: two epitopes have been mapped to within 20 residues, one has been mapped to 36 residues, and the last to 80 residues. We used polyclonal antisera to identify 16 overlapping clones of varying lengths of apoB-100 cDNAs extending from the C-terminus of apoB-100 cloned in the expression vector, lambda gt11. These clones were then tested against individual Mabs. By nucleotide sequence analysis of overlapping clones that show differential reactivities to different Mabs, we have mapped the individual epitopes of each Mab to within about 50-150 amino acid residues predicted from the DNA sequences. Confirmation and further fine mapping were accomplished by competition for LDL binding using partially purified fusion proteins and chemically synthesized oligopeptides. Two epitopes (Mabs 7 and 22) were mapped to the C-terminal 20 amino acids of apoB-100, one (Mab 16) to residues 4154-4189, and another (Mab 20) to residues 3926-4005. Mab 16 precipitates more than 80% of LDL particles. Mab 20 precipitates only denatured apoB but not native LDL apoB [Milne et al. (1987) Mol. Immunol. 24, 435]. Mabs 7 and 22 are unique in that they precipitate LDL apoB modified by storage much better than freshly isolated LDL-apoB. Although epitope expression and trypsin-accessibility represent two useful probes for the study of protein conformation, there was no obvious correlation between these two parameters when applied to LDL apoB for the antibodies we have examined.     

Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles.

The involvement of possible cytoplasmic factors in ATP-dependent postttranslational translocation of proteins into Escherichia coli membrane vesicles was examined. The precursor of OmpA protein was partially purified by DEAE-cellulose chromatography, and its translocation was found to require material from the soluble cytoplasmic fraction. The fractionated active cytoplasmic translocation factor (CTF) was protease sensitive, micrococcal nuclease insensitive, N-ethylmaleimide resistant, and heat labile. The heat sensitivity of the CTF allowed its specific and preferential inactivation in the crude-precursor synthesis mixture, which provided a simple and rapid assay procedure for the factor during purification. Two active fractions were detected upon further fractionation: the major one was about 8S in sucrose gradient centrifugation and 120 kilodaltons by Sephadex filtration, whereas the other was about 4S and 60 kilodaltons in sucrose gradient centrifugation and by Sephadex filtration, respectively. The active fractions could also be fractionated by DEAE-Sepharose chromatography. These CTFs are apparently different from the previously reported 12S export factor (M. Muller and G. Blobel, Proc. Natl. Acad. Sci. USA 81:7737-7741, 1984).     

A dye release assay for determination of lysostaphin activity

We describe a method for determination of lysostaphin activity using Remazol Brilliant Blue R (RBB)-dyed staphylococcal cells or RBB-dyed staphylococcal peptidoglycan as substrate. The dyed substrates are easy to prepare and are stable for at least 6 months. Soluble hydrolytic products released by lysostaphin are measured spectrophotometrically at 595 nm after the insoluble substrate is removed by filtration or centrifugation. The dye release assay is more sensitive and more accurate than the previously described turbidimetric assay.     

Construction and analysis of monomobile DNA junctions

Immobile DNA junctions are complexes of oligomeric DNA strands that interact to yield branched structures in which the branch point cannot migrate. This is achieved by minimizing the sequence symmetry in the flanking arms, so that base pairs lock at the branch site. Here, we report the design, synthesis, and analysis of two semimobile junctions, structures in which a controlled extent of branch point migratory freedom is deliberately introduced. We have constructed two minimally symmetric four-arm semimobile junctions from synthetic deoxy 17-mers. These junctions, termed "monomobile", contain a single pair of base pairs (A-T or C-G) which can migrate at the site of branching, while the rest of the junction is immobile. We have demonstrated by gel electrophoresis techniques that these junctions form and that they have the predicted 1:1:1:1 stoichiometry. We have compared these junctions with the immobile junction on which they are based, by means of hydroxyl radical protection experiments. From these data, both migratory conformers can be seen to coexist in solution. The semimobile junction with the C-G base pair has the same crossover and stacking pattern observed for the immobile junction, while the junction with the A-T base pair has the opposite pattern. We conclude that crossover and stacking patterns are a direct consequence of the base pairs which flank the junction. In addition, the data indicate that the crossover pattern biases for these junctions are much greater than are the migratory biases.     

Fibrin(ogen) peptide B beta 15-42 inhibits platelet aggregation and fibrinogen binding to activated platelets

The binding of fibrinogen to activated platelets leads to platelet aggregation. Fibrinogen has multiple binding sites to platelet membrane glycoprotein IIb-IIIa complex. At least two well-defined sequences in fibrinogen, Arg-Gly-Asp sequence of A alpha 95-97 and A alpha 572-574 and gamma 400-411, have been shown to interact with glycoprotein IIb-IIIa. A possible binding site on the amino-terminal end of fibrinogen to platelet glycoprotein IIb-IIIa has also been reported. In this paper the effect of synthetic peptides derived from the amino-terminal end of the B beta chain on platelet aggregation and fibrinogen binding has been examined. B beta 15-42 peptide inhibits platelet aggregation and 125I-fibrinogen binding to activated platelets in a dose-dependent manner. Since B beta 15-42 contains a previously identified fibrinogen binding site, B beta 15-18, exposed by thrombin cleavage of native fibrinogen, we also examined the effect of B beta 15-18, B beta 19-42, and B beta 1-14 (fibrinopeptide B) on platelet aggregation and fibrinogen binding. Synthetic fibrinopeptide B and B beta 15-18 had no effect on platelet aggregation and fibrinogen binding while B beta 19-42 retained the inhibitory effect. When fibrinogen is chromatographed on a column of agarose-bound B beta 15-42, a cation-dependent retention of fibrinogen on the peptide column was observed, and fibrinogen was eluted from the column by B beta 15-42 but not by B beta 1-14. Under the same conditions, platelet glycoprotein IIb-IIIa was not retained in the column. Thus, the observed inhibitory effect is due to its interaction with fibrinogen rather than to platelet glycoprotein IIb-IIIa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of duck growth hormone and cloning of the complementary DNA

Duck growth hormone (GH) was isolated and purified from duck pituitaries by salt precipitation and HPLC on reverse-phase C18 columns. The duck GH was homogeneous as shown by SDS-polyacrylamide gel electrophoresis with a molecular weight of 22,000. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The positive clones were selected and sequenced. The full-length duck GH cDNA contains 820 nucleotide pairs with an open reading frame coding for the precursor form duck GH of 216 amino-acid residues. The partial amino-acid sequence from the protein completely agrees with that derived from the cDNA, with Phe as the first residue in mature duck GH preceded by a 27-residue hydrophobic signal peptide. The duck GH is almost completely homologous to the chicken GH, with only three conservative substitutions (Ser for Thr, His for Tyr and Lys for Arg) and one deletion (Ala) in the duck GH sequence. Comparison of amino-acid sequence of duck GH with that of various species reveals 56%, 73% and 40% homologies with GHs of human, rat and salmon, respectively.     

Alternate pathways of DNA replication in Escherichia coli

We have described the pcbA1 mutation which enables E. coli cells to replicate DNA in the absence of a functional dnaE gene product if DNA polymerase I (the polA gene product) is present. The pcbA1 mutation phenotypically suppresses multiple dnaEts and dnaEam alleles. The pcbA1/PolI replication pathway differs from normal in sensitivity to certain DNA-damaging agents such as methylmethane sulfonate (MMS) and a lack of damage-directed mutagenesis. We report here cloning of the pcbA1 gene in a multicopy plasmid. The pcbA1 mutation is detected only in cis; therefore, cloning necessitated gene eviction. The pcbA1 gene lies closely- linked to gyrB. We have demonstrated the physical presence of DNA polymerase I in the replicating holoenzyme complex by immunoblotting using dnaEam strains. We conclude that E. coli has two alternate replisome structures: REP-A, in which DNA polymerase I is the functional synthetic subunit; and REP-E, in which the alpha-subunit, product of the dnaE gene, is functional. To investigate further the role of individual DNA polymerases in replication, we have isolated the polB gene on multicopy plasmids.     

Kinetics of enzyme-mediated reduction of lipid soluble nitroxide spin labels by living cells

Nitroxide spin labels can be reduced to the corresponding hydroxylamines in cells. The selective action of inhibitors, and thermal and chemical inactivation demonstrate that the reduction of nitroxides in cells is an enzymatic or enzyme-mediated process. The kinetics of reduction of doxylstearates are affected by the position of the doxyl moiety along the stearic acid chain. The doxyl moiety of 5-doxylstearate is close to the membrane surface, and its reduction is first order with respect to the nitroxide, whereas the doxyl moieties of 10- and 12-doxylstearate are in the membrane hydrocarbon region and their reduction is a zero-order process. The reduction of 16-doxylstearate which usually has a mixture of first- and zero-order kinetics becomes zero order with addition of an extracellular broadening agent, potassium trioxalatochromiate(III). These results suggest that the rate of reduction of doxyl moieties is controlled by their accessibility to reducing equivalents, i.e., the rate-limiting step for the reduction of the doxyl moiety deep in the membrane is the diffusion of reducing equivalents within or into the membrane. The reduction of doxylstearates in cells is inhibited by rotenone but not antimycin A, cyanide, propyl gallate or SKF-525A. It appears that the reduction of doxylstearates takes place at the level of the ubiquinone in the respiratory chain in mitochondria in these cells.     

Use of primary deuterium and 15N isotope effects to deduce the relative rates of steps in the mechanisms of alanine and glutamate dehydrogenases

We have used deuterium and 15N isotope effects to study the relative rates of the steps in the mechanisms of alanine and glutamate dehydrogenases. The proposed chemical mechanisms for these enzymes involve carbinolamine formation, imine formation, and reduction of the imine to the amino acid [Grimshaw, C.E., Cook, P.F., & Cleland, W.W. (1981) Biochemistry 20, 5655; Rife, J.E., & Cleland, W.W. (1980) Biochemistry 19, 2328]. These steps are almost equally rate limiting for V/Kammonia with alanine dehydrogenase, while with glutamate dehydrogenase carbinolamine formation, imine formation, and release of glutamate after hydride transfer provide most of the rate limitation of V/Kammonia. Release of oxidized nucleotide is largely rate limiting for Vmax for both enzymes. When beta-hydroxypyruvate replaces pyruvate, or 3-acetylpyridine NADH (Acpyr-NADH) or thio-NADH replaces NADH with alanine dehydrogenase, nucleotide release no longer limits Vmax, and hydride transfer becomes more rate limiting. With glutamate dehydrogenase, replacement of alpha-ketoglutarate by alpha-ketovalerate makes hydride transfer more rate limiting. Use of Acpyr-NADPH has a minimal effect with alpha-ketoglutarate but causes an 8-fold decrease in Vmax with alpha-ketovalerate, with hydride transfer the major rate-limiting step. In contrast, thio-NADPH with either alpha-keto acid causes carbinolamide formation to become almost completely rate limiting. These studies show the power of multiple isotope effects in deducing details of the chemistry and changes in rate-limiting step(s) in complicated reaction mechanisms such as those of alanine and glutamate dehydrogenases.