Signal peptide of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by the removal of the NH2-terminal 41 amino acid sequence (41 amino acid leader sequence). DNA fragments coding for short sequences consisting of 28 (Pro as the COOH terminus) 29 (Ala), 31 (Ala), and 33 (Ala) amino acids from the translation initiator, Met, in the leader sequence were prepared and fused in frame to the DNA encoding the mature alpha-amylase. The secretion activity of the 33 amino acid sequence was nearly twice as high as that of the parental 41 amino acid sequence, whereas the activity of the 31 amino acid sequence was 75% of that of the parent. In contrast, almost no secretion activity was observed with the 28 and 29 amino acid sequences. The signal peptide cleavage site of the precursor expressed from the plasmid encoding the 33 amino acid sequence was located between Ala and Leu at positions 33 and 34 and that from the 31 amino acid sequence between Thr and Ala at positions 33 and 34. The NH2-terminal amino acid from the latter corresponded to the 3rd amino acid of the mature enzyme. These results indicated that the functional signal peptide of the B. subtilis beta-amylase consists of the first 33 amino acids from the initiator, Met.     

Interaction of bilirubin with small unilamellar vesicles of dipalmitoylphosphatidylcholine

Interaction of bilirubin with phospholipid bilayers was studied at physiological pH above and below the gel-liquid crystalline phase transition of small unilamellar vesicles of dipalmitoylphosphatidylcholine. Chromatographic, calorimetric and 1H-NMR evidences strongly suggest that dianion form of bilirubin binds to the polar heads of the phosphatidylcholines protruding from the outer leaflet of the vesicles, whilst acid bilirubin, which is insoluble in water, is hydrophobically inserted into the lipophilic region of the bilayers. The surface-bound bilirubin is promptly removed from vesicles, whilst the acid form hydrophobically inserted into the vesicles is firmly bound to the membrane in the gel state. This pool of bilirubin could perturb the chemico-physical properties of the membrane (i.e., fluidity, phase transition, etc. ...) thus contributing to perturbation of the biological properties of living cells.     

Autoregulation of alpha-amylase formation in Bacillus subtilis

The phenomenon of self-regulation of alpha-amylase formation in Bacillus subtilis is discovered. The dependence of regulatory effect upon the phase of culture development in shown.     

NMR of molecules interacting with lipids in small unilamellar vesicles

Detailed characterization of protein, peptide or drug interactions with natural membrane is still a challenge. This review focuses on the use of nuclear magnetic resonance (NMR) for the analysis of interaction of molecules with small unilamellar vesicles (SUV). These phospholipid vesicles are often used as model membranes for fluorescence or circular dichroism experiments. The various NMR approaches for studying molecule-lipid association are reviewed. After a brief survey of the SUV characterization, the use of heteronuclear NMR (phosphorous, carbon, fluorine) is described. Applications of proton NMR through transferred nuclear Overhauser effect to perform structural determination of peptide are presented. Special care is finally given to the influence of the kinetic of the interactions for the proton NMR of bound molecules in SUV, which can constitute a good model for the study of dynamical processes at the membrane surface. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Crystal structure of Bacillus subtilis alpha-amylase in complex with acarbose

The crystal structure of Bacillus subtilis alpha-amylase, in complex with the pseudotetrasaccharide inhibitor acarbose, revealed an hexasaccharide in the active site as a result of transglycosylation. After comparison with the known structure of the catalytic-site mutant complexed with the native substrate maltopentaose, it is suggested that the present structure represents a mimic intermediate in the initial stage of the catalytic process.     

Stabilization of small unilamellar phospholipid vesicles during spray-drying

In the presence of 10% (0.3 M) sucrose in the aqueous medium, small unilamellar phospholipid vesicles are preserved during freeze-drying and spray-drying. Moreover, the bilayer integrity and permeability barrier are maintained during these processes.     

Antibody directed targeting of methotrexate-containing small unilamellar vesicles

Summary The potential of antibody-linked SUVs containing MTX in anticancer therapy was investigated. The SUVs, mean diameter 50±20 nm, were prepared by probe sonication of MTX-containing MLVs and were covalently linked either to a RAMG or NRG. After incubation with M21 melanoma cells for 2 h, RAMG-linked SUVs showed 2 and 4 times more binding than NRG-linked MTX-containing SUVs or MTX-containing SUVs unlinked to any Ig. Furthermore, on incubating M21 melanoma cells with RAMG-linked ³H MTX-containing SUVs for 2, 4, and 8 h at 4° C or 37° C, a higher radioactivity was associated with cells at 37° C than at 4° C. Membrane immunofluorescence revealed aggregation of and cap formation by RAMG-linked SUVs after 2 h (37° C) and endocytosis at 4 and 8 h at 37° C. Electron microscopic and autoradiographic studies confirmed aggregation of ³H MTX-containing SUVs around and on the surface of M21 cells. Electron microscopy also revealed these SUVs inside invaginations of and under the plasma membrane of melanoma cells. A colony inhibition assay showed that RAMG-linked, MTX-containing SUVs were 60 times, 8 times, and 4.5 times more growth inhibitory than free MTX, NRG-linked MTX-containing SUV, and MTX-containing SUVs unlinked to any Ig, but not toxic to a human kidney cancer line (that did not react with RAMG). Abbreviations used: DPPC, DL- -dipalmitoyl phosphatidylcholine; DTT, dithiothreitol; MTX, methotrexate; (MTX)SUV or MLV, MTX-containing SUV or MLV; MLV, multilamellar vesicle; NRG, normal rabbit immunoglobulin G; RAMG, rabbit antimelanoma IgG; SA, stearylamine; SPDP, N-succinimidy1-3-(2-pyridyldithio)propionate; SUV, small unilamellar vesicle; CHOL, cholesterol; LUV, large unilamellar vesicle; Ig, immunoglobulin; PDP-SA, N-[3-(2-pyridyldithio)-propinyl]stearylamine     

NH2-terminal processing of Bacillus subtilis alpha-amylase

Mature alpha-amylase of Bacillus subtilis is known to be formed from its precursor by removal of the NH2-terminal 41-amino acid sequence. To study the mechanism of this processing, the extracellular forms of alpha-amylase were analyzed for B. subtilis N7 alpha-amylase cloned and expressed in B. subtilis. The major form (form N34) isolated from log phase cultures in L-broth had an NH2 terminus corresponding to position 34 from the initiator Met but appeared to be microheterogeneous, as judged by native gel electrophoresis. The major forms from stationary phase cultures had NH2 termini at positions 40 (form N40) or 42 (form N42) and were homogeneous. The conversion of the larger to smaller forms could be achieved in culture supernatants or partially purified samples. The process N34----N40 was inhibited by EDTA; N40----N42 was facilitated by Ca2+. Phenylmethylsulfonyl fluoride inhibited the former but not the latter process. These results suggest that the signal peptidase cleavage site 30 decreases 35 is -Ala-Ala-Ala-Ser-Ala-Glu-Thr- (arrow or further upstream) and that proteolytic maturation occurs after secretion, which involves at least two different processing enzymes.     

Preparation of ready-to-use small unilamellar phospholipid vesicles by ultrasonication with a beaker resonator

Lipid vesicles are widely used as models to investigate the interactions of proteins, peptides, and small molecules with lipid bilayers. We present a sonication procedure for the preparation of well-defined and ready-to-use small unilamellar vesicles composed of phospholipids with the aid of a beaker resonator. This indirect but efficient sonication method does not require subsequent centrifugation or other purification steps, which distinguishes it from established sonication procedures. Vesicles produced by this method reveal a unimodal size distribution and are unilamellar, as demonstrated by dynamic light scattering and ³¹P nuclear magnetic resonance spectroscopy, respectively.     

The interaction of apolipoprotein A-I with small unilamellar vesicles of L-alpha-dipalmitoylphosphatidylcholine

The interaction between apolipoprotein A-I and small unilamellar vesicles of dipalmitoylphosphatidylcholine at the lipid phase transition resulted in complete release of vesicle contents at molar ratios of lipid to protein from 4000:1 down to 50:1. This indicated the existence of two types of stable complexes: a vesicular apo-A-I complex with a maximum of two to three apo-A-Is/vesicle, and a micellar complex (disc) with a stoichiometry of about 50 phosphatidylcholines/apo-A-I (mol/mol). We characterized the complexes by density gradient centrifugation, by gel filtration, and by immunoprecipitation using an anti-apo-A-I antibody. The morphology of the discs was similar to that of previously reported discs. Apo-A-I-induced release of vesicle contents was monitored by the relief of self-quenching of vesicle-encapsulated carboxyfluorescein. Using this assay we characterized the nature of the interaction between apo-A-I and phospholipid vesicles. The formation of complexes between vesicles and apo-A-I followed a two-step process; below or above the lipid phase transition temperature (Tc), apo-A-I bound to phosphatidylcholine vesicles but caused little leakage of contents. Kinetic analysis of the interaction between apo-A-I and dipalmitoylphosphatidylcholine vesicles below Tc indicated that about 1 in 500 collisions leads to a stable apo-A-I-vesicle complex. The second step involved passage of those complexes through Tc, which resulted in a very rapid transition into discs or vesicular complexes. Vesicular complexes contain apo-A-I which was no longer capable of interacting with pure lipid. Discs, on the other hand, interacted with vesicles at their phase transition.     

N-terminal amino acid sequence of Bacillus licheniformis alpha-amylase: comparison with Bacillus amyloliquefaciens and Bacillus subtilis Enzymes.

The thermostable, liquefying alpha-amylase from Bacillus licheniformis was immunologically cross-reactive with the thermolabile, liquefying alpha-amylase from Bacillus amyloliquefaciens. Their N-terminal amino acid sequences showed extensive homology with each other, but not with the saccharifying alpha-amylases of Bacillus subtilis.     

Kinetics of melittin-induced fusion of dipalmitoylphosphatidylcholine small unilamellar vesicles

We have studied the kinetics of fusion of dipalmitoylphosphatidylcholine small unilamellar vesicles at 51 degrees C which is induced by bee venom melittin at a protein-to-lipid molar ratio of 1/60. This was done by following with a stopped-flow fluorometer the reduction in the ratio of the excimer to monomer fluorescence intensities of 1-palmitoyl-2-(10-pyrenyldecanoyl)-sn-glycero-3-phosphorylcholine that accompanies fusion. At a low melittin concentration and low ionic strength, for which case the protein is monomeric, the value of the rate constant for fusion is 0.006 s-1. This is much smaller than that of 0.06 s-1 obtained for a high melittin concentration at low ionic strength, i.e. for the protein in the tetrameric form which is not induced by a high salt concentration. The value of the rate constant for fusion for a low melittin concentration in the presence of 2 M NaCl, i.e. for the protein in the tetrameric form which is induced by a high salt concentration, is 0.12 s-1. This is twice as large as that for fusion induced by the tetramer in a low ionic strength solution. These findings show that the state of aggregation of the protein in solution and, to a lesser extent, electrostatic interactions play an important role in the kinetics of melittin-induced fusion of vesicles.     

Identification of flagellin associated with the Bacillus subtilis folded chromosome.

We have analyzed the nature and contents of a major protein, P36, in the nucleoid of the Bacillus subtilis wild type and an isogenic mutant devoid of flagella. It appears that deoxyribonucleic acid-P36 complex is flagellin present as membrane-associated flagella.     

Action of Bacillus subtilis alpha-amylase on native wheat starch

Native starch granules from wheat have been subjected to enzymatic depolymerization with an alpha-amylase from Bacillus subtilis. Crystallites made from short-chain amylose and residues from mild acid hydrolysis have been also tested. Electron microscopy, particle size analysis, DSC, and x-ray diffractometry reveal that enzymatic degradation occurs granule by granule. Gel permeation chromatography shows off the macromolecular nature of the remaining material. In contrast, acid erodes simultaneously all the granules, leading to a splitting into small particles. Crystalline fractions are completely degraded by alpha-amylase. These results support evidence for an active disentanglement of chains, carried out by the different subsites of alpha-amylase molecules. A simple mathematical treatment is proposed to explain the results of the kinetics.     

Kinetics of melittin binding to phospholipid small unilamellar vesicles

We have used the decrease in the fluorescence intensity of the single tryptophan residue of bee venom melittin at long emission wavelengths that accompanies binding of the peptide to phospholipid small unilamellar vesicles to determine the rate of binding through the use of stopped-flow fluorometry in the millisecond range. We have found the rate to depend on the degree of saturation of the lipid acyl chains as well as on the physical state of the bilayer, the net electric charge of the polar headgroups, and the lipid-to-melittin molar ratio R. For zwitterionic lipids (i) the binding process is found to exhibit negative cooperativity, and (ii) the rate-limiting step appears to be penetration of the protein into the hydrophobic region of the bilayer. For negatively charged lipids the results show that binding is a very fast process that seems to be electrostatic in nature.