Reconstitution of nicotinic acetylcholine receptors into gel-protected lipid membranes.

We report the functional reconstitution of nicotinic acetylcholine receptors into gel-protected bilayer lipid membranes using two different methods. In the first case, reconstitution was achieved by direct membrane formation from an emulsion of glycerol monooleate, hexane, and a membrane receptor extract. In the second case, incorporation was achieved via the fusion of vesicles from a preparation of membrane-bound receptors into preformed membranes after diffusion through the protective front gel layer. Measurement of the dc conductivity of the membranes in the presence of either acetylcholine or alpha-bungarotoxin was used to test for the functional activity of incorporated receptors.

Sonoelectrochemical effects in electro-organic systems.

This paper describes recent studies in organic sonoelectrochemistry at Coventry University, including the oxidation of thiophene monoxides, degradation of dye pollutants, formation of conducting polymers and electrosynthetic modification of proteins.

Specific electrochemical nitration of horse heart myoglobin.

Earlier findings on electronitration of hen egg-white lysozyme demonstrated a product which was mononitrated at Tyr23, by ion-exchange chromatography, absorbance at 430 nm, dithionite reduction, and Edman sequencing of a nitrated proteolytic peptide. However, the whole protein was not sequenced; therefore, although the enzyme remained active upon nitration, reaction at other residues could not be completely eliminated. This study has now been extended to the redox protein myoglobin. We demonstrate the novel electronitration (electrooxidation in the presence of nitrite) of a specific tyrosine residue in horse heart myoglobin and also in apomyoglobin. Production of the yellow chromophore, 3-nitrotyrosine (3-NT), was apparent in apomyoglobin from A430 but was masked in holomyoglobin by the Soret band. In both cases, the presence of 3-NT in the electronitrated samples was further indicated by the binding of antibody to 3-NT in Western blots. High-resolution electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry revealed a reaction product at [M + 45] (consistent with substitution of NO2 for H), indicating that the nitration reaction is the only reaction occurring which gives rise to a change in mass in the electrooxidation. Fragmentation mass spectrometry identified the nitration site as Tyr103, with no nitration at Tyr146. The procedure may be useful in preparing model nitrated proteins for the study of disease mechanisms.

Detection of complement activity by using a polysaccharide-protected membrane.

The complement system of mammalian blood is a nonspecific part of the immune system involved in a number of disease conditions. We report the observation of pore creation caused by its activation in blood applied to the front gel layer of a bilayer membrane formed from dioleoylphosphatidyl choline and protected by a polysaccharide gel. The pores were detected by measuring the DC conductivity between nonblocking Ag/AgCl electrodes. The thickness of the protective gel was approximately 100 µm, and the complement response was seen within 3 min after application of activator. The lifetime of such membranes is limited only by hydrolysis of the phospholipid constituting the membrane. This easily prepared system is suitable for examining the kinetics of complement component interactions with inhibitors.

The effects of tyrosine nitration on the structure and function of hen egg-white lysozyme.

We have investigated the effects of tyrosine nitration (to form the weak acid, 3-nitrotyrosine) at positions 23 or 20 plus 23, on the structure and function of hen egg-white lysozyme. Enzyme activity against Micrococcus luteus cell-wall fragments or soluble substrates exhibits two phenomena. (a) A decrease in Km and kcat for the hydrolysis of soluble oligo- and poly-saccharides, resulting in only minor changes in the catalytic efficiency (kcat/Km) upon nitration. (b) The hydrolysis of M. luteus cell-wall fragments appeared to be dominated by electrostatic interactions with the protein, giving a decrease in enzyme activity as the 3-nitrotyrosyl group became ionized. Removal of the cell-wall anionic polymer, teichuronic acid, from M. luteus abolished this effect. The 3-nitrotyrosine group was also found to act as a fluorescence quencher of exposed tryptophan residues in lysozyme.

Electrochemical modification of lysozyme: anodic reaction of tyrosine residues.

Preparative electrooxidation of lysozyme at copper electrodes held at potentials around 1.2 V vs. a saturated calomel reference electrode induces the formation of a yellow chromophore with a concomitant decrease in the pI of the protein. Ion-exchange high-performance liquid chromatography revealed two new lysozyme species with pI values of 10.8 and 10.7 (lysozyme-11.0) which bear the chromophore. Sequence analysis of these two species showed that protein with lower pI was modified at both Tyr 23 and Tyr 20 and the other exclusively at Tyr 23. ribonuclease A, subtilisin BPN', and BSA were also found to produce the same chromophore using similar electrochemical reaction schemes. Characterization of the chromophore by a variety of techniques revealed that it is apparently 3-nitrotyrosine.

Purification of the major endoglucanase from Aspergillus fumigatus Fresenius.

Aspergillus fumigatus (Fresenius), IMI 246651, A.T.C.C. 46324, produces two beta-glucosidase enzymes, cotton-solubilizing activity, xylanase and endoglucanase enzymes which can be separated by gel-filtration chromatography. The major endoglucanase does not bind to concanavalin A-Sepharose and does not stain with periodic acid/Schiff reagent. It is homogeneous on polyacrylamide isoelectric focusing (pI = 7.1) and has a mol.wt. of 12500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The endoglucanase produces glucose and a mixture of oligosaccharides from cellulose; the purified enzyme has a small dextranase activity. It is stable at 50 degrees C and pH 6.

The enzymic synthesis of beta-[32P]UDP-N-acetylglucosamine.

Cells of Micrococcus sp. 2102 incorporate inorganic [32P]phosphate from the medium into the sugar-phosphate polymer of the wall. Controlled acid hydrolysis of sodium dodecyl sulphate-extracted cells gives N-acetylglucosamine 6-[32P]-phosphate which can be purified by ion-exchange chromatography and incubated with UTP in the presence of crude preparations of phosphoacetylglucosamine mutase from Neurospora crassa and UTP:N-acetylglucosamine 1-phosphate phosphotransferase from Bacillus licheniformis which act in concert to synthesise beta-[32P]UDP-N-acetylglucosamine.

The linkage of sugar phosphate polymer to peptidoglycan in walls of Micrococcus sp. 2102.

1. Protein-free walls of Micrococcus sp. 2102 contain peptidoglycan, poly-(N-acetylglucosamine 1-phosphate) and small amounts of glycerol phosphate. 2. After destruction of the poly-(N-acetylglucosamine 1-phosphate) with periodate, the glycerol phosphate remains attached to the wall, but can be removed by controlled alkaline hydrolysis. The homogeneous product comprises a chain of three glycerol phosphates and an additional phosphate residue. 3. The poly-(N-acetylglucosamine 1-phosphate) is attached through its terminal phosphate to one end of the tri(glycerol phosphate). 4. The other end of the glycerol phosphate trimer is attached through its terminal phosphate to the 3-or 4-position of an N-acetylglucosamine. It is concluded that the sequence of residues in the sugar 1-phosphate polymer-peptidoglycan complex is: (N-acetylglucosamine 1-phosphate)24-(glycerol phosphate)3-N-acetylglucosamine 1-phosphate-muramic acid (in peptidoglycan). Thus in this organism the phosphorylated wall polymer is attached to the peptidoglycan of the wall through a linkage unit comprising a chain of three glycerol phosphate residues and an N-acetylglucosamine 1-phosphate, similar to or identical with the linkage unit in Staphylococcus aureus H.

Cell surface protein of Pseudomonas (Hydrogenomonas) facilis.

Intact cells of Pseudomonas facilis contain one major molecular weight class of protein that is exposed at the cell surface as revealed by lactoperoxidase-catalyzed iodination with (125)I. All molecular weight classes of protein in derived cell envelope preparations are apparently saturated by iodination by lactoperoxidase after prolonged sonic treatment. The molecular weight of the predominantly exposed protein in intact cells is approximately 16,000, which is the minimal molecular weight of a cell envelope protein that precipitates as a complex with phospholipid from extracts of P. facilis. The isolation of labeled phospholipoprotein (PLP) after labeling intact cells with (125)I corroborates previous experiments which suggested a surface location for the protein portion of the phospholipoprotein (P(PLP)). Solvent extraction of cells and immunological evidence, including studies with ferritin-coupled antibodies, indicate that P(PLP) is located at the cell surface and may also be within the cell envelope. These experiments suggest that P(PLP) is the major cell surface protein in P. facilis.

Effect of growth conditions on morphology of Hydrogenomonas facilis and on yield of a phospholipoprotein.

Hydrogenomonas facilis grown heterotrophically on fructose with very low aeration eventually ceased to divide and produced elongated forms. Short forms were obtained from fructose-grown long forms by increasing the availability of oxygen to the organisms. A phospholipoprotein, the protein moiety of which is known to be present in the cell envelope, precipitated upon lowering the ionic strength of extracts from cells in the earlier stages of elongation (i.e., in the middle and late log phase of growth). The maximal yield of the protein moiety of the phospholipoprotein precipitate (i.e., grams of protein/grams of soluble protein x 100) was 2%. Poly-beta-hydroxybutyric acid accumulated as growth on fructose progressed, the accumulation being more marked with lower aeration.

Pathways leading to and from serine during growth of Pseudomonas AM1 on C1 compounds or succinate.

1. The following enzymes of the phosphorylated pathway of serine biosynthesis have been found in methanol- and succinate-grown Pseudomonas AM1: phosphoglycerate dehydrogenase, phosphoserine-alpha-oxoglutarate aminotransferase and phosphoserine phosphohydrolase. Their specific activities were similar in the organism grown on either substrate. 2. A procedure for preparation of auxotrophic mutants of Pseudomonas AM1 is described involving N-methyl-N'-nitro-N-nitrosoguanidine as mutagen and a penicillin enrichment step. 3. A mutant, M-15A, has been isolated that is unable to grow on methanol and that lacks phenazine methosulphate-linked methanol dehydrogenase. The mutant is able to grow on methylamine, showing that the amine is not oxidized by way of methanol. 4. Loss of methanol dehydrogenase activity in mutant M-15A led to loss of phenazine methosulphate-linked formaldehyde dehydrogenase activity showing that the same enzyme is probably responsible for both activities. 5. A mutant, 20B-L, has been isolated that cannot grow on any C(1) compound tested but can grow on succinate. 6. Mutant 20B-L lacks hydroxypyruvate reductase, and revertants that regained the ability to grow on methanol, methylamine and formate contained hydroxypyruvate reductase activity at specific activities similar to that of the wild-type organism. This shows that hydroxypyruvate reductase is necessary for growth on methanol, methylamine and formate but not for growth on succinate. 7. The results suggest that during growth of Pseudomonas AM1 on C(1) compounds, serine is converted into 3-phosphoglycerate by a non-phosphorylated pathway, whereas during growth on succinate, phosphoglycerate is converted into serine by a phosphorylated pathway.