Conformation of brain proteolipid apoprotein

The conformation of brain proteolipid apoprotein (PLA) has been investigated using infrared spectroscopy and freeze-fracture electron microscopy. For this purpose, spectroscopic samples consisting of a mixture of liquid paraffin and wet protein have been prepared. These systems have allowed us to record the infrared spectra of PLA at neutral pH. The amide I and III regions reveal the existence of a predominantly -helical structure, as well as the presence of minor -strands and random coil forms. The effect of sonication and a non-denaturing detergent, (n-octyl--d-glucopyranoside), on the structure of the protein have also been investigated. Sonication produces an increase of the and unordered structures at the expense of the -helical conformation. These structural changes are enhanced in the presence of the non-ionic detergent n-octyl--d-glucopyranoside. Lipids protect the native protein structure from the effects of sonication. The aforementioned detergent changes the PLA conformation by increasing the -helical content at the expense of -sheet and random coil forms. Therefore the PLA structure seems to be similar to the structures of other proteins intrinsic to non-neural membranes. The effects investigated also suggest that PLA behaves in a conformationally flexible manner.     

A study on the separation of reconstituted proteoliposomes and unincorporated membrane proteins by use of hydrophobic affinity gels, with special reference to band 3 from bovine erythrocyte membranes

Alkyl-Sepharose 4B with octyl, decyl, or dodecyl groups as an alkyl chain was a good adsorbent for any type of detergents and a variety of proteins, but not for phospholipids in a vesicle form. When these gels were added to the mixtures of reconstituted proteoliposomes prepared by using bovine band 3 and the protein unincorporated into liposomes, free band 3 in solution was adsorbed onto the gels and the proteoliposomes could be recovered by filtration, suggesting that this procedure, when applicable, permits a rapid isolation of proteoliposomes without loss and dilution of the sample. In addition, the results indicated that Bio-Beads SM-2 resin, which is virtually nonadsorbing for most proteins, can be used in removing any kind of detergents from those protein-detergent mixtures.     

Effect of an anionic detergent—sodium dodecyl sulphate exposure on club cells in the epidermis of Clarias batrachus

When treated with detergent club cells showed degenerative changes and released their contents to plug the intercellular spaces. This mechanism could act as an efficient protective barrier substantiating the role of club cells in assisting fish to overcome adverse conditions. A general decrease in the dimensions of club cells was associated with discharge of their contents. The appearance of juvenile club cells reflected their differentiation in response to increased demands to meet the challenge. Increases in the number of club cells indicated the differentiation of these cells at a rate faster than their degeneration. Localization of glycogen in club cells at the start of detergent treatment and, in general, its absence later and, in both control and juvenile club cells, is discussed in relation to the metabolic status of the cells. No marked shift was observed in the mucopolysaccharide and protein moieties of club cells.     

Automated on-line ionic detergent removal from minute protein/peptide samples prior to liquid chromatography-electrospray mass spectrometry

An automated on-line ionic detergent removal pre-column system coupled to capillary liquid chromatography-electrospray mass spectrometry is described. The system involves two micro precolumns, composed of a specific ionic detergent trapping column and a preconcentration column, respectively, and a packed 300 μm I.D. analytical column. Sample loading to the micro precolumns and regeneration of the detergent trapping column were performed at a flow-rate of 50 μl/min, while the flow-rate through the analytical column was set at 5.0 μl/min. Ionic detergent-containing tryptic-digested protein samples were directly applied to the micro precolumns without sample pretreatment and were analysed by UV absorption detection and electrospray mass spectrometry. The presented system allows for the fully automated removal of SDS with virtually no loss in protein/peptides. Maximum SDS load and breakthrough have been determined. Excellent protein recovery and complete removal of SDS is found. The chromatographic separation after SDS removal was completely restored and equalled the reference chromatogram. Mass spectral data confirm these findings. Finally, this technique allows for SDS removal from minute protein samples without the need for any sample handling.     

Identification of compendial nonionic detergents for the replacement of Triton X-100 in bioprocessing

We have systematically investigated six compendial nonionic detergents as potential replacements for Triton ×-100 in bioprocessing applications. Use of compendial raw materials in cGMP bioprocessing is advantageous for a variety of reasons including material specifications developed to meet stringent pharmaceutical product quality requirements, regulatory familiarity and comfort, and availability from vendors experienced supplying the biopharmaceutical industry. We first examine material properties of the detergents themselves including melting point and viscosity. Process performance and product contact in real-world bioprocess applications are then investigated. Lastly, we test the detergents in virus inactivation (VI) experiments with recombinant proteins and adeno-associated virus. Two of the detergents tested, PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides, showed favorable properties that make them attractive for use as potential Triton X-100 replacements. Process performance testing indicated negligible impact of the detergents on product yield, purity, and activity compared to a control with no detergent. Importantly, both PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides demonstrated very fast VI kinetics with complete inactivation of XMuLV observed in less than 1 min at a target 1% detergent concentration. Potential advantages and disadvantages of both candidate detergents for use in cGMP bioprocessing are summarized and discussed.     

NMR study of the tetrameric KcsA potassium channel in detergent micelles

Nuclear magnetic resonance (NMR) studies of large membrane-associated proteins are limited by the difficulties in preparation of stable protein-detergent mixed micelles and by line broadening, which is typical of these macroassemblies. We have used the 68-kDa homotetrameric KcsA, a thermostable N-terminal deletion mutant of a bacterial potassium channel from Streptomyces lividans, as a model system for applying NMR methods to membrane proteins. Optimization of measurement conditions enabled us to perform the backbone assignment of KcsA in SDS micelles and establish its secondary structure, which was found to closely agree with the KcsA crystal structure. The C-terminal cytoplasmic domain, absent in the original structure, contains a 14-residue helix that could participate in tetramerization by forming an intersubunit four-helix bundle. A quantitative estimate of cross- relaxation between detergent and KcsA backbone amide protons, together with relaxation and light scattering data, suggests SDS-KcsA mixed micelles form an oblate spheroid with approximately 180 SDS molecules per channel. K(+) ions bind to the micelle-solubilized channel with a K(D) of 3 +/- 0.5 mM, resulting in chemical shift changes in the selectivity filter. Related pH-induced changes in chemical shift along the "outer" transmembrane helix and the cytoplasmic membrane interface hint at a possible structural explanation for the observed pH-gating of the potassium channel.     

Effects of inclusion of neutral detergent soluble fibre sources in diets varying in forage particle size on feed intake, digestive processes, and performance of mid-lactation Holstein cows

The aim of this study was to evaluate effects of partial replacement of neutral detergent soluble fibre (NDSF) for starch in diets varying in particle size (PS) of alfalfa hay on chewing activities, ruminal fermentation, nutrient digestibility and performance in mid-lactation dairy cows. Eight multiparous Holstein cows (146 ± 6.0 d in milk; 36.7 ± 2.57 kg milk/d) were used in a replicated 4 × 4 Latin square design with four 21 d periods with the last 7 d for data collection. The experiment was a 2 × 2 factorial arrangement with 2 levels of NDSF (low = 85 g/kg or high = 130 g/kg diet dry matter) each combined with 2 PS (short = 20 mm or long = 40 mm) of alfalfa hay. Results show that forage PS alone, or in combination with NDSF inclusion, had no effect on dry matter (DM) intake. Although total chewing, eating and ruminating times were not affected by treatments, eating time per kg of neutral detergent fibre (NDF) ingested was higher in long versus short alfalfa hay-based diets (P<0.05). Feeding long forage PS increased sorting of the diet against particles >19 mm, and in favor of those <8 mm (P<0.05). Feeding diets high in NDSF lowered DM intake (P<0.05), but increased apparent digestibility of all nutrients including NDF (P<0.05) independent of forage PS. Ruminal pH and concentrations of total volatile fatty acids were unaffected by dietary treatments, however the proportion of butyrate was higher in ruminal fluid of cows fed high NDSF diets (P<0.05). Changes in milk composition included lower milk crude protein content in high NDSF diets and higher lactose content for short hay-based diets (P<0.05). That milk yield and milk energy output were similar in low versus high NDSF diets suggests that high NDSF-fed cows had higher energy efficiency due to lower DM intake. Results suggest that, independent of forage PS, NDSF sources can be successfully included to partly replace starchy grains in diets exceeding minimum fibre recommendations.     

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

Cell‐free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis‐based Escherichia coli cell‐free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light‐driven proton pump bacteriorhodopsin, consisting of seven transmembrane α‐helices. The cell‐free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3–0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent‐lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.