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Membrane Fractionation (membrane + fractionation)
Selected AbstractsExpression of Na+/HCO3, co-transporter proteins (NBCs) in rat and human skeletal muscleACTA PHYSIOLOGICA, Issue 1 2004J. M. Kristensen Abstract Aim:, Sodium/bicarbonate co-transport (NBC) has been suggested to have a role in muscle pH regulation. We investigated the presence of NBC proteins in rat and human muscle samples and the fibre type distribution of the identified NBCs. Methods and results:, Western blotting of muscle homogenates and sarcolemmal membranes (sarcolemmal giant vesicles) were used to screen for the presence of NBCs. Immunohistochemistry was used for the subcellular localization. The functional test revealed that approximately half of the pH recovery in sarcolemmal vesicles produced from rat muscle is mediated by bicarbonate-dependent transport. This indicates that the NBCs are preserved in the vesicles. The western blotting experiments demonstrated the existence of at least two NBC proteins in skeletal muscle. One NBC protein (approximately 150 kDa) seems to be related to the kidney/pancreas/heart isoform NBC1, whereas the other protein (approximately 200 kDa) is related to the NBC4 isoform. The two NBC proteins represent the electrogenic isoforms named NBCe1 and NBCe2. Membrane fractionation and immunofluorescence techniques confirmed that the two NBCs are located in the sarcolemmal membrane as well as in some internal membranes, probably the T-tubules. The two NBCs localized in muscle have distinct fibre type distributions. Conclusions:, Skeletal muscle possesses two variants of the sodium/bicarbonate co-transporter (NBC) isoforms, which have been called NBCe1 and NBCe2. [source] Flk prevents premature secretion of the anti-, factor FlgM into the periplasmMOLECULAR MICROBIOLOGY, Issue 3 2006Phillip Aldridge Summary The flk locus of Salmonella typhimurium was identified as a regulator of flagellar gene expression in strains defective in P- and l -ring formation. Flk acts as a regulator of flagellar gene expression by modulating the protein levels of the anti-,28 factor FlgM. Evidence is presented which suggests that Flk is a cytoplasmic-facing protein anchored to the inner membrane by a single, C-terminal transmembrane-spanning domain (TMS). The specific amino acid sequence of the TMS is not essential for Flk activity, but membrane anchoring is essential. Membrane fractionation and visualization of protein fusions of green fluorescent protein derivatives to Flk suggested that the Flk protein is present in the membrane as punctate spots in number that are much greater than the number of flagellar basal structures. The turnover of the anti-,28 factor FlgM was increased in flk mutant strains. Using FlgM,,-lactamase fusions we show the increased turnover of FlgM in flk null mutations is due to FlgM secretion into the periplasm where it is degraded. Our data suggest that Flk inhibits FlgM secretion by acting as a braking system for the flagellar-associated type III secretion system. A model is presented to explain a role for Flk in flagellar assembly and gene regulatory processes. [source] Haem utilization in Vibrio cholerae involves multiple TonB-dependent haem receptorsMOLECULAR MICROBIOLOGY, Issue 3 2001Alexandra R. Mey Vibrio cholerae has multiple iron transport systems, one of which involves haem uptake through the outer membrane receptor HutA. A hutA mutant had only a slight defect in growth using haemin as the iron source, and we show here that V. cholerae encodes two additional TonB-dependent haem receptors, HutR and HasR. HutR has significant homology to HutA as well as to other outer membrane haem receptors. Membrane fractionation confirmed that HutR is present in the outer membrane. The hutR gene was co-transcribed with the upstream gene ptrB, and expression from the ptrB promoter was negatively regulated by iron. A hutA, hutR mutant was significantly impaired, but not completely defective, in the ability to use haemin as the sole iron source. HasR is most similar to the haemophore-utilizing haem receptors from Pseudomonas aeruginosa and Serratia marcescens. A mutant defective in all three haem receptors was unable to use haemin as an iron source. HutA and HutR functioned with either V. cholerae TonB1 or TonB2, but haemin transport through either receptor was more efficient in strains carrying the tonB1 system genes. In contrast, haemin uptake through HasR was TonB2 dependent. Efficient utilization of haemoglobin as an iron source required HutA and TonB1. The triple haem receptor mutant exhibited no defect in its ability to compete with its Vib, parental strain in an infant mouse model of infection, indicating that additional iron sources are present in vivo. V. cholerae used haem derived from marine invertebrate haemoglobins, suggesting that haem may be available to V. cholerae growing in the marine environment. [source] PREPARATION AND CHARACTERIZATION OF MODIFIED WHEAT GLUTEN BY ENZYMATIC HYDROLYSIS-ULTRAFILTRATIONJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2008JIN-SHUI WANG ABSTRACT The present work was aimed to investigate the characteristics of the modified wheat gluten by enzymatic hydrolysis followed ultrafiltration membrane fractionation. An 8% aqueous dispersion of wheat gluten was hydrolyzed by papain, and then subjected to ultrafiltration membrane for fractionation. The molecular mass of the peptides released during enzymatic hydrolysis of gluten was in the range of 5,000,15,000 D according to gel permeation chromatography profiles. The resultant hydrolysate (retentate fraction) after membrane fractionation produced the desired products with a protein yield of about 89%. The modified glutens had higher surface hydrophobicity compared with the original gluten. Moreover, the modified gluten protein had superior solubility to the original gluten in pH 3,10. The enhanced foaming capacity in the pH ranges studied had been found in the modified gluten. The improved functional properties of the modified gluten were related to the well-balanced distribution of hydrophilic and hydrophobic domains. The modified gluten was rich in glutamic acid and proline. PRACTICAL APPLICATIONS Gluten is an economically important byproduct during processing of wheat starch. The expanded utilization of gluten in food and nonfood applications had been limited for lack of desirable functional properties. To extend the range of available functional properties it needs to be investigated further. The limited enzymatic hydrolysis and membrane ultrafiltration had been extensively studied and had been shown to be very effective for improving the functional properties of the proteins. In this present study, wheat gluten was modified by enzymatic hydrolysis , membrane ultrafiltration fractionation. This method was proved to be an efficient way to modify wheat gluten protein. The functional properties of the modified glutens were significantly improved compared to the original gluten. It is expected that the modified gluten with enhanced functional properties could be used in a wide range of foods as new components to enhance their nutritional value and functional properties in food processing. [source] DLGdifferentially localizes Shaker K+ -channels in the central nervous system and retina of DrosophilaJOURNAL OF NEUROCHEMISTRY, Issue 6 2002C. Ruiz-Caņada Abstract Subcellular localization of ion channels is crucial for the transmission of electrical signals in the nervous system. Here we show that Discs-Large (DLG), a member of the MAGUK (membrane-associated guanylate kinases) family in Drosophila, co-localizes with Shaker potassium channels (Sh Kch) in most synaptic areas of the adult brain and in the outer membrane of photoreceptors. However, DLG is absent from axonal tracts in which Sh channels are concentrated. Truncation of the C-terminal of Sh (including the PDZ binding site) disturbs its pattern of distribution in both CNS and retina, while truncation of the guanylate kinase/C-terminal domain of DLG induces ectopic localization of these channels to neuronal somata in the CNS, but does not alter the distribution of channels in photoreceptors. Immunocytochemical, membrane fractionation and detergent solubilization analysis indicate that the C-terminal of Sh Kch is required for proper trafficking to its final destination. Thus, several major conclusions emerge from this study. First, DLG plays a major role in the localization of Shchannels in the CNS and retina. Second, localization of DLG in photoreceptors but not in the CNS seems to depend on its interaction with Sh. Third, the guanylate kinase/C-terminal domain of DLG is involved in the trafficking of Shaker channels but not of DLG in the CNS. Fourth, different mechanisms for the localization of Sh Kch operate in different cell types. [source] | ||||||||||