Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements

Members of the SLC20 family or type III Na⁺-coupled P_i cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular P_i homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic P_i cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na⁺-dependent ³²P_i uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li⁺ substituted for Na⁺. The dependence of the P_i-induced current on P_i concentration was Michaelian, and the dependence on Na⁺ concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent P_i affinity constant showed a minimum in the pH range 6.2–6.8 of 0.05 mM and increased to 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual ²²Na-³²P_i uptake and suggested a 2:1 Na⁺:P_i stoichiometry. A correlation of ³²P_i uptake and net charge movement indicated one charge translocation per P_i. Changes in oocyte surface pH were consistent with transport of monovalent P_i. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na⁺:H₂PO₄^–:Na⁺. Significantly, in contrast to type II Na⁺-P_i cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity. Na⁺-P_i cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor     

NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa

Dextrans are the main exopolysaccharides produced by Leuconostoc species. Other dextran-producing lactic acid bacteria include Streptococci, Lactobacilli, and Weissella species. Commercial production and structural analysis has focused mainly on dextrans from Leuconostoc species, particularly on Leuconostoc mesenteroides strains. In this study, we used NMR spectroscopy techniques to analyze the structures of dextrans produced by Leuconostoc citreum E497 and Weissella confusa E392. The dextrans were compared to that of L. mesenteroides B512F produced under the same conditions. Generally, W. confusa E392 showed better growth and produced more EPS than did L. citreum E497 and L. mesenteroides B512F. Both L. citreum E497 and W. confusa E392 produced a class 1 dextran. Dextran from L. citreum E497 contained about 11% alpha-(1-->2) and about 3.5% alpha-(1-->3)-linked branches whereas dextran from W. confusa E392 was linear with only a few (2.7%) alpha-(1-->3)-linked branches. Dextran from W. confusa E392 was found to be more linear than that of L. mesenteroides B512F, which, according to the present study, contained about 4.1% alpha-(1-->3)-linked branches. Functionality, whether physiological or technological, depends on the structure of the polysaccharide. Dextran from L. citreum E497 may be useful as a source of prebiotic gluco-oligosaccharides with alpha-(1-->2)-linked branches, whereas W. confusa E392 could be a suitable alternative to widely used L. mesenteroides B512F in the production of linear dextran.     

New enzyme-based method for analysis of water-soluble wheat arabinoxylans

Arabinoxylans (AX) are the predominant cell-wall polysaccharides in wheat flour. Water-extractable AX are essential for dough and bread properties and performance. However, there is no specific and accurate way of determining the content and structure of AX. An enzyme-assisted method employing an efficient enzyme mixture for the total hydrolysis of AX was developed in the present work. Enzymatic hydrolysis (EH) is a gentle method during which no unwanted sugar destruction occurs. Following EH, liberated monosaccharides were analysed by gas chromatography (GC) and liquid chromatography using HPAEC-PAD. The results were compared with acid methanolysis (AM) and acid hydrolysis (AH). EH performed better on commercially isolated AX samples than the reference method AM. Its action in the water extract from wheat flour was also more efficient than that of AM and comparable to the efficiency of AH. HPAEC-PAD revealed a significant amount of fructose in the water extract following EH, originating from fructans in wheat flour not detected in the GC analysis. The wheat flour examined contained 0.29% water-extractable AX. The arabinose/xylose ratio was 0.32. The enzyme-based method developed is applicable for comparison of different wheat flours and can be used in evaluating the effect of processing on the content and structure of water-extractable AX.     

Lipid content in 19 brackish and marine microalgae: influence of growth phase, salinity and temperature

Algal lipids provide essential fatty acids for higher trophic levels in the marine food web, and understanding the fatty acid composition in phytoplankton is critical for evaluating its value as a diet. Nineteen microalgal species, mainly originating from the Baltic Sea, covering major algal classes were grown in different growth conditions. Samples were taken during both the exponential and stationary growth phases and analysed regarding their fatty acid methyl esters and free fatty acids. Our results show that across all screened species, total fatty acids increased significantly from exponential to stationary growth phase. Furthermore, it was observed that warm-water species contained more lipids and differed in their lipid profile as compared with the cold-water species. Brackish water species also showed a slightly higher lipid content than the marine species, but their lipid profile was not significantly different. Plotting changes in lipids against changes in cell nitrogen revealed a significant dependency between decrease in cell nitrogen and increase in lipids across all tested species.     

Manipulating the genetic code for membrane protein production: What have we learnt so far?

With synthetic gene services, molecular cloning is as easy as ordering a pizza. However choosing the right RNA code for efficient protein production is less straightforward, more akin to deciding on the pizza toppings. The possibility to choose synonymous codons in the gene sequence has ignited a discussion that dates back 50years: Does synonymous codon use matter? Recent studies indicate that replacement of particular codons for synonymous codons can improve expression in homologous or heterologous hosts, however it is not always successful. Furthermore it is increasingly apparent that membrane protein biogenesis can be codon-sensitive. Single synonymous codon substitutions can influence mRNA stability, mRNA structure, translational initiation, translational elongation and even protein folding. Synonymous codon substitutions therefore need to be carefully evaluated when membrane proteins are engineered for higher production levels and further studies are needed to fully understand how to select the codons that are optimal for higher production. This article is part of a Special Issue entitled: Protein Folding in Membranes.