Kinetic Measurements of Di- and Tripeptide and Peptidomimetic Drug Transport in Different Kidney Regions Using the Fluorescent Membrane Potential-Sensitive Dye, DiS-C3-(3).

Tri- and dipeptides are transported in the kidney by PEPT1 and PEPT2 isoforms. The aim of this study was to investigate differences in transport kinetics between renal brush border (BBMV) and outer medulla (OMMV) membrane vesicles (where PEPT1 and PEPT2 are sequentially available) for a range of di- and tripeptides and peptidomimetic drugs. This was accomplished through the use of the potential-sensitive fluorescent dye 3,3'-dipropylthiacarbocyanine iodide [DiS-C3-(3)]. BBMV and OMMV were prepared from the rat kidney using standard techniques. The presence of PEPT1 in BBMV and PEPT2 in OMMV was confirmed using Western blotting. Fluorescence changes were measured when extravesicular medium at pH 6.6 containing 0-1 mM substrates was added to a cuvette containing vesicles pre-equilibrated at pH 7.4 and 2.71 μM DiS-C3-(3). An increase in fluorescence intensity occurred upon substrate addition reflecting the expected positive change in membrane potential difference. Of the range of substrates studied, OMMV manifested the highest affinity to cefadroxil and valacyclovir (K m 4.3 ± 1.2 and 11.7 ± 3.2 µM, respectively) compared to other substrates, whilst the BBMV showed a higher affinity to Gly-His (K m 15.4 ± 3.1 µM) compared to other substrates. In addition, OMMV showed higher affinity and capacity to Gly-Gln (K m 47.1 ± 9.8 µM, 55.5 ± 2.8 ΔF/s/mg protein) than BBMV (K m 78.1 ± 13.3 µM and 35.5 ± 1.7 ΔF/s/mg protein, respectively). In conclusion, this study successfully separated the expression of PEPT1 and PEPT2 into different vesicle preparations inferring their activity in different regions of the renal proximal tubule

Première occurrence du Vetulicolia problématique Skeemella clavula dans la Formation cambrienne de Marjum Formation d'Utah, É.U.A.

The Cambrian Marjum Formation of western Utah (USA) preserves a diverse soft-bodied fauna from the upper Drumian that is slightly younger than the well-known Burgess Shale. While the Marjum is dominated by arthropods, animals belonging to a variety of phyla have been found. Here, we document the second occurrence of the rare, enigmatic taxon Skeemella clavula, which was previously thought to be restricted to the Pierson Cove Formation of the Drum Mountains. The occurrence in the Marjum represents a new preservational setting, as well as a slightly younger deposit. The new specimens also expand the number of known specimens to three. In addition, they improve understanding of the morphology of this representative of the problematic phylum Vetulicolia.Dans l'ouest de l'Utah (É.U.A.), la Formation cambrienne de Marjum préserve une faune diversifiée d'animaux à corps mou dans le Drumien supérieur, une faune légèrement plus jeune que celle des fameux Schistes de Burgess. Alors que la Formation de Marjum est dominée par les arthropodes, des animaux appartenant à différents phylums ont également été découverts. Nous illustrons ici la seconde occurrence de Skeemella clavula, un rare et énigmatique taxon dont on pensait précédemment qu'il n'était présent que dans la Formation de Pierson Cove des Montagnes de Drum (N Utah). Leur présence dans la Formation de Marjum représente un nouveau milieu de conservation, ainsi qu'un dépôt légèrement plus jeune. Les nouveaux spécimens portent à trois le nombre de spécimens connus. Enfin, ces spécimens contribuent à une meilleure compréhension de la morphologie de ce représentant du phylum problématique des Vetulicolia

Evidence for a specific phosphoryl binding site in swine kidney phosphofructokinase

Phosphofructokinase (PFK) from swine kidney was purified by a procedure which included affinity chromatography on Cibacron blue F3GA-Sepharose 4B and ATP-Sepharose 413 columns in order to examine its binding properties. The homogeneous enzyme was purified more than 3 000-fold with a yield of 30% and it had a specific activity of 39.8 µmol/min/ mg of protein at 25°C. The molecular weight of the native enzyme was 360 000 and it contained 4 identical subunits of molecular weight 88 000. The principal catalytically reacting form of the enzyme had a S20,w of 13.7 S which corresponds to a molecular weight of 360 000 ± 6 000. The initial velocity patterns in the forward and reverse directions suggested a sequential mechanism for the reaction. The Km values for fructose 6-phosphate, ATP, fructose, 1,6-bisP and ADP were 33 μM, 8.3 μM, 460 μM, and 110 μM, respectively. The homogeneous native enzyme binds specifically to phosphoryl groups immobilized in cellulose phosphate columns. ATP and fructose 6-phosphate interacted with the enzyme and decreased its affinity for phosphoryl binding sites. Other metabolites including fructose 1,6-bisP, glucose 6-phosphate and various nucleotides, alone or in various combinations, were ineffective in promoting the dissociation of the enzyme. Allosteric effectors of the enzyme, such as citrate and AMP were also inactive. However, they cooperatively altered the eoncentration of ATP required to dissociate the enzyme from phosphoryl groups. The bound enzyme was enzymatically inactive. The enzyme was also inactivated when it was treated with pyridoxal 5'-phosphate and reduced with sodium borohydride and the inactive enzyme no longer bound to cellulose phosphate. These effects were not observed when treatment with pyridoxal 5'-phosphate was carried out in the presence of fructose 6-phosphate. These observations and the results of similar studies with swine kidney fructose 1,6-bisphosphatase (FBPase) show that both enzymes share the unique property of binding specifically to phosphoryl groups. FBPase interacts through its allosteric AMP binding site and PFK binds through its fructose 6-P binding site. This specific binding of both enzymes through these sites result in the inactivation of PFK and the desensitization of FBPase to allosteric inhibition by AMP. In the unbound state PFK may be active and FBPase can be inhibited by AMP. Taken collectively, these binding effects could play a role in the reciprocal regulation of these enzymes during gluconeogenesis in kidney