From pyruvate to PEP...the unknown pathway

Abstract only availableSoybean is a major crop in the United States. Its survival and relationship with its environment are closely monitored. For example, we are looking at the symbiotic relationship between soybean and the soil bacteria. These bacteria can convert nitrogen in the air into usable nitrogen for the soybean plant. This process occurs when the rhizobium forms a nodule on the soybean root. These nodules are complex, hyperplastic tissue masses derived from cortical cells that transport nitrogen as uerides. The soybean plant then in turn acts as a carbon and energy source for the bacteria. The metabolic pathway for the bacteria's reception and consumption of this carbon is unknown. Our specific focus is to use in gel assays to identify the presence of glyceraldehydes-3-P-dehydrogenase, phosphoglycerate kinase, phosohpoglycerate mutase and enolase, used by the plant to synthesize a reaction to convert glyceraldehydes-3-phosphate into PEP (phosphoenolpyruvate) which will be conducted after my departure from the lab. We first conduct a protein extraction to isolate only the desired material from the soybean plant. We then use a one-dimensional gel enzyme assay to determine whether our four main enzymes are active (we received positive results for all four). Also, a two-dimensional gel enzyme assay to determine whether the enzyme we have found is indeed the one we think we have identified in one dimension (we have found positive results for all except phosphoglycerate mutase). Finally, we use an enzyme assay to determine if there is enzyme activity by measuring the absorbance of a solution containing substrate and introducing the enzyme. We found the presence and activity of all of the crucial enzymes involved in the pathway. We have pretty much concluded that this pathway, formerly thought to be a part of the Alanine Transport model, is more likely to be a part of the plant's metabolism.MU Monsanto Undergraduate Research Fellowshi

Human FXYD2 G41R mutation responsible for renal hypomagnesemia behaves as an inward-rectifying cation channel

A mutation in the human FXYD2 polypeptide (Na-K-ATPase γ subunit) that changes a conserved transmembrane glycine to arginine is linked to dominant renal hypomagnesemia. Xenopus laevis oocytes injected with wild-type FXYD2 or the mutant G41R cRNAs expressed large nonselective ion currents. However, in contrast to the wild-type FXYD2 currents, inward rectifying cation currents were induced by hyperpolarization pulses in oocytes expressing the G41R mutant. Injection of EDTA into the oocyte removed inward rectification in the oocytes expressing the mutant, but did not alter the nonlinear current-voltage relationship of the wild-type FXYD2 pseudo-steady-state currents. Extracellular divalent ions, Ca2+ and Ba2+, and trivalent cations, La3+, blocked both the wild-type and mutant FXYD2 currents. Site-directed mutagenesis of G41 demonstrated that a positive charge at this site is required for the inward rectification. When the wild-type FXYD2 was expressed in Madin-Darby canine kidney cells, the cells in the presence of a large apical-to-basolateral Mg2+ gradient and at negative potentials had an increase in transepithelial current compared with cells expressing the G41R mutant or control transfected cells. Moreover, this current was inhibited by extracellular Ba2+ at the basolateral surface. These results suggest that FXYD2 can mediate basolateral extrusion of magnesium from cultured renal epithelial cells and provide new insights into the understanding of the possible physiological roles of FXYD2 wild-type and mutant proteins