Prefractionation procedures facilitate the identification of lower‐abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one‐tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal‐regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two‐dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK‐activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.

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Interactions betweenβ‒amyloid (Aβ) peptides and neuronal membranes play an important role in Alzheimer's disease (AD). Using surface plasmon resonance we assayed a kinetic model to study the interactions of Aβ25‒35, Aβ40 and Aβ42 with surfaces containing single glycolipids (Asialo‒GM1, GM1, GD1a or GT1b). The larger peptides interacted with gangliosides stronger than Aβ25‒35, which showed some significant bindings solely at high concentrations under acidic conditions. Only the interactions at low Aβconcentrations were useful to calculate the kinetic constants. The affinities increased at low pH. The specificity, but not the affinity correlated with the number of sialic acids in the ganglioside sugar moiety. The most important finding in this study, was a special group of sensorgrams with linear association phases that appeared for the interactions of Aβwith the membranes containing gangliosides, due to the following process: when Aβis injected at a critical concentration, the first molecules that interact with the gangliosides remain fixed on the membrane. Next Aβmolecules bind to these fixed molecules, so that for each Aβmolecule bound, new binding sites are activated on the surface in a linear ratio, which explains the linear shape of the sensorgrams. This way a laminar‒arranged Aβaccumulate is progressively formed on the membrane surface and fixed there. These linear sensorgrams were not observe with asialo‒GM1 or DMPC, which indicates the main role of sialic acid in these interactions. This model for progressive Aβdeposition could simulate the initial stage of the Aβpeptide accumulation on cell surfaces.

The effects of phytate on the hydrolysis of lactose with /?-galactosidases from bovine liver and Escherichia coli were investigated. The activities of both /?-galactosidases were decreased to the same extent by increased concentrations of phytate. The rates of inhibition of /?-galactosidase activity from E. coli in a reaction mixture containing 10mMof phytate were 78.9% and 64.4%, respectively, in the absence of and with 4mMof Mg2+. Therefore, it was found that the stimulatory effect ofMg2+ was hardly affected by the presence ofphytate in the range from 2 to lOmM.The /?-galactosidase activity was also not influenced by preincubating /?-galactosidase or lactose with phytate. Kinetic studies showed that the inhibition of /?-galactosidase activity by phytate was of an uncompetitive type with a Ki value of 3.46 mM.Therefore, it is considered that phytate may interact with a complex of /?-galactosidase and lactose.