A second magnesium ion is critical for ATP binding in the kinase domain of the oncoprotein v-Fps

The activity of the kinase domain of the oncoprotein v-Fps was found to be sensitive to the concentration of magnesium ions. Plots of initial velocity versus free magnesium concentration are hyperbolic and do not extrapolate to the origin at stoichiometric ATP-Mg, indicating that there are two sites for metal chelation on the enzyme and the second is nonessential for catalysis. The second metal is strongly activating and increases the reaction rate constant almost 20-fold from 0.5 to 8.3 s-1 using 0.2 mM ATP-Mg and 1 mM peptide, EAEIYEAIE. This increase in rate is due to a large increase in the apparent affinity of ATP-Mg at high magnesium concentrations. At 0.5 and 10 mM free Mg2+, KATP-Mg is 3.6 and 0.22 mM, respectively. Extrapolation of the observed affinity of ATP-Mg to zero and infinite free metal indicates that KATP-Mg is greater than 8 mM in the absence of the second metal and 0.1 mM in the presence of the second metal, a minimum 80-fold enhancement. By comparison, free levels of the divalent ion do not influence maximum turnover (kcat) and have only a 2-fold effect on the Km for the peptide substrate between 0.5 and 20 mM free Mg2+. Viscosometric studies indicate that free Mg2+ does not influence the rates of phosphoryl transfer or net product release above 0.5 mM but does affect directly the dissociation constant for ATP-Mg. The Kd for ATP-Mg in the absence and presence of the second metal ion is >32 and 0.4 mM, respectively. At high magnesium concentrations, ATP-Mg and the peptide substrate bind independently, while at lower concentrations (0.5 mM), there is significant negative binding synergism suggesting that the second metal may help to reduce charge repulsion between ATP-Mg and the peptide. The data indicate that the first metal is sufficient for phosphoryl transfer. While the second metal could have some influence on phosphoryl transfer or product binding, it is a potent activator that functions minimally by controlling ATP-Mg binding.     

Factors facilitating or impeding older adults’ creative contributions in the collaborative design of a novel DTV-based application

This article describes some of the challenges of supporting older adults’ creative input to the design of an (as yet) undefined digital television application. Focusing on commonly used brainstorming and paper prototyping techniques, constructive insights are gathered from users with limited experience of interactive technology. Encouraging older participants to move beyond what they are familiar with, the research reports on how fragmented conceptual knowledge of familiar technologies can either facilitate or impede the creative development of new ideas. This includes bias or contradictory ideas that can emerge in the elicitation process. As reported, other factors, such as the self-perception of inadequate drawing skills, demonstrate further barriers for some older adults to articulate their visions and aspirations for suitably designed technology.     

Determination of partition coefficients and aqueous solubilities by reverse phase chromatography—I: Theory and background

Water solubilities and octanol/water partition coefficients are widely used to predict partitioning and bioconcentration phenomena for hydrophobic organic pollutants in aqueous systems. This paper is the first in a two part series describing the application of high performance reverse phase liquid chromatography (HPRPLC) for indirect estimation of these two physicochemical parameters to facilitate environmental fate and transport predictions for organic compounds. In the first part, thermodynamic factors which control partitioning processes, water solubilities, and reverse phase retention behavior are discussed, and models for interlinking these three properties are summarized. The second part presents the results of aqueous solubility and octanol/water partition coefficient predictions for a number of organic contaminants from measurements of their HPRPLC behavior, and compares the modeling capabilities of some of the theoretical partitioning/solubility equations developed in the first paper.     

A membrane filter method for the enumeration of aquatic ammonifiers

A standard procedure for the enumeration of heterotrophic bacteria, utilizing heterotrophic plate count medium (m-HPC), has been modified to allow bacterial ammonifier populations also to be counted. Nessler's reagent was included as an outside indicator to ascertain what portion of the population is capable of ammonification. Typically the ammonifier population varied among aquatic habitats, ranging from 0.08 to 37.0% of the heterotrophic population in river water samples, 0.2 to 10.6% for irrigation water samples, and 1.4 to 3.0% for pond and lake water samples.