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Cu + -ATPases are integral membrane proteins belonging to the IB subfamily of the P-type ATPases that couple Cu + transport to the hydrolysis of ATP. As some structural and functional particularities arise for Cu + -ATPases, several authors suggest that some of the reaction steps of the Albers-Post model postulated for other P-ATPases may be different. In this work we describe a functional characterization of Legionella pneumophila Cu + -ATPase (LpCopA), the first P IB -ATPase whose structure was determined by X-ray crystallography. Cu + -ATPase activity of the enzyme presents a maximum at ∼37 °C and pH 6.6-6.8. Phospholipids enhance LpCopA Cu + -ATPase activity in a non-essential mode where optimal activity is achieved at an asolectin molar fraction of 0.15 and an amphiphile-protein ratio of ~30,000. As described for other P-ATPases, Mg 2+ acts as an essential activator. Furthermore, Cu + -ATPase activity dependence on [Cu + ] and [ATP] can both be described by a sum of two hyperbolic functions. Based on that, and the [Cu + ] and [ATP] dependencies of the best fitting parameters of the hyperbolae pointed above, we propose a minimal reaction scheme for the catalytic mechanism that shares the basic reaction steps of the Albers-Post model for P-type ATPases. The reaction scheme postulated contemplates two different binding affinities for a single ATP (apparent affinities of 0.66 and 550 μM at [Cu + ] → ∞) and binding of at least 2 Cu + with different affinities as well (apparent affinities of 1.4 and 102.5 μM at [ATP] → ∞)., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The present paper describes a quenching-and-washing chamber (QWC) to be used with a rapid-mixing apparatus (RMA) for the study of processes in the millisecond time scale. The QWC enables fast, nondestructive quenching by cooling and dilution of reactants in particulate systems that can be trapped on a filter. The reaction mixture (e.g., at 25 degrees C) is injected from the RMA into the QWC where it is immediately mixed with a stream of ice-cold solution flowing at a rate of 15-40 ml s-1. Quenching requires that the process studied is slowed considerably by cooling to 0-2 degrees C and/or by removal of reactants by dilution. The equipment was characterized through a study of the tight binding (occlusion) of 86Rb+ to purified, membrane-bound Na+/K+-ATPase. Millipore filters of 0.22-0.80 microm pore size trapped close to 100% of the enzyme protein. Enzyme with occluded 86Rb+ was formed in the RMA under conditions where the rate constant for release of Rb+ at 25 degrees C is up to 25 s-1 and then injected into the QWC. The high off-rate constant is due to the presence of 2.5 mM ATP, which accelerates release of Rb+. The recovery of occluded 86Rb+ on the filter was at least 90%, indicating that both cooling of the reactants and dilution of ATP are fast enough to stop the reaction. The quenching time was 3-4 ms., (Copyright 1999 Academic Press.)