Quantifying enzyme activity in living cells.

For over a century, enzymatic activity has been studied , assuming similar activity in the crowded cellular milieu. Here, we determined in real time the catalytic activity of TEM1-β-lactamase inside living cells and compared the values to those obtained We found the apparent catalytic efficiency, / , to be lower than , with significant cell-to-cell variability. Surprisingly, the results show that inside the cell the apparent catalytic efficiency decreases, and increases with increasing enzyme concentration.

Direct determination of enzyme kinetic parameters from single reactions using a new progress curve analysis tool.

With computer-based data-fitting methods becoming a standard tool in biochemistry, progress curve analysis of enzyme kinetics is a feasible, yet seldom used tool. Here we present a versatile Matlab-based tool (PCAT) to analyze catalysis progress curves with three complementary model approaches. The first two models are based on the known closed-form solution for this problem: the first describes the required Lambert W function with an analytical approximation and the second provides a numerical solution of the Lambert W function.

Microtubule assembly-derived by dimerization of TPPP/p25. Evaluation of thermodynamic parameters for multiple equilibrium system from ITC data.

Background: The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system. In this paper the role of dimerization in its microtubule-related functions is established, and an approach is proposed to evaluate thermodynamic constants for multiple equilibrium systems from ITC measurements.

Methods: For structural studies size exclusion chromatography, SDS-PAGE, chemical cross-linking, circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry were used; the functional effect was analyzed by tubulin polymerization assay.

Zn²+-induced rearrangement of the disordered TPPP/p25 affects its microtubule assembly and GTPase activity.

Tubulin polymerization promoting protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays crucial role in the myelination of oligodendrocytes. Here we showed by CD, fluorescence, and NMR spectroscopies that Zn(2+) is the first ligand that induces considerable rearrangement of the disordered TPPP/p25. Zinc finger motif (His(2)Cys(2)) (His(61)-Cys(83)) was identified within the flexible region of TPPP/p25 straddled by extended unstructured N- and C-terminal regions.

Disordered TPPP/p25 binds GTP and displays Mg2+-dependent GTPase activity.

The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays a crucial role in differentiation of oligodendrocytes. Here we first demonstrated by multinuclear NMR that the extended disordered segments are localized at the N- and C-terminals straddling a flexible region. We showed by affinity chromatography, fluorescence spectroscopy and circular dichroism that GTP binds to TPPP/p25 likely within the flexible region; neither positions nor intensities of the peaks in the assigned terminals were affected by GTP.

Tubulin polymerization promoting proteins (TPPPs): members of a new family with distinct structures and functions.

TPPP/p25 is a brain-specific protein, which induces tubulin polymerization and microtubule (MT) bundling and is enriched in Lewy bodies characteristic of Parkinson's disease [Tirián et al. (2003) Proc. Natl.