Experimental part
Strains and growth conditions,
Estimation of glucose concentrations, Bacterial conjugation, and DNA methods
and sequencing were described previously (1), in details. The Glucose Uptake
Assays Protocol was as follows: Cells were harvested at the mid-exponential
phase, washed with phosphate buffer (100 mM, pH 6.5) and resuspended in the
same buffer essentially as described by Walsh et al. (2). Uptake was measured
using D-[U-14C]glucose (Amersham, England, 291 mCi/mmol) at concentrations
ranging from 0.25 to 50 mM. Z. mobilis
cells (50 μl) and 5-fold-concentrated radiolabeled glucose (12.5 μl)
were preincubated separately at 200C, mixed together to give the
appropriate glucose concentration and vortexed immediately. Uptake was stopped
by adding cold (-2.50C) 10 ml phosphate buffer (100 mM, pH 7.5)
containing 500 mM unlabelled glucose. Cells were immediately filtered and
washed with 10 ml of the same buffer. The uptake rate was expressed as nanomoles
of glucose taken up per min per mg of total protein.
Results and Discussion
Attempts to fit all collected series of glucose uptake
experimental data by nonlinear regression to a one kinetic component nonlinear
equation i.e. an analogous to the Michaelis-Menten equation [1] were failed.
In several cases, additional parameters were determined by nonlinear curve
fitting of the following equations, [2] and [3] to the experimental data.
v(uptake) = 
[1]
v(uptake) = 
[2]
v(uptake) = 
+ 
[3]
Equation [2] denotes that there is one molecule of
proteinaceous transporter, which can bind two Glucose molecules, while equation
[3] denotes that there are two different molecules of proteinaceous transporters,
which can bind only one molecule of Glucose. In equations, [2] and [3] Km1, and Km2 are the apparent affinity constants, and Vmax1, and Vmax2 are the respective maximal rates of Glucose uptake;
[S] is the concentration of Glucose. In Table
1 are depicted all series of glucose uptake data. Some of them were exhibited
Michaelis-Menten like behavior, while other were exhibited biphasic kinetics
in Eadie – Hofstee plots.
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