The maltose transport system of Escherichia coli is a high affinity, periplasmic-protein dependent transport system. The transport system provides for the uptake of both maltose and maltodextrins (up to seven glucose residues in length) into the cytoplasm. At the present time, we are investigating the stoichiometric relationship between the ability of the inner membrane component of this transport system to hydrolyse ATP in the presence of various substrates with its ability to transport these substrates. Previous work in our lab has shown that the rate of ATP hydrolysis by the inner membrane component of this transport system depends upon the substrate which is being translocated. Maltodextrins, such as maltotetraose, stimulate this protein complex's ATP activity more than maltose. However, whether this stimulation is due to more maltotetraose being transported into the cytoplasm via the protein complex, or due to more ATP per maltodextrin being needed to transport a single maltodextrin moiety is not known. To determine the nature of this increase in stimulation compared to maltose, we plan to measure the uptake of both maltose and maltotetraose directly. The methodology of this uptake experiment is as follows. Proteoliposomes (containing the inner membrane protein complex) are prepared in the presence of ATP such that ATP is trapped inside the proteoliposomes upon their formation. Once formed, the proteoliposomes are incubated with both maltose-binding protein (the periplasmic component of the transport system which binds all substrates, interacts with the inner membrane complex when in the MBP-substrate form, and is essential for substrate transport into the cytoplams to occur) and tritiated substrate. After MBP and labelled substrate have been added, aliquots will be removed at various time points and substrate uptake will be measured by liquid scintillation counting. We expect either of two results. Maltotetraose may be transported at the same rate as maltose. This would imply that maltodextrins require more ATP per maltodextrin transported than does maltose. If maltotetraose is transported at a quicker rate than maltose, it implies that maltotetraose, and other maltodextrins, stimulate the inner-membrane complex more than maltose such that they are taken up into the cytoplasm more readily.
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