Substrate Analogs, Membrane Vesicles, and Bacterial Mutants Are Used to Study Transport

Isotopes, Substrate Analogs, Membrane Vesicles, and Bacterial Mutants Are Used to Study Transport 

To measure rates of transport, methods must be available for identifying molecules that have crossed the membrane. Radioactively labeled substrates are commonly used for this purpose. In a typical experiment, the substrate is added to a suspension of cells, time is allowed for transport to occur, the cells are collected and rinsed rapidly by centrifugation or filtration, and the amount of labeled substrate in the cells is measured. Control measurements usually are made with a labeled substrate that the cells do not transport. 

To study transport in the absence of complicating metabolic processes, it often is advantageous to work with isolated membrane vesicles rather than with whole cells. Cytoplasmic membrane vesicles can be obtained from either eukaryotic or bacterial cells after homogenization or osmotic lysis. Transport proteins that have been solubilized with detergents also can be reincorporated into synthetic phospholipid vesicles (fig. 17.27). 

The amino acid sequences of numerous transport proteins have been elucidated during the last 10 years. As with conventional enzymes, molecular biological techniques now make it possible to modify individual amino acid residues in the proteins and thus to probe the roles these residues play in catalysis of facilitated diffusion or active transport. 

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