Preparation and Assay of Activities in Subcellular Samples

In group I, we considered the question of obtaining an activity from a tissue or organ, from a biological fluid, or from cultured cells. The primary task in all these samples was the separation of the extracellular and cellular compartments. Next, the problem of separation of the different cell types within the cellular compartment was considered. In the section that follows, we will open the cell for a look inside. However, let us first consider briefly the surface of the intact cell and the problems associated with the assay of any activities that might be located there. 

Setting up an HPLC assay for activities on intact cells requires only one major change since the reaction mixture will contain cells, the samples for HPLC analysis cannot be injected directly onto the analytical column thus the reactions must be terminated and any precipitated proteins removed (see Chapter 4). Termination can be accomplished by centrifugation at a low speed or by filtration (see Fig. 4.9B). However, care should be taken to avoid any cell breakage, particularly if the product of the primary or even secondary reactions can also be found as a naturally occurring intracellular component. 

Consider an assay for a cell surface ATPase where the reaction product is ADP. A reaction mixture is prepared that contains ATP as the substrate. The addition of the cells will start the reaction, and ADP will be produced directly into the extracellular solution. Lysis of the cell during this assay will release cellular ADP into the incubation medium, altering the results of the assay. 

In the absence of any specific information about the nature of such activities, it is often best to mix a cocktail containing several or all of the available proteolytic inhibitors. These include such compounds as 1,10-phenanthroline, soybean trypsin inhibitor, leupeptin, benzamidine, antipain, aprotinin, phenyl-methanesulfanyl fluoride, and diisopropylfluorophosphate. As a precaution against any enzyme destruction, such a cocktail is best added to the buffer in which the cellular lysis will be carried out (Fig. 5.6). 

Opening the cells can have other consequences. For example, many enzymes require cofactors for catalytic activity. If, as a result of the lysis of cells and organelles, the enzymes become separated from these cofactors, a loss 

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