Gearhead26

While it is commonly understood that ATP contains a "high-energy" phosphate bond that allows it to do useful work in the cell, in reality this is only part of the story. The spontaneity of the reaction ATP --> ADP + Pi is very much dependent upon the ratio of ATP/ADP that exists within the cell. A high ratio means that ATP hydrolysis is very favorable, and the delta-G is quite negative, and work can be done by coupling ATP hydrolysis to various reactions that require energy input to drive them forward. So, essentially, it is not a property of the phosphate bond in the ATP that allows it to be a useful energy currency, but rather its relative abundance compared to the hydrolyzed form ADP. The cell has to be sure to maintain the proper ratio of ATP/ADP in order for the ATP to be useful at all.

Hey guys/gals, This is my first time going to an internet forum on account of an issue in the lab. I'm no biochemist, but have decided (for some masochistic reason) that the best way to identify a particular enzyme whose activity can be measured but for which no gene has been identified, is to try to purify this enzyme "from scratch". We have an FPLC, and I've used ion-exchange and gel filtration to purify proteins, however in those cases I always knew the characteristics of those proteins (pI, cofactors, etc) and they were overexpressed. In this case, I have a sensitive assay for the activity of this "new" enzyme, however I obviously know nothing about its characteristics. In order to remove bulk impurities, I have tried ammonium sulfate precipitation. The enzyme in question salts out in the 45-55% fraction, however it looses tons of activity (The overall yield after AS precipitation is 1.8% of the original cell-free extract). I don't know what I should do at this point. My gut tells me that it's not the overnight dialysis (post-precipitation) that is causing loss of activity, because I have dialyzed cell-free extract overnight and loose only 50% of the activity compared to just leaving the extract in the fridge overnight. Is it worth trying to optimize this step, or should I try to use another approach entirely? Is it "normal" for an enzyme to loose this much activity during AS precipitation? I had always thought that most enzymes were quite stable in an AS pellet... Thanks!