GLYCOLYSIS TOXINS AND INHIBITORS

This will come in two parts. It might work to skip part one, and go directly to part two. However, if you do part one, which changes the yeast into a cell-free mass, you will not have to be concerned with the ability of various reagents' being able to penetrate the membranes. For this first time around, I suggest that you skip part one, and jump right to part 2 with your quarter lump of yeast cake slurried in 15 ml of 7.5% sucrose solution.

  1. Preparation of Cell-Free Yeast Extract.

    (This was first done by Buchner, which led to his getting the first Nobel Prize ever - for demonstrating cell-free fermentation, and not for inventing the porcelain collander!).

    Blend 80 gm of 20-40 dried yeast with 334 ml of 0.1 M KHCO3 in a blendor for 60 seconds. Allow the slurry to stand for 5 minutes and then blend again for an additional 30 seconds. It will then autolyze while sitting at 37°c for 4 hrs. In the cold, centrifuge for 15 min at 25,000×G (10,000 rpm or more; note: a clinical centrifuge has max of 3,600 rpm). Decant off the clear supernate and use immediately if possible. However, this will retain activity for up to 24 hrs if stored at 1-3°c. DO NOT FREEZE! Trick: float it in a large container of half ice - half liquid water; put container in refrigerator.

    Pre-incubation: to 100 ml of above cold extract, add 10 ml of 0.2 M MgCl2 and 20 ml of 1 M glucose. (Addition of 20 ml of 0.2 M FDP also helps "prime the pump.") Incubate at 37°c for 45 minutes. The prep ought to bubble vigorously if drawn up into a pipette. It is now ready to use. Go on to Part Two, next.

  2. The Experiment

    To every 14 ml of the above, before you pour it onto the two layers of paper towel, mix in 1 ml of any ONE of the following toxins to test their effects:

    • 0.1 M NaH2AsO4 (arsenate)
    • 0.4 M iodoacetic acid (IAA)
    • 0.2 M KF or NaF (fluoride)
    • 0.2 M NaHSO3 (sodium bisulfite)

    Immediately pour onto paper towels so that they are homogeniously moist, accordianate, stuff into reaction vessel, and connect up the gas collection devices, and begin recording CO2 evolution data.

    IMPORTANT. Before the students actually add their slurries onto the towels and put them into the flasks, have the students do a "wet practice run" - 15 ml of water onto two towels, accordianate, stuff into flask, unfurl, get all the gas collection equipment set up ready to work. (One thing college students like to refuse to do is PRACTICE, and then when the chips are down they find they are lacking something. So, next time you are at an auction, buy a bull whip! Maybe the sound of lion's roar might also help!!!)

    SUSPECTED RESULTS

    Arsenate. When GAP normallly goes to P2GA with the uptake of an inorganic phosphate, what happens now is that arsenate is taken up instead and the PAsGA that is made spontaneously breaks down. Hence rather than finding that glycolysis gives a P/O = 2 / 0, you get 0 / 0 (no ATP made). Glycolysis thus becomes a runaway reaction that amounts to nothing.

    Fluoride and IAA should cause cause glycolysis to more or less stop as a major enzyme is inactivated by either losing its substrate (which is phosphate as Fl- binds with it), or it active site is blocked (IAA).

    Bisulfite precipitates the acetaldehyde at the next to the last step of alcoholic fermentation. Hence NADH is not able to be recycled on that pathway. However live yeast have an alternative route it can take - though it is no good for making a net gain of ATP. What it does is takes GAP to glycerol phosphate, and then recovers the phosphate leaving glycerol. (There are two important commercial applications here in the biosynthesis of glycerol and for acetaldehyde.)

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