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\protocol\ppi
J. Walton. April 13, 1993
ATP/PPi assay for cyclic peptide synthetases
Enzyme extraction buffer:
50 mM KH2PO4 pH 7
10% glycerol
4 mM DTT
0.2 M KCl
2 mM EDTA
DTT is important; phosphate buffer is the best, according to work with GS. Glycerol
concentration maybe should be higher - this might be the trick to keeping HTS together during
purification but I haven't systematically tested it. The Berlin group uses 40-50% glycerol to get
out cyclosporin synthetase intact, but such a high conc. must be hard to work with.
With GS they add 0.2 M KCl and after grinding the cells stir the extract for half-hour in
the cold. I do this routinely but haven't tested its importance.
I have found EDTA to be essential to minimize proteolysis. I routinely add PMSF but
don't know if it's important. I haven't seen any effect of other proteolysis inhibitors such as
leupeptin and pepstatin.
In my protocol, I centrifuge once to get rid of cell wall debris, then add polyethylenimine
to a final conc. of 0.2%. This is essential for me and probably even more so with bacteria. It gets
rid of lots of nucleic acid and other garbage. Make the PEI as a 13% stock (must be
NEUTRALIZED with conc HCl) and store cold. Stir for 5 min in the cold, spin 10,000, 10 min.
After PEI I always get a large gloppy pellet.
Then I do an ammonium sulfate precipitation. HTS comes down at 40% saturation and so
this is a great purification step. I think all CPS's, because they are so large and hydrophobic, will
ppt. at low amm. sulfate concs. For me it gets rid of the majority of non-specific exchange
activity.
I just measure the volume of the post-PEI supernatant and add solid amm. sulfate directly.
I let it stir for about 30 min, then centrifuge 15,000 for 15 min.
2
Assay reaction mix has four components in equal ratios. Since you then add one part
enzyme, each of the assay reaction components (as written) is 5x the actual final concentration in
the assay.
50 ml
1. Buffer: (store frozen)
100 mM TES, pH 7
1.15 gm
25 mM MgSO4
25% sucrose
12.5 gm
10 mM DTT
0.07 gm
10 mM ATP (2Na, 3H20: MW 605) .303 gm
Add all but ATP, adjust pH with 5M NaOH. Add ATP, readjust pH with 5M NaOH. I found that
a final Mg conc. of 5 mM was slightly better than 10 mM for my enzyme.
2. amino acid stock - Km's of HTS1 for L-pro: 17 mM; HTS-2 for D-ala: 3 mM; for L-ala: 100
mM. So for HTS-1 use stock of ca. 100 mM L-pro. For HTS-2 use stock of D-ala of ca. 50 mM
and stock of
L-ala of 500 mM. Store frozen.
3. Bovine serum albumin
0.4% dissolved in 0.01 N NaOH. Store in the refrigerator or frozen.
4. 5 mM sodium [32P]pyrophosphate (store frozen). The precise cpm per reaction doesn't matter
since the conc. of unlabelled PPi predominates. I usually use 200,000 to 500,000 cpm per
reaction, so the stock should be 5x this. You can use it for two weeks or more; there doesn't seem
to be any "inhibition" by decayed PPi. [32]PPi is sold by NEN (# NEX 019). I make a stock of 40
mM unlabelled PPi.
To make the working enzyme reaction mix, mix one part of each of the four ingredients. I
usually make four ml of each solution containing a different amino acid. Don't forget the water
control - add water instead of amino acid. This is essential to correct for "nonspecific" exchange
activity which can be quite high especially in crude extracts. You can also add all the amino acids
together in one tube - since the activations are independent, the total activity will be sum of all
the individual amino acids.
I do the reactions in 1.5 ml Eppendorf tubes without caps (you can buy them this way). I
add 100 ul of reaction mix to each tube, then 25 ul of enzyme mix. I usually have a problem with
too much activity rather than too little, so frequently I have to dilute the enzyme 5 or more times.
In the final volume of 125 ul all the components are at the final correct conc.
I run the reaction at 30 or 37 C in a water bath, for 20 to 30 min. Stop the reaction by
adding 0.5 ml of charcoal suspension. This is made by adding 16 g charcoal (Sigma activated
acid-washed # C-4836) plus 44.6 gm sodium pyrophosphate plus 50 ml 70% perchloric acid to
water, one liter final volume. This kills the reaction and the charcoal binds the ATP and not the
3
pyrophosphate. Since the charcoal settles out, during this part of the assay I keep the charcoal
suspension in a small beaker on a stir plate.
Filter the samples. You can use the same pipet tip over and over. I use a Millipore 12place filter manifold. Any type of filter (almost) will work - GF/A or equivalent is the least
expensive. I wet the filters in perchlorate/pyrophosphate before adding the charcoal
suspension/reaction mix, but I don't know if this reduces background or not. Wash the filters
twice with 5 ml of 40 mM pyrophosphate plus 1.4% perchloric acid, then once with 5 ml water. I
have found this sufficient. Count the filters. Because the charcoal causes quite a bit of quenching
I use a scintillation cocktail (for aqueous samples) but you could probably get by with just water
(Cerenkov counting).
In my experience the assay is remarkably sensitive and reproducible. Routinely, at least
for HPLC fractions, we do not do duplicates.
April 2003: I assayed some C. carbonum partially purified HTS1 preps that were >10
years old. They had been stored at -80C. They were still very active.