amp deaminase purification and assay via coupled glutamate dehydrogenase reaction in saccharomyces cerevisiae

1: AMP deaminasepurification and assay via coupled glutamate dehydrogenase reaction in Saccharomyces cerevisiae Jennifer Cahpluk Jinnie Towey Eric Hernandez Jerry Mensah Luke O�Neill

3: Introduction

6: Normally when a drop in [ATP] occurs because of increased demand AMPP and AMPD are inhibited - which facilitates the production of ATP Conversely when [ATP] is high, AMPP and AMPD actively remove AMP from the adenylate pool Ice worms exhibit counterintuitive increases in ATP as temperature decreases. Reductions in the activity of these enzymes would result in higher [ATP] Serves as �an arguably easier molecular adaptation compared with the more typical challenges of maintaining enzymatic activity at low temperature� (Napolitano & Shain 2005)

7: Preparation for Experiment

8: Overview of the Experiment Grow Yeast, use a mortar and pestle to grind up the yeast cells, releasing the proteins inside the cells to give us crude extracts Extract AMPD from yeast cells via AS precipitation Perform enzyme assay on crude extracts Use varying Ammonium Sulfate solution concentrations to purify AMPD Assess each AS trial for enzyme activity, narrow down to find ideal AS% solution Once we obtain the best fractionation possible we can check to see how much we have and what other impurities are present by running an SDS-PAGE with a ladder and our sample, looking for a band around 37 kDa

9: Yeast Preparing overnight liquid cultures: Take fresh colonies from a working stock plate, and use one large colony per 5 mL of medium. Use several colonies for larger volumes Vortex the medium for approximately 1 minute to rigorously disperse the cells (clumps will cause the culture to grow slower) Incubate at 30�C for 16-18 hrs on a shaking apparatus at 230-270 rpm Should produce a stationary phase culture with OD(600) > 1.5

10: Yeast S. cerevisiae will need to grow on YPD medium which is comprised of peptone, yeast extract and dextrose. YPD is commercially available but can also be prepared with: 20 g/L Difco peptone 10 g/L Yeast extract 20 g/L Agar (for plates only) Add H20 to 950 mL, adjust pH to 6.5 if necessary then autoclave. Allow medium to cool to ~55�C and then add dextrose to 2% (50 mL of sterile stock solution). Adjust final volume to 1 L The protein extracts will need protection from endogenous proteases. Protease inhibitor solution: 0.1 mg/mL Pepstatin A(carboxyl proteases) 0.03 mM Leupeptin (some thiol and serine proteases) 145 nM Benzamidine (Trypsin, plasmin, and thrombin) 0.37 mg/mL Aprotinin (some serine proteases) Prepare right before use and keep on ice. Adopted from Clontech Laboratories Yeast protocol handbookAdopted from Clontech Laboratories Yeast protocol handbook

11: Protein Purification Issues with protein stabilization to be mindful of when conducting experiment: Many proteins become denatured by contact with air-water interface so minimize frothing (each bubble represents denaturing proteins) Oxidation of cysteine residues to form disulfide bonds Heavy metal contaminants may irreversibly bind with protein to be purified Salt concentration and polarity of solution must be in the stable range for the protein Watch out for microorganisms which may try to eat your protein(s). Prevent by performing at cold temperatures or introducing a toxic substance which won�t affect the protein

12: Ammonium Sulfate Precipitation Our level of purity will depend on where AMPD precipitates out of AS solution Salting In/Out Solubility of proteins at low ionic strength generally increases with the [salt] (the addition of counter ions effectively shields the proteins multiple ionic charges) Solubility of proteins decreases at high ionic strengths (competition between the added salt ions and the other dissolved solutes for finite # of molecules of solvation)

13: Manipulating Protein Solubility By adjusting the [salt] in a solution containing a mixture of proteins to just below the precipitation point of the protein of interest, many unwanted proteins can be eliminated from the solution. Then, after the precipitate is removed by filtration/centrifugation, the [salt] of the remaining solution is increased so as to precipitate the desired protein. Ions that decrease the solubility of proteins stabilize their native structures, so that proteins which have been salted out are not denatured

14: Ammonium sulfate precipitation is a technique used to purify proteins based on solubility properties. First, we will add a low concetration of (NH4)2SO4 to our sample containing AMPD. Hopefully, this will cause most of the othe proteins and molecules to precipitate out leaving our desired enzyme in the supernatant. Then we will remove the pellet and increase the concentration of the (NH4)2SO4 in the supernatant to cause our enzyme AMPD to precipitate out. This pellet should contain most of the AMPD and then it can be dissolved in a buffer. ���� ����-increasing the ionic strength of the solution decreases the� �����solubility of the proteins ����-higher concentration of salt yields more protein �����precipitation � Ammonium Sulfate Precipitation

15: Procedure: Obtain crude cytosol containing AMPD & measure volume. Add (NH4)2SO4 to bring salt conc to 30% saturation. Mix and measure volume. Allow to sit on ice for 30 min. Centrifuge 10 min @ 15000 rpm. Remove and save supernatant. Measure volume. Add (NH4)2SO4 to increase salt conc by 10% higher satur. Allow to sit on ice for 30 min. Centrifuge 10 min @ 15000 rpm. Remove pellet and save. ~90% of AMPD should be in pellet. Desolve pellet in 10 mM Tris- HCl buffer pH 7.4. Assay activity using UV-Vis as discussed. ���� Ammonium Sulfate Precipitation

16: AMP Deaminase Assay

17: Coupled GDH Reaction The products of AMPD couldn�t be directly quantitated, so further chemical treatment is required to yield an observable product (assay of enzyme activity). Several ways to accomplish this, the safest is to perform a coupled glutamate dehydrogenase reaction. AMPD will produce IMP and ammonia, we can find the ammonia released by measuring NADH oxidation via GDH

18: The oxidation of NADH should produce an observable change in absorbance at 340 nm Ideally one NAD+ will be produced by GDH for every one NH3 liberated by AMPD Glutamate Dehydrogenase

19: AMP Deaminase Spectrophotometric Assay

20: AMP Deaminase Spectrophotometric Assay