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Crude cAMP Dependent Protein Kinase with Different ATP Photoaffinity Analogs

by J. Curt Pendergrass Ph.D.
President, ALT Inc.

The purpose of these experiments was to demonstrate the dramatic effect that the choice of buffer and pH can have on photolabeling of cAMP dependent protein kinase (PKA). Never assume the buffer and pH that you have been using in the past to assess catalytic activity will be the same as the optimum conditions for photolabeling. Many times they not and assuming otherwise may mean the difference between success and failure when it comes to photolabeling a particular protein. In addition, the choice of nucleotide photoaffinity analog can have a dramatic effect on the photolabeling efficiency. For example, base modified ATP photoaffinity analogs such as 8N₃ATP and 2N₃ATP may exhibit a dramatically different photolabeling profiles than the gamma phosphate modified analogs ATP[g]AA and ATP[g]BP. In general, we have found that BisTris, MES, sodium phosphate and Tris-HCl all give good results when attempting to photolabel most proteins.

TABLE OF CONTENTS

	Materials
	Photolabeling Procedures
	Experiment 1. Effect of Buffer and pH on 2-Azidoadenosine 5'-[g³²P]triphosphate ([g³²P]2N₃ATP) Photolabeling of Crude PKA Preparation
	Experiment 2. Effect of Buffer and pH on 8-Azidoadenosine 5'-[g³²P]triphosphate ([g³²P]8N₃ATP) Photolabeling of Crude PKA Preparation
	Experiment 3. Effect of Buffer and pH on Adenosine 5'-[g³²P] triphosphate [g] 4-Azidoanilide ([g³²P]ATP[g]AA) Photolabeling of Crude PKA Preparation
	Experiment 4. Effect of Buffer and pH on Adenosine 5'-[g³²P] triphosphate [g] Benzophenone ([g³²P]ATP[g]BP) Photolabeling of Crude PKA Preparation
	Experiment 5. [g³²P]-8-Azidoadenosine 5'-triphosphate Versus [a³²P]-8-Azidoadenosine 5'-triphosphate Photolabeling of Purified PKA Catalytic Subunit: Effect of Buffer and pH on Photolabeling

Materials:

Protein Kinase A bovine heart

	From Sigma-Aldrich Company (http://www.sigmaaldrich.com), Product Number P5511
	Synonyms: Protein Kinase, 3',5'-cycl-AMP-dependent bovine heart
	Enzyme Commission (EC) Number: 2.7.1.37, CAS Number: 9026-43-1
	Miscellaneous: One unit will transfer 1.0 picomole phosphate from g-³²P-ATP to hydrolyzed and partially dephosphorylated casein, per min at pH 6.5 at 30°C in the presence of 0.006 mM cyclic AMP.
	Physical form: Crude (very, see Figure 1A) lyophilized powder, 1-2 units/µg protein. Protein approx. 80% Protein determined by Biuret method. Contains 10% EDTA and 10% potassium phosphate, pH 7.0. Fractionated essentially by procedure of Gilman, A., Proc. Natl. Acad. Sci. USA, 67, 305 (1970).
	Unit definition: One unit will transfer 1.0 picomole phosphate from g-³²P-ATP to hydrolyzed and partially dephosphorylated casein, per min at pH 6.5 at 30°C in the presence of 0.006 mM cyclic AMP (activity is reduced at least 10-fold in the absence of cAMP)
	Biochemical/physiological actions: Many 3',5'-cyclic AMP dependent protein kinases have been reported. Structural studies (Traugh, J.A., et al., Meth. Enzymol., Vol. 38, p. 290 [1974]) show the presence of at least two subunits, the regulatory subunit and the catalytic subunit. When both units are linked together, the catalytic activity is inhibited. However, when the cyclic-AMP binds to the regulatory subunit, the catalytic subunit is released and can then catalyze the transfer of phosphate from ATP to various proteins.

Protein Kinase A Catalytic Subunit from bovine heart

	From Sigma-Aldrich Company (http://www.sigmaaldrich.com), Product Number P2645
	Comments: Lyophilized powder, 30-65 units/µg protein (cyclic-AMP is not required for this activity.)
	Physical form: Lyophilized powder containing < 1% protein with sucrose and phosphate buffer salts as stabilizer
	Miscellaneous: Prepared from protein kinase A (P 5511)
	Unit definition: One unit will transfer 1.0 picomole of phosphate from ATP to a synthetic substrate per min at pH 7.4 at 30°C.

ATP Photoaffinity Analogs:

2-Azidoadenosine 5'-[g³²P]triphosphate ([g³²P]2N₃ATP)

gamma2N3ATP.gif (4068 bytes)

	Specific activity: 10-15 Ci/mmol: 370-555 GBq/mmol; 1-4 mCi/ml; 37-148 MBq/ml
	Molecular Formula: C₁₀H₁₅N₈0₁₃P₃ for the free acid
	Molecular Weight: 548.2 for the free acid
	Description: Triethylammonium salt solution in absolute methanol

8-Azidoadenosine 5'-[g³²P]triphosphate ([g³²P]8N₃ATP)

gamma8N3ATP.gif (4000 bytes)

	Specific activity: 10-15 Ci/mmol: 370-555 GBq/mmol; 1-4 mCi/ml; 37-148 MBq/ml
	Molecular Formula: C₁₀H₁₅N₈0₁₃P₃ for the free acid
	Molecular Weight: 548.2 for the free acid
	Description: Triethylammonium salt solution in absolute methanol

Adenosine 5'-[g³²P] triphosphate [g] 4-Azidoanilide, ([g³²P]ATP[g]AA)

gammaATPAA.gif (4544 bytes)

	Specific activity: 10-15 Ci/mmol: 370-555 GBq/mmol; 1-4 mCi/ml; 37-148 MBq/ml
	Molecular Formula: C₁₆H₂₀N₉0₁₂P₃ for the free acid
	Molecular Weight: 623.3 for the free acid
	Description: Triethylammonium salt solution in absolute methanol

Adenosine 5'-[g³²P] triphosphate [g] Benzophenone, ([g³²P]ATP [g]BP)

	Specific activity: 10-15 Ci/mmol: 370-555 GBq/mmol; 1-4 mCi/ml; 37-148 MBq/ml
	Molecular Formula: C₁₆H₂₀N₉0₁₂P₃ for the free acid
	Molecular Weight: 700.4 for the free acid
	Description: Triethylammonium salt solution in absolute methanol

Buffers: All buffers and metals were from Sigma-Aldrich Chemical Company, St. Louis, MO (http://www.sigmaaldrich.com)

	Tris: 2-Amino-2-(hydroxymethyl)-1,3-propanediol, THAM, Tris base, Trometamol, MDL number: MFCD00004679, 77-86-1, minimum 99% (titration), Powder T1378
	BIS-TRIS: 2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol, Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane, MDL number: MFCD00002853, 6976-37-0, SigmaUltra, >98% (titration) B7535
	MES monohydrate: 4-Morpholineethanesulfonic acid monohydrate, 2-(N-Morpholino)ethanesulfonic acid, MDL number: MFCD00006181, 145224-94-8, minimum 99.5% (titration) M8250
	Sodium phosphate monobasic: Monosodium phosphate, Sodium dihydrogen phosphate, mono-Sodium phosphate, MDL number: MFCD00003527, 7558-80-7, minimum 99.0% S0751
	Sodium phosphate dibasic: Disodium hydrogen phosphate, , MDL number: MFCD00003496, 7558-79-4, SigmaUltra, minimum 99% S7907

Photolabeling Procedure:

20 mM stock buffer solutions were prepared in sterile, distilled and deionized water and the pH adjusted to the desired value by addition of 1.0 N HCl with stirring by pH meter. For sodium phosphate buffers, 20 mM stock solutions of NaH₂PO₄ and Na₂HPO₄ were admixed together to the desired pH.
Either 20 µg of total protein in the crude PKA preparation (Experiments #1-4) or 0.2 µg of purified PKA catalytic subunit (Experiment #5) was incubated for 30 min on ice at 4°C with 6 µM 3',5'-cAMP (crude PKA only) in 20 mM BisTris, MES, Tris, or sodium phosphate buffer at the indicated pH in a 50 µl reaction volume in 1.7 ml microcentrifuge tubes.
Afterwards, 10 µl of stock [g³²P] labeled ATP photoaffinity analog prepared in ddH₂O was added to a final test concentration of 10 µM and samples gently vortexed to mix.
PKA was incubated with the photoprobe for 30 sec. followed by 60 sec of irradiation 4,000 µW/cm² at 254 nm with a hand-held UV lamp (UV Products, Model UVG-11) held 4 cm from the surface of the test mixture.
For [g³²P]ATP[g]BP photolabeling, PKA was incubated with the photoprobe for 1 min. followed by 6 min. of irradiation 4,600 µW/cm² at 366 nm with a hand-held UV lamp (UV Products, Model UVL-21) held 4 cm from the surface of the test mixture.
Immediately after photolysis, samples were quenched with 20 µl of protein solubilizing mixture containing 3.6 M urea, 10% SDS, 2.5% (w/v) DTT, 125 mM Tris-HCl, pH 6.8 and 0.05% (w/v) pyronin Y (tracking dye).
Proteins were resolved on a 7-11% discontinuous SDS-PAGE, stained with Coomassie brilliant blue and destained overnight.
The gel was air dried between cellophane and ³²P photoincorporation determined using a Packard Biosciences Cyclone Phosphoimager (2-5 min exposure to screen).

Experiment #1. Effect of Buffer and pH on [g³²P]2N₃ATP, Photolabeling of Crude PKA Preparation.

Figure 1A. Coomassie brilliant blue stained 7-11% discontinuous gradient SDS-PAGE analysis of (very) crude Protein Kinase A preparation photolabeled with 10 µM [g³²P]2N₃ATP in the presence of 6 µM cAMP as described above.

Figure 1B. Cyclone Phosphoimager scan of SDS-PAGE analysis of crude Protein Kinase A preparation photolabeled with 10 µM [g³²P]2N₃ATP in the presence of 6 µM cAMP as described above.

Figure 1C. Plot of the quantified data from Figure 1A above.

Experiment #2. Effect of Buffer and pH on [g³²P]8N₃ATP Photolabeling of Crude PKA Preparation.

Figure 2A. Cyclone Phosphoimager scan of SDS-PAGE analysis of crude Protein Kinase A preparation photolabeled with 10 µM [g³²P]8N₃ATP in the presence of 6 µM cAMP as described above.

Figure 2B. Plot of the quantified data from Figure 2A above.

Experiment #3. Effect of Buffer and pH on [g³²P]ATPgAA Photolabeling of Crude PKA Preparation.

Figure 3A. Cyclone Phosphoimager scan of SDS-PAGE analysis of crude Protein Kinase A preparation photolabeled with 10 µM [g³²P]ATP[g]AA in the presence of 6 µM cAMP as described above.

Figure 3B. Plot of the quantified data from Figure 3A above.