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[32P] Azido ATP Versus Biotinylated Azido ATP Photolabeling of Purified cAMP Dependent Protein Kinase Catalytic Subunit: Effect of Buffer and pH on Photolabeling

 

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

The purpose of these experiments was to determine if biotinylated azido ATP photoaffinity analogs could be used to photomodify a true protein kinase. The cAMP dependent protein kinase (PKA) from bovine heart was chosen for this purpose.  Two preparations of PKA were used. A purified PKA catalytic subunit which does not require cAMP for activation and a crude PKA preparation which requires cAMP for regulatory subunit dissociation and subsequent catalytic subunit activation.  We also wanted to compare photobiotinylation of PKA with various gamma phosphate and ribose modified biotinylated azido ATP analogs with P-32 photolabeling of purified PKA catalytic subunit.  In addition, we wanted to compare the efficiency of photobiotinylation reagents with the photoactive azido moiety in the 2 and 8 position on the purine ring.  Results from a previous set of experiments had shown that the ATP photoprobes with the photoactive moiety attached off the gamma phosphate (i.e.. ATP[g]AA and ATP[g]BP) were poor substrates for PKA so we decided not to try their corresponding biotinylated analogs.  Lastly, we wanted to demonstrate once again the dramatic effect that the choice of buffer and pH can have on photolabeling of PKA. 

TABLE OF CONTENTS

 Materials
Photolabeling Procedure
Experiment 1.  [g32P]-8-Azidoadenosine 5'-triphosphate Versus [a32P]-8-Azidoadenosine 5'-triphosphate  Photolabeling of Purified PKA Catalytic Subunit: Effect of Buffer and pH on Photolabeling
Experiment 2.  2-Azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine Versus 2-Azidoadenosine 5'-triphosphate 2',3'-biotin-long chain-hydrazone Photolabeling of Purified PKA Catalytic Subunit: Effect of Buffer and pH on Photolabeling
Experiment 3.  8-Azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine Versus 8-Azidoadenosine 5'-triphosphate 2',3'-biotin-long chain-hydrazone Photolabeling of Purified PKA Catalytic Subunit: Effect of Buffer and pH on Photolabeling
Experiment 4: 8-Azidoadenosine 5'-triphosphate [g]--biotinyl-3,6,9-trioxaundecanediamine Versus 2-Azidoadenosine 5'-triphosphate [g]--biotinyl-3,6,9-trioxaundecanediamine  Photolabeling of Purified PKA Catalytic Subunit: Effect of Buffer and pH on Photolabeling
Experiment 5.  Effect of Buffer and pH on 2N3ATP[g]Biotinpentylamine and 2N3ATP-2',3'-biotin-LC-Hydrazone   Photolabeling of Crude PKA Preparation in the Presence of 6然 cAMP
Experiment 6.  Effect of Buffer and pH on 8N3ATP[g]Biotinpentylamine and 8N3ATP-2',3'-biotin-LC-Hydrazone   Photolabeling of Crude PKA Preparation in the Presence of 6然 cAMP

Materials:

 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/痢 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蚓. 
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蚓 in the presence of 0.006 mM cyclic AMP.
Physical form: Crude (very, see Figure 1A) lyophilized powder, 1-2 units/痢 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蚓 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.
 

 

 Biotinylated ATP Photoaffinity Analogs:
2-Azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine (2N3ATP[g]Biotinpentylamine)
Absorbance maximum (l max) = 271 at pH 7.0
Molar extinction coefficient (e) = 15,500

 
2-Azidoadenosine 5'-triphosphate 2',3'-biotin-long chain-hydrazone (2N3ATP-2'3'-Biotin-LC-Hydrazone)
Absorbance maximum (l max) = 271 at pH 7.0
Molar extinction coefficient (e) = 15,500

  

 

8-Azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine (8N3ATP[g]Biotinpentylamine)
Absorbance maximum (l max) = 281 at pH 7.0
Molar extinction coefficient (e) = 13,300

8-Azidoadenosine 5'-triphosphate 2',3'-biotin-long chain-hydrazone (8N3ATP-2'3'-Biotin-LC-Hydrazone)
    Absorbance maximum (l max) = 281 at pH 7.0
    Molar extinction coefficient (e) = 13,300

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 

 

Western Reagents:
Avidin耍lkaline Phosphatase hen egg white/calf intestinal
Physical form: Lyophilized powder containing Tris aspartate buffer with trace magnesium and zinc salts Labeled with highly purified alkaline phosphatase, Protein approx. 35% by modified Warburg-Christian
Alkaline phosphatase 600-1,200 DEA units/mg, 
From Sigma-Aldrich Company (http://www.sigmaaldrich.com), Product number: A2527 
1-Step NBT/BCIP
From Pierce Biotechnology, Inc. Prod. No. 34042   0560TB-1.PDF 
BCIP (5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt) and NBT (Nitro-Blue Tetrazolium Chloride).
Tris Buffered Saline + Tween (TTBS)
50mM Tris-HCl, pH 7.4, 200 mM NaCl, 0.05% Tween-20
Immobilon-P PVDF membrane
From Millipore Corporation

 

 

 Photolabeling Procedure:

  1. 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.

  2. Either 20 痢 of total protein in the crude PKA preparation (Experiments #1-4) or 0.2 痢 of purified PKA catalytic subunit (Experiment #5) was incubated for 30 min on ice at 4蚓 with 6 然 3',5'-cAMP (crude PKA only) in 20 mM BisTris, MES, Tris, or sodium phosphate buffer at the indicated pH in a 50 痞 reaction volume in 1.7 ml microcentrifuge tubes.

  3. Afterwards, 10 痞 of stock biotinylated azido ATP photoaffinity analog prepared in ddH2O was added to a final test concentration of 10 然 and samples gently vortexed to mix.

  4. PKA was incubated with the photoprobe for 30 sec. followed by 60 sec of irradiation 4,000 琺/cm2 at 254 nm with a hand-held UV lamp held 4 cm from the surface of the test mixture.

  5. Immediately after photolysis, samples were quenched with 20 痞 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).

  6. Proteins were resolved on a 7-11% discontinuous SDS-PAGE

  7. Proteins were transferred to PVDF using 25 mM Tris, 192 mM glycine, 10% (v/v) methanol at 30V/100 mA for 12 hrs.

  8. The membrane was blocked with 5% powdered dry milk in TTBS for 1 hr at room temperature followed by 3 successive washes with TTBS.

  9. After washing, avidin-alkaline phosphatase diluted 1:10,000 in TTBS was added to the blot and incubated for 1 hr. at room temperature.

  10. The blot was washed 3 times with TTBS and then developed with 25 ml of 1 step NBT/BCIP solution to visualize the photobiotinylation of PKA.

  11. For P-32 labeled proteins, the gel was stained with Coomassie brilliant blue, and destained overnight.  The stained gel was air dried between cellophane and 32P photoincorporation determined using a Packard Biosciences Cyclone Phosphoimager (2-5 min exposure to screen).

 

Experiments:
  

Experiment 1. [g32P]-8-Azidoadenosine 5'-triphosphate Versus [a32P]-8-Azidoadenosine 5'-triphosphate  Photolabeling of Purified PKA Catalytic Subunit: Effect of Buffer and pH on Photolabeling
Figure 1A. Coomassie brilliant blue stained 7-11% discontinuous gradient SDS-PAGE analysis of purified  Protein Kinase A catalytic subunit photolabeled with 10 然 [g32P]8N3ATP as described above.

 

Figure 1B. Cyclone Phosphoimager scan of SDS-PAGE analysis of purified  Protein Kinase A catalytic subunit  photolabeled with 10 然 [g32P]8N3ATP as described above.

 

Figure 1C. Plot of the quantified data from Figure 5B above.

 

Figure 1D. Cyclone Phosphoimager scan of SDS-PAGE analysis of purified  Protein Kinase A catalytic subunit  photolabeled with 10 然 [a32P]8N3ATP as described above.

 

Figure 1E. Plot of the quantified data from Figure 1D above.

 

Experiment 2: 2-Azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine Versus 2-Azidoadenosine 5'-triphosphate 2',3'-biotin-long chain-hydrazone Photolabeling of Purified PKA Catalytic Subunit: Effect of Buffer and pH on Photolabeling
Figure 2.  Avidin-alkaline phosphatase NBT/BCIP colorimetric development of PVDF immobilized purified Protein Kinase A catalytic subunit photolabeled with 10 然 2-Azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine (2N3ATP[g]Biotinpentylamine, lanes 1-9) versus 2-Azidoadenosine 5'-triphosphate 2',3'-biotin-long chain-hydrazone (2N3ATP-2',3'-biotin-LC-hydrozone, lanes 10-18) as described above.

 

Experiment 3: 8-Azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine Versus 8-Azidoadenosine 5'-triphosphate 2',3'-biotin-long chain-hydrazone Photolabeling of Purified PKA Catalytic Subunit: Effect of Buffer and pH on Photolabeling
Figure 3.  Avidin-alkaline phosphatase NBT/BCIP colorimetric development of PVDF immobilized purified Protein Kinase A catalytic subunit photolabeled with 10 然 8-Azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine (8N3ATP[g]Biotinpentylamine, lanes 1-9) versus 8-Azidoadenosine 5'-triphosphate 2',3'-biotin-long chain-hydrazone (8N3ATP-2',3'-biotin-LC-hydrazone, lanes 10-18) as described above.

 

 

Experiment 4: 8-Azidoadenosine 5'-triphosphate [g]--biotinyl-3,6,9-trioxaundecanediamine Versus 2-Azidoadenosine 5'-triphosphate [g]--biotinyl-3,6,9-trioxaundecanediamine  Photolabeling of Purified PKA Catalytic Subunit: Effect of Buffer and pH on Photolabeling
Figure 4.  Avidin-alkaline phosphatase NBT/BCIP colorimetric development of PVDF immobilized purified Protein Kinase A catalytic subunit photolabeled with 10 然 8-Azidoadenosine 5'-triphosphate [g]--biotinyl-3,6,9-trioxaundecanediamine  (8N3ATP[g]Biotin-LC-PEO-Amine, lanes 1-9) versus 2-Azidoadenosine 5'-triphosphate [g]--biotinyl-3,6,9-trioxaundecanediamine (2N3ATP[g]Biotin-LC-PEO-Amine, lanes 10-18) as described above.

 

 

Experiment 5.  Effect of Buffer and pH on 2N3ATP[g]Biotinpentylamine and 2N3ATP-2',3'-LC-Hydrazone   Photolabeling of Crude PKA Preparation in the Presence of 6然 cAMP
Figure 5A.  Avidin-alkaline phosphatase NBT/BCIP colorimetric development of PVDF immobilized crude Protein Kinase A preparation photolabeled with 10 然 2-Azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine (2N3ATP[g]Biotinpentylamine) in the presence of 6 然 cAMP as described above. 

 

Figure 5B.  Avidin-alkaline phosphatase NBT/BCIP colorimetric development of PVDF immobilized crude Protein Kinase A preparation photolabeled with 10 然 2-Azidoadenosine 5'-triphosphate 2',3'-biotin-long chain-hydrazone (2N3ATP-2',3'-Biotin-LC-Hydrazone) in the presence of 6 然 cAMP as described above.

 
 

 

Experiment 6.  Effect of Buffer and pH on 8N3ATP[g]Biotinpentylamine and 8N3ATP-2',3'-Biotin-LC-Hydrazone   Photolabeling of Crude PKA Preparation in the Presence of 6然 cAMP
Figure 6A.  Avidin-alkaline phosphatase NBT/BCIP colorimetric development of PVDF immobilized crude Protein Kinase A preparation photolabeled with 10 然 8-Azidoadenosine 5'-triphosphate [g]-5(biotinamido)pentylamine (8N3ATP [g]Biotinpentylamine) in the presence of 6 然 cAMP as described above.

 

Figure 6B.  Avidin-alkaline phosphatase NBT/BCIP colorimetric development of PVDF immobilized crude Protein Kinase A preparation photolabeled with 10 然 8-Azidoadenosine 5'-triphosphate 2',3'-biotin-long chain-hydrazone (8N3ATP-2',3'-Biotin-LC-Hydrazone) in the presence of 6 然 cAMP.

Technical Information or General Inquiries

Contact Dr. Anjan Bhattacharyya, Ph.D.
Radiochemicals Laboratory Director
E-mail: ajbhatta@Photoprobe.com

 
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