[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:
-
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 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.
-
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.
-
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.
-
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).
-
Proteins
were resolved on a 7-11% discontinuous
SDS-PAGE
-
Proteins
were transferred to PVDF using 25
mM Tris, 192 mM glycine, 10% (v/v)
methanol at 30V/100 mA for 12 hrs.
-
The
membrane was blocked with 5% powdered
dry milk in TTBS for 1 hr at room
temperature followed by 3 successive
washes with TTBS.
-
After
washing, avidin-alkaline phosphatase
diluted 1:10,000 in TTBS was added
to the blot and incubated for 1 hr.
at room temperature.
-
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.
-
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.
| | |
