Photoaffinity labeling of cAMP-dependent protein kinase by 4-azido-2-nitrophenyladenylyl pyrophosphate
A photoaffinity analogue of ATP, 4-azido-2-nitrophenyl-adenylyl pyrophosphate (ANAP) has been synthesized to investigate the topographical interaction between the catalytic and the regulatory subunits of the bovine heart type II cAMP-dependent protein kinase. The synthesis involves coupling of 4-azido-2-nitrophenyl phosphate with adenosine 5'-monophosphomorpholidate. ANAP has an absorption maximum at 260 nm (molar absorptivity = 35.4 x 10/sup 3/ M/sup -1/ cm/sup -1/) and a shoulder at 320 nm. Kinetically, ANAP inhibits the enzyme competitively against ATP with a Ki of 0.37 mM. The catalytic subunit is inactivated by ANAP upon photolysis in the presence of magnesium ion. ATP protects the enzyme from photoinactivation but the regulatory subunit does not. Gel electrophoretic analysis of the enzyme labeled by (/sup 14/C)ANAP shows that the photoincorporated ANAP is associated mainly with the catalytic subunit, even when the regulator dimer is in twelve fold excess. Little or no ANAP is found incorporated into the regulator subunit. The data suggest that the photoreactive portion of ANAP does not lie within reach of the regulatory protein when the analogue is bound to the catalytic subunit.
- Research Organization:
- Univ. of Lowell, MA
- OSTI ID:
- 5283183
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AFFINITY
ALKALINE EARTH METAL COMPOUNDS
AMP
ANIMALS
ATP
BIOSYNTHESIS
BODY
CARBON 14 COMPOUNDS
CARDIOVASCULAR SYSTEM
CATTLE
CHEMICAL REACTIONS
DECOMPOSITION
DOMESTIC ANIMALS
ELECTROPHORESIS
ENZYMES
HEART
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LABELLING
MAGNESIUM COMPOUNDS
MAMMALS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANS
OXYGEN COMPOUNDS
PHOSPHORUS COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
PYROPHOSPHATES
RUMINANTS
SYNTHESIS
TRACER TECHNIQUES
TRANSFERASES
VERTEBRATES