Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate
The photoaffinity analogue 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz/sub 2/ATP) contains the photoreactive benzophenone group esterified at the 2' or 3' hydroxyl groups of ribose. MgBz/sub 2/ADP has a single binding site on skeletal myosin chymotryptic subfragment-one (SF/sub 1/) with a binding constant of 3.2 x 10/sup 5/ M/sup -1/. Bz/sub 2/ATP is also a substrate for the ATPase activity of SF/sub 1/ in the presence of different cations. The irradiation of SF/sub 1/ with (/sup 3/H)Bz/sub 2/ATP photoinactivates the ATPase activity with concomitant incorporation of the analogue into the enzyme. Polyacrylamide gel electrophoresis of photolabeled SF/sub 1/ after milk trypsin digestion shows that all three tryptic peptides, 25 K, 50K, and 20 K, and both light chains are labeled. The presence of ATP during irradiation reduces labeling of the 50 K peptide only indicating that the other peptides are non-specifically labeled. To reduce the non-specific labeling (/sup 3/H)Bz/sub 2/ATP is trapped on SF/sub 1/ by cross-linking the two reactive thiols, SH/sub 1/ and SH/sub 2/, by N,N'-p-phenylene dimaleimide or Co(II)/Co(III) phenanthroline complexes. The Co(II)/Co(III) phenanthroline modified (/sup 14/C)Bz/sub 2/ATP-SF/sub 1/, after proteolytic digestion, yields five labeled peptides which were purified by gel filtration and high performance liquid chromatography.
- Research Organization:
- Washington State Univ., Pullman (USA)
- OSTI ID:
- 6527049
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
Similar Records
Serine-324 of myosin's heavy chain is photoaffinity-labeled by 3 prime (2 prime )-O-(4-benzoylbenzoyl)adenosine triphosphate
Identification of the ATP binding sites of the carbamyl phosphate synthetase domain of the Syrian hamster multifunctional protein CAD by affinity labeling with 5 prime -(p-(fluorosulfonyl)benzoyl)adenosine
Related Subjects
ATP-ASE
INACTIVATION
MYOSIN
MOLECULAR STRUCTURE
BENZOPHENONE
CARBON 14 COMPOUNDS
ELECTROPHORESIS
ENZYME ACTIVITY
PHOTOCHEMICAL REACTIONS
TRITIUM COMPOUNDS
ACID ANHYDRASES
CHEMICAL REACTIONS
ENZYMES
GLOBULINS
HYDROLASES
KETONES
LABELLED COMPOUNDS
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
PROTEINS
550201* - Biochemistry- Tracer Techniques