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Title: Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate

Abstract

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.

Authors:
Publication Date:
Research Org.:
Washington State Univ., Pullman (USA)
OSTI Identifier:
6527049
Resource Type:
Thesis/Dissertation
Resource Relation:
Other Information: Thesis (Ph. D.)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 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

Citation Formats

Mahmood, R. Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate. United States: N. p., 1985. Web.
Mahmood, R. Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate. United States.
Mahmood, R. Tue . "Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate". United States.
@article{osti_6527049,
title = {Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate},
author = {Mahmood, R.},
abstractNote = {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.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {1985},
month = {1}
}

Thesis/Dissertation:
Other availability
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