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Title: Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate

Abstract

The active-site topology of smooth muscle myosin has been investigated by direct photoaffinity-labeling studies with ({sup 3}H)ADP. Addition of vanadate (V{sub i}) and Co{sup 2+} enabled ({sup 3}H)ADP to be stably trapped at the active site. The extraordinary stability of the myosin{center dot}Co{sup 2+}{center dot}({sup 3}H)ADP{center dot}V{sub i} complex allowed it to be purified free of excess ({sup 3}H)ADP before irradiation began and ensured that only active-site residues became labeled. Following UV irradiation, approximately 10% of the trapped ({sup 3}H)ADP became covalently attached at the active site. All of the ({sup 3}H)ADP incorporated into the 200-kDa heavy chain, confirming earlier results using untrapped ({alpha}-{sup 32}P)ATP. After extensive trypsin digestion of labeled subfragment 1, HPLC separation methods combined with alkaline phosphatase treatment allowed two labeled peptides to be isolated. Sequence analysis of both labeled peptides indicated that Glu-185 was the labeled residue. Since Glu-185 has been previously identified as a residue at the active site of smooth myosin using ({sup 3}H)UDP as a photolabel, these results provide further evidence that Glu-185, located immediately adjacent to the glycine-rich loop, is located in the purine binding pocket of the active site of smooth muscle myosin.

Authors:
;  [1]
  1. (Washington State Univ., Pullman (United States))
Publication Date:
OSTI Identifier:
5488546
Resource Type:
Journal Article
Journal Name:
Biochemistry; (United States)
Additional Journal Information:
Journal Volume: 30:42; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; MYOSIN; LABELLING; PEPTIDES; BIOLOGICAL RADIATION EFFECTS; ADENOSINE; ADP; COBALT CHLORIDES; GLUTAMIC ACID; MOLECULAR MODELS; SODIUM COMPOUNDS; TRITIUM COMPOUNDS; ULTRAVIOLET RADIATION; VANADATES; ALKALI METAL COMPOUNDS; AMINO ACIDS; BIOLOGICAL EFFECTS; CARBOXYLIC ACIDS; CHLORIDES; CHLORINE COMPOUNDS; COBALT COMPOUNDS; ELECTROMAGNETIC RADIATION; GLOBULINS; HALIDES; HALOGEN COMPOUNDS; HYDROGEN COMPOUNDS; MATHEMATICAL MODELS; NUCLEOSIDES; NUCLEOTIDES; ORGANIC ACIDS; ORGANIC COMPOUNDS; OXYGEN COMPOUNDS; PROTEINS; RADIATION EFFECTS; RADIATIONS; RIBOSIDES; TRANSITION ELEMENT COMPOUNDS; VANADIUM COMPOUNDS; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Garabedian, T.E., and Yount, R.G. Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate. United States: N. p., 1991. Web. doi:10.1021/bi00106a008.
Garabedian, T.E., & Yount, R.G. Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate. United States. doi:10.1021/bi00106a008.
Garabedian, T.E., and Yount, R.G. Tue . "Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate". United States. doi:10.1021/bi00106a008.
@article{osti_5488546,
title = {Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate},
author = {Garabedian, T.E. and Yount, R.G.},
abstractNote = {The active-site topology of smooth muscle myosin has been investigated by direct photoaffinity-labeling studies with ({sup 3}H)ADP. Addition of vanadate (V{sub i}) and Co{sup 2+} enabled ({sup 3}H)ADP to be stably trapped at the active site. The extraordinary stability of the myosin{center dot}Co{sup 2+}{center dot}({sup 3}H)ADP{center dot}V{sub i} complex allowed it to be purified free of excess ({sup 3}H)ADP before irradiation began and ensured that only active-site residues became labeled. Following UV irradiation, approximately 10% of the trapped ({sup 3}H)ADP became covalently attached at the active site. All of the ({sup 3}H)ADP incorporated into the 200-kDa heavy chain, confirming earlier results using untrapped ({alpha}-{sup 32}P)ATP. After extensive trypsin digestion of labeled subfragment 1, HPLC separation methods combined with alkaline phosphatase treatment allowed two labeled peptides to be isolated. Sequence analysis of both labeled peptides indicated that Glu-185 was the labeled residue. Since Glu-185 has been previously identified as a residue at the active site of smooth myosin using ({sup 3}H)UDP as a photolabel, these results provide further evidence that Glu-185, located immediately adjacent to the glycine-rich loop, is located in the purine binding pocket of the active site of smooth muscle myosin.},
doi = {10.1021/bi00106a008},
journal = {Biochemistry; (United States)},
issn = {0006-2960},
number = ,
volume = 30:42,
place = {United States},
year = {1991},
month = {10}
}