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Title: Direct photoaffinity labeling of gizzard myosin with ( sup 3 H)uridine diphosphate places Glu185 of the heavy chain at the active site

Abstract

The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with ({sup 3}H)UDP. ({sup 3}H) UDP was stably trapped at the active site by addition of vanadate (Vi) and Co{sup 2+}. The extraordinary stability of the myosin.Co2+.(3H)UDP.Vi complex (t1/2 greater than 5 days at 0{degrees}C) allowed it to be purified free of extraneous ({sup 3}H)UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped ({sup 3}H)UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results using ({sup 3}H)UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a zero-length cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by ({sup 3}H)UTP photolabeling in Acanthamoeba myosin II.

Authors:
;  [1]
  1. (Washington State Univ., Pullman (USA))
Publication Date:
OSTI Identifier:
5988342
Resource Type:
Journal Article
Journal Name:
Journal of Biological Chemistry; (USA)
Additional Journal Information:
Journal Volume: 265:36; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; MYOSIN; LABELLING; AFFINITY; AMINO ACID SEQUENCE; BIOCHEMICAL REACTION KINETICS; CHEMICAL RADIATION EFFECTS; CHICKENS; GLUTAMIC ACID; LIQUID COLUMN CHROMATOGRAPHY; MUSCLES; TRACER TECHNIQUES; TRITIUM COMPOUNDS; ULTRAVIOLET RADIATION; URIDINE; AMINO ACIDS; ANIMALS; AZINES; BIRDS; CARBOXYLIC ACIDS; CHEMISTRY; CHROMATOGRAPHY; ELECTROMAGNETIC RADIATION; FOWL; GLOBULINS; HETEROCYCLIC COMPOUNDS; HYDROGEN COMPOUNDS; HYDROXY COMPOUNDS; ISOTOPE APPLICATIONS; KINETICS; MOLECULAR STRUCTURE; NUCLEOSIDES; NUCLEOTIDES; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; PROTEINS; PYRIMIDINES; RADIATION CHEMISTRY; RADIATION EFFECTS; RADIATIONS; REACTION KINETICS; RIBOSIDES; SEPARATION PROCESSES; URACILS; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques; 560120 - Radiation Effects on Biochemicals, Cells, & Tissue Culture

Citation Formats

Garabedian, T.E., and Yount, R.G. Direct photoaffinity labeling of gizzard myosin with ( sup 3 H)uridine diphosphate places Glu185 of the heavy chain at the active site. United States: N. p., 1990. Web.
Garabedian, T.E., & Yount, R.G. Direct photoaffinity labeling of gizzard myosin with ( sup 3 H)uridine diphosphate places Glu185 of the heavy chain at the active site. United States.
Garabedian, T.E., and Yount, R.G. Tue . "Direct photoaffinity labeling of gizzard myosin with ( sup 3 H)uridine diphosphate places Glu185 of the heavy chain at the active site". United States.
@article{osti_5988342,
title = {Direct photoaffinity labeling of gizzard myosin with ( sup 3 H)uridine diphosphate places Glu185 of the heavy chain at the active site},
author = {Garabedian, T.E. and Yount, R.G.},
abstractNote = {The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with ({sup 3}H)UDP. ({sup 3}H) UDP was stably trapped at the active site by addition of vanadate (Vi) and Co{sup 2+}. The extraordinary stability of the myosin.Co2+.(3H)UDP.Vi complex (t1/2 greater than 5 days at 0{degrees}C) allowed it to be purified free of extraneous ({sup 3}H)UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped ({sup 3}H)UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results using ({sup 3}H)UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a zero-length cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by ({sup 3}H)UTP photolabeling in Acanthamoeba myosin II.},
doi = {},
journal = {Journal of Biological Chemistry; (USA)},
issn = {0021-9258},
number = ,
volume = 265:36,
place = {United States},
year = {1990},
month = {12}
}