Direct photoaffinity labeling of gizzard myosin with ( sup 3 H)uridine diphosphate places Glu185 of the heavy chain at the active site
- Washington State Univ., Pullman (USA)
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.
- OSTI ID:
- 5988342
- Journal Information:
- Journal of Biological Chemistry; (USA), Vol. 265:36; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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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