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

Journal Article · Tue Oct 22 04:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00106a008· OSTI ID:5488546

Garabedian, T E; Yount, R G ^[1]

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1. Washington State Univ., Pullman (United States)

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.

• OSTI ID: 5488546
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:42; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ADENOSINE
ADP
ALKALI METAL COMPOUNDS
AMINO ACIDS
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CARBOXYLIC ACIDS
CHLORIDES
CHLORINE COMPOUNDS
COBALT CHLORIDES
COBALT COMPOUNDS
ELECTROMAGNETIC RADIATION
GLOBULINS
GLUTAMIC ACID
HALIDES
HALOGEN COMPOUNDS
HYDROGEN COMPOUNDS
LABELLING
MATHEMATICAL MODELS
MOLECULAR MODELS
MYOSIN
NUCLEOSIDES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PEPTIDES
PROTEINS
RADIATION EFFECTS
RADIATIONS
RIBOSIDES
SODIUM COMPOUNDS
TRANSITION ELEMENT COMPOUNDS
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION
VANADATES
VANADIUM COMPOUNDS