Title

The photoaffinity probe 8-azidoadenosine 5'-triphosphate selectively labels the heavy chain of Chlamydomonas 12 S dynein

UMMS Affiliation

Department of Cell Biology

Date

7-10-1984

Document Type

Article

Subjects

Adenosine Triphosphatases; Adenosine Triphosphate; Affinity Labels; Azides; Binding, Competitive; Chlamydomonas; Dynein ATPase; Flagella; Kinetics; Macromolecular Substances; Protein Binding

Abstract

Chlamydomonas 12 S dynein, which makes up part of the outer arm of the flagellar axoneme, consists of three polypeptides of 330,000, 22,000, and 18,000 daltons. We have used 8-azidoadenosine 5'-triphosphate (8-N3ATP), a photoaffinity analog of ATP, to investigate which of the dynein polypeptides contains the site of ATP hydrolysis. 8-N3ATP is a competitive inhibitor of the hydrolysis of ATP by 12 S dynein and is hydrolyzed by 12 S dynein in an ATP- and vanadate-sensitive fashion, indicating that it binds to the 12 S dynein hydrolytic site in the same way as ATP. When dynein was incubated with [gamma-32P]- or [alpha-32P]8-N3ATP in the presence of UV light to activate the azido moiety, the analog was incorporated into 12 S dynein's heavy polypeptide chain, but not its light chains. The incorporation was UV-dependent, was blocked by addition of ATP or vanadate plus ADP to the reaction mixture, and did not occur in heat-denatured dynein. These results strongly suggest that the hydrolytic site of 12 S dynein is contained in its heavy chain.

Rights and Permissions

Citation: J Biol Chem. 1984 Jul 10;259(13):8499-504.

Related Resources

Link to Article in PubMed

Journal Title

The Journal of biological chemistry

PubMed ID

6234314