Journal article
Binding of 2-azidoadenosine [.beta.-32P]diphosphate to the receptor on intact human blood platelets which inhibits adenylate cyclase
Biochemistry (Easton), Vol.21(3), pp.544-549
02/01/1982
DOI: 10.1021/bi00532a020
PMID: 6279146
Abstract
[β-32P]-2-Azido-ADP was prepared with a specific activity up to 10000 Ci/mol and used to investigate the ADP receptors on human blood platelets. 2-Azido-ADP was about 5-fold more potent than ADP as an aggregating agent and 10–20-fold more potent as an inhibitor of prostaglandin E1 stimulated cyclic AMP accumulation in intact platelets. 2-Azido-ADP exists at neutral pH as a mixture of azide and tetrazole forms, the former being favored by low pH: the tetrazole isomer was 2-fold less potent than the azide isomer both as an aggregating agent and as an inhibitor of cyclic AMP accumulation, and the products of photolysis of 2-azi-do-ADP were also 10–20-fold weaker. Equilibrium binding measured by a centrifugal technique revealed about 500 noninteractive binding sites per platelet with an affinity close to the concentration for half-maximal inhibition of cyclic AMP accumulation. Binding, and dissociation in the presence of excess ADP, were complete in less than 30 s. Both ADP and ATP inhibited the binding of 2-azido-ADP competitively (half-maximal inhibition at 5–10 µM). UDP, GDP, IDP, and AMP were at least 100-fold less effective. Binding was blocked by p-mercuribenzenesulfonate, a thiol agent which blocks the action of ADP on the cyclase but does not block the ability of ADP to induce the platelet shape change. When washed platelets were photolyzed with [β-32P]-2-azido-ADP, several macromolecules were covalently labeled, but none of this labeling was suppressed by ADP or ATP. It is concluded that the binding site is the receptor for ADP which regulates the adenylate cyclase, and because this site is blocked by p-mercuribenzenesulfonate—an agent which does not inhibit the shape change induced by ADP—it cannot be the receptor responsible for the induction of shape change.
Details
- Title: Subtitle
- Binding of 2-azidoadenosine [.beta.-32P]diphosphate to the receptor on intact human blood platelets which inhibits adenylate cyclase
- Creators
- Donald E MacfarlaneDavid C. B MillsPrem C Srivastava
- Resource Type
- Journal article
- Publication Details
- Biochemistry (Easton), Vol.21(3), pp.544-549
- Publisher
- American Chemical Society
- DOI
- 10.1021/bi00532a020
- PMID
- 6279146
- ISSN
- 0006-2960
- eISSN
- 1520-4995
- Language
- English
- Date published
- 02/01/1982
- Academic Unit
- Hematology, Oncology, and Blood & Marrow Transplantation; Internal Medicine
- Record Identifier
- 9984094672002771
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