191, No. 1, November,





interactions J. WOLFF,



of Arthritis,

with Thyroid C. LONDOS,


Metabolism and Digestive Bethesda, Maryland Received


19, 1978; revised




Diseases, 20014



of Health,

June 6, 1978

Adenosine and certain adenosine analogues inhibit beef thyroid membrane adenylate cyclase. The inhibition has a rapid onset, is not directly on the catalytic or nucleotide regulatory sites, occurs with all activators tested (ITP, Gpp(NH)p, TSH, and F-), and is seen also in mouse and human thyroid membranes. Addition of manganous ion, which activates adenylate cyclase, markedly enhances the inhibition by adenosine analogues. The order of potencies is: 2’,5’-dideoxyadenosine > 5’-deoxyadenosine > 2’-deoxy-3’-phosphoadenosine > 2’-deoxyadenosine > adenosine > adeninenyloside > adenine arabinoside. Purinemodified analogues are either inactive or stimulate slightly at high concentrations. This chemical specificity, the Mn*+ requirement, and the lack of reversal by theophylline, suggest that these membranes have little “R” site activity (stringent for the ribose moiety) and primarily contain a “P” site that has stringent purine requirement but permits changes in the ribose moiety. This site appears to be associated with the catalytic unit since it persists in solubilized adenylate cyclase.

Among the many actions of adenosine on cells, a great deal of attention has recently been devoted to the effects of this nucleoside on adenylate cyclase (EC Some adenylate cyclases are stimulated, some inhibited, and yet others show a biphasic effect characterized by stimulation at low concentrations of the nucleoside and inhibition at higher concentrations. In an attempt to resolve these paradoxical effects of adenosine on adenylate cyclases, we have compared purine and ribose-modified analogues of adenosine (1). This has led to the identification of two different sites in the adenylate cyclase system with which adenosine can interact: an “R” site, requiring an unmodified ribose moiety, occupancy of which usually leads to activation of adenylate cyclase; and a “P” site, requiring the intact purine ring, and which generally inhibits the enzyme system when occupied. Additional differences between these two sites were that theophylhne competes for the “R” site and not the “P” site (2-8), whereas analogues interacting with the “P” site are much more sensitive to changes in divalent metal concentration (1, 9-11). Indirect studies suggest the “R” site is located

on the external cell surface (12-14), whereas the location of the “P” site has not been established. In the present study, we identify the presence of “P” type sites in beef thyroid membanes and suggest that the “R” site is virtually absent in these preparations. We also compare membrane-bound and solubilized adenylate cyclase in an effort to localize this site. MATERIALS



Adenosine analogues were obtained as follows: the deoxyribose analogues, 9-fi-n-arabinofuranosyl adenosine, and N’-methyl adenosine from P-L Laboratories (the 2’-deoxyadenosine contained (

Adenosine interactions with thyroid adenylate cyclase.

ARCHIVES Vol. OF BIOCHEMISTRY AND 191, No. 1, November, Adenosine BIOPHYSICS 161-168, 1978 interactions J. WOLFF, National Institute of A...
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