Date of Award
Master of Science (MS)
2-Azidoadenosine has previously been shown to be a potent activator of adenylate cyclase in human platelet particulate fractions. In the present study, the activation of adenylate cyclase was enhanced by: (a) reducing Mg²⁺ concentration in the assay mixture, (b) adding excess adenosine deaminase during the incubation, and (c) using rabbit platelets instead of human platelets. Under these optimal conditions a 200% increase in adenylate cyclase activity was achieved with 10 μM 2-azidoadenosine.
2-Azidoadenosine was stable in aqueous buffer systems in the absence of light. At pH 7.4, 2-azidoadenosine exists as two tautomers, the azide form and the tetrazole form which were shown to interconvert in a first order fashion with a half-life of 15.3 min at 22°C. These two tautomers appear to be equipotent in their activation of platelet particulate fraction adenylate cyclase. However, on irradiation at 313 nm only the azide form appears to be photolysed to a mixture of products, presumably via a nitrene intermediate.
Although the photolysis of 2-azidoadenosine in the presence of platelet particulate fraction produced activation of platelet adenylate cyclase, evidence was obtained that this was not the result of photoaffinity labeling. The individual effects of pre-exposure to 2-azidoadenosine and irradiation at 313 nm were additive.
Activation of adenylate cyclase by 2-azidoadenosine added to the enzyme assay was reduced if the particulate fraction had been exposed to UV radiation. The same reduction in activation by 2-azidoadenosine added to the assay was seen when the prior irradiation of the enzyme was carried out in the presence of 2-azidoadenosine.
The ³H covalently bound to the platelet particulate fraction upon irradiation in the presence of 2-azido[³H] adenosine was shown to be radiation dependent. However, pre-irradiation of 2-azido-[³H] adenosine generated a product that covalenty labeled platelet particulate fraction as effectively as irradiation of 2-azido[³H]-adenosine in the presence of particulate fraction. This indicates that affinity labeling rather than photoaffinity labeling had occurred. Some inhibition of this labeling was produced by the addition of unlabeled 2-azidoadenosine, 2-chloroadenosine and theophylline. However, neither adenosine nor N⁶-cyclohexyladenosine caused inhibition of labeling when present during photolysis, suggesting that adenosine receptors were not detected. Inhibition of labeling by dipyridamole and coformycin suggested reactions with adenosine transport proteins and adenosine deaminase, respectively. Further studies with various combinations of ligands are required to determine the nature of sites labeled by the photolysis products of 2-azido[³H]adenosine.
Lemmex, Byron William Gary, "Activation of Platelet Adenylate Cyclase by 2-Azidoadenosine: A Potential Photoaffinity Label" (1980). Open Access Dissertations and Theses. Paper 2853.