There were 2 problems to be overcome: Tinyest amounts of cyclic AMP had to be detected and quantitated, and ATPases had to be "neutralized" because they would eat up ATP so fast, there was nothing left for the adenylyl cyclase.
Enzyme preparation: We worked with fat cells. Homogenizing cells often destroyed the enzyme activity - so we treated fat pads with collagenase to obtain free cells that could then be lysed in hypotonic buffer, yielding membrane "ghosts" that proved to retain activity of membrane-associated enzymes like the cyclase.
Radioactive substrate - p32-labeled ATP: This was obtained from a manufacturer that was able to custom produce ATP that had radioactive P32 only in the alpha postition, the one that of course was retained in the cAMP product. It had to have highest specific radioactivity to produce the sensitivity we needed. This was a new market for the manufacturer, and we managed to make them improve their product in terms of purity - initial 2-dimensional chromatography studies we did showed them and us that the preps contained radioactive impurities exceeding what we expected our enzyme product, P32 labeled cyclic AMP, to be.
New enzyme technology: To date (1967) enzyme studies always involved incubates of 1-10 ml - but that would waste radioactive substrates immensely - so we developed microassays in volumes of 50 uL. Fortuitously, the Eppendorf company was in the process of pioneering pushbutton pipetting devices with disposable tips for pipetting and transferring amounts down to 5 microliter to special propylene incubation vessels (before that we had to use tiny test tubes that needed siliconizing the glass surface so that the tiny volumes would mix and form one volume rather than scattering inside the tube). These pipettes were intended for hospital labs - and we may have been the first to use them for enzyme assays in research.
The assay: Now we combined, in 5-10 uL increments to a total of 50 uL, a Mg ion containing buffer solution, ATP-alpha-P32, various other components to be tested (hormone solutions), high concentrations of creatinine kinase and creatinine phosphate (supposed to regenerate any ATP hydrolyzed by ATPases) and enzyme preparations (fat cell ghosts) - the latter added last to start the reaction. To stop the reaction we initially used immersion in boiling water - but then we just added 10 uL of a mix of high concentrations of Assays and evaluate ATP/ADP/AMP and unlabeled cyclic AMP plus EDTA (a Mg complexing agent). In the next step we used a chromatographic separation method on thin-layer cellulose plates with poly-imine that served as an ion exchange system. After chromatographic separation we identified the location of cyclic AMP and of the other nucleotides uinder UV light, marked them with pencil and then cut out the areas containing these. The cut out strips were immersed in scintillation vials and counted in a common scintillation medium to count the radioactivity levels. This way we could determine how much of the radioactive ATP had been transformed into cyclic AMP and calculate the enzyme rates in proper units - pmol/min/mg protein.
The exciting thing was then that indeed the enzyme rates proved to be affected by various hormones such as epinephrine, glucagon, TSH, ACTH, FSH and others produced dose-dependent activation of the enzyme.
I cannot believe today that we went through all this trouble to study this enzyme, needing such an elaborate setup. Once you are able to develop a technology to measure something new and reproducibly so, you can crank out data and generate publications because every enzyme assay produces new findings and results!
And here is a little question for you: How come fat cells seem to respond to so many different hormones with lipolysis (mediated always by cyclic AMP? This is not seen in any other tissue or cell.
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