Abstract
The interaction of various spin-labeled compounds with the murine thymocyte adenylate cyclase-cyclic AMP system was investigated. Electron paramagnetic resonance spectra from spin-labeled compounds were used to calculate the order parameter, S, and indicated that the thymocyte plasma membrane is a relatively rigid structure. Increasing concentrations of spin-labeled stearates, but not their corresponding methyl esters, resulted in increased membrane fluidity, partial lysis, and concomitant complete inhibition of cholera toxin-mediated increases in cyclic AMP content. Upon subsequent isolation of plasma membranes from these cells, cholera toxin-stimulated adenylate cyclase activity was also completely inhibited. Direct addition of spin-labeled stearates, but not spin-labeled methyl stearates, to thymocyte homogenates caused a dramatic reduction of basal, cholera toxin-, isoproterenol-, NaF-, and prostaglandin E1-stimulated adenylate cyclase activity. Inhibition was complete within the first minute of addition to homogenates and required approximately 0.2 mM spin-labeled stearate I(12,3) for half-maximal inhibition. This inhibition occurred in the presence or absence of an ATP-regenerating system and was not readily reversible. Furthermore, since the membrane cyclic phosphodiesterase activity was not altered by spin-labeled stearates, their inhibition was attributed to a direct action of stearate spin labels on adenylate cyclase. Neither stearate, methyl stearate, spin-labeled methyl stearates nor 2,2,6,6,-tetramethylpiperidine-1-oxyl (Tempo) altered cell viability or enzyme activities at the concentrations studied. Spin-labeled stearates seemed to intercalate into different areas of the plasma membrane than their corresponding methyl esters. Furthermore, the action of spin-labeled stearates appeared to be on the exterior of the plasma membrane rather than the interior. These results illustrate the presence of multilipid domains and the importance of selected lipids and lipid-protein interactions in the adenylate cyclase-cyclic AMP system. Thymocyte adenylate cyclase is described in terms of a current model for membrane proteins.
Highlights
The interaction of various spin-labeled compounds with the murine thymocyte adenylate cyclasecyclic AMP system was investigated
Current knowledge of the adenylate cyclase-cyclic AMP system is being interpreted in terms of this fluid mosaic mode1 of membrane structure
Time course study of 1(12,3) inhibition of murine thymocyte adenylate cyclase activity. 1(12,31 or 3.3% ethanol control were added to thymocyte homogenates at 4” and immediately assayed for adenylate cyclase activity at 30” in the presence of 10 mM NaF at the indicated times
Summary
Materials - [(u-32P]ATP, cyclic [G-“HIAMP, and Riafluor were purchased from New England Nuclear, Boston, Mass. Cells were incubated in the presence or absence of 250 rig/ml of cholera toxin This dose of cholera toxin has previously been shown to maximally activate murine thymocyte adenylate cyclase after 30 min at 37” [24]. Different spin-labeled compounds were added during the last 20 min. Cells were processed for determination of adenylate cyclase activity or cyclic AMP content. Prior to determination of enzymatic activity, membranes were incubated for 20 min at 4” with varying concentrations of spin-labeled compounds while the per cent of ethanol remained constant. Concentrations of labeled compounds are expressed as that amount present during the determination of enzymatic activity unless stated otherwise. The two-step procedure of Thompson and Appleman [29] for measuring cyclic phosphodiesterase activity was used with the first step being linear for 10 min at. None of the compounds tested interfered with the cyclic AMP binding protein assay
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
