Effect of adenylate cyclase activators and Mg2+ on the binding and the electron spin resonance spectra of N-methylmaleimide nitroxide in membrane particles from the liver fluke Fasciola hepatica.
Optimal conditions for activation of adenylate cyclase in membrane particles were studied. Enzyme activation with serotonin (5-hydroxytryptamine). NaF, and guanosine 5'-(3-O-thio)-triphosphate (GTP gamma S) was time-and temperature-dependent. Mg2+ was required for enzyme activation. Adenylate cyclase that was activated by NaF or GTP gamma S was gradually inhibited by N-methylmaleimide while enzyme activated with serotonin and GTP responded faster to inhibition by the same sulfhydryl reagent. Th enzyme responded in a similar fashion to a spin-labeled N-methylmaleimide analog 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrolidinyloxyl (i.e., N-methylmaleimide nitroxide). Binding of the spin label was enhanced following enzyme activation by serotonin, NaF, or GTP gamma S in the presence of Mg2+. Activation of the enzyme was accompanied by an increase in the strong immobilization peaks in the EPR spectra. Both effects, the increase in binding and in the strong immobilization peaks, can be induced by Mg2+ alone. The results indicate that a general conformational induced by Mg2+ may be essential for adenylate cyclase activation.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D005210:Fasciola hepatica / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008301:Maleimides / Q000378:metabolism* / Q000494:pharmacology; D012701:Serotonin / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D008301:Maleimides; D013873:Thionucleotides; D012701:Serotonin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C034994:N-methylmaleimide nitroxide; D006160:Guanosine Triphosphate; D012969:Sodium Fluoride; D000262:Adenylyl Cyclases; D008274:Magnesium