Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain.
Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37 degrees C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480 000. Lubrol PX, hydroxylamine, or NaN3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN3 plus catalase, N-methyl-N'-nitro-N-nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn2+ for its activity; the maximum activity was observed at 3-5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2 mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(beta,gamma-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN3 plus catalase or N-methyl-N'-nitro-N-nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.
D000818:Animals; D001386:Azides / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D006152:Cyclic GMP / Q000494:pharmacology; D004164:Diphosphonates; D006150:Guanine Nucleotides; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000302:isolation & purification / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006898:Hydroxylamines / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D051381:Rats; D013574:Synaptosomes / Q000201:enzymology*
D001386:Azides; D004164:Diphosphonates; D006150:Guanine Nucleotides; D006898:Hydroxylamines; D008769:Methylnitronitrosoguanidine; D009599:Nitroprusside; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase; D006152:Cyclic GMP