Rat brain guanylate cyclase. Purification, amphiphilic properties and immunological characterization.
Soluble guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) has been purified to apparent homogeneity from rat brain by chromatography on Blue-Sepharose CL-6B, precipitation with (NH4)2SO4, preparative isoelectric focusing and gel-filtration on Ultrogel AcA-34. On sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis the purified enzyme showed a single band with an apparent molecular weight 59 000, when stored in buffer without glycerol and 2-mercaptoethanol. Purified enzyme has been found to be very unstable; inactivation can however be partially reversed by an endogenous heat-stable activator fraction. A monospecific antiserum obtained by immunization of rabbits was found to precipitate guanylate cyclase. This antibody also reacted with membrane-bound enzyme, indicating a close similarity to the soluble enzyme. Metal divalent cations were in general found to be strong inhibitors of the enzyme activity, though Ca2+ had no effect. ATP, CTP or UTP were shown to be competitive inhibitors of purified guanylate cyclase. Sodium nitroprusside increased cyclic GMP formation by the purified enzyme. Lysophosphatidylcholine and oleic acid, at low concentration, activated guanylate cyclase. Other unsaturated fatty acids, particularly arachidonic acid, dramatically inhibited the enzyme activity. Lipids may regulate the enzyme activity by binding to an apolar domain, as suggested by charge-shift electrophoresis. The mechanism by which guanylate cyclase is regulated in the cell appears to be a complex phenomenon. It may occur through oxidative reductive processes, and/or depend on other effectors, such as triphospho-nucleotides, divalent cations and lipid microenvironment.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002712:Chlorides; D004789:Enzyme Activation / Q000187:drug effects; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005260:Female; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D005779:Immunodiffusion; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D017895:Manganese Compounds; D009599:Nitroprusside / Q000494:pharmacology; D051381:Rats
D002712:Chlorides; D005231:Fatty Acids, Unsaturated; D008244:Lysophosphatidylcholines; D017895:Manganese Compounds; D009599:Nitroprusside; D008345:Manganese; D006162:Guanylate Cyclase; C025340:manganese chloride