Activation of purified soluble guanylate cyclase by arachidonic acid requires absence of enzyme-bound heme.
The mechanism by which arachidonic acid activates soluble guanylate cyclase purified from bovine lung is partially elucidated. Unlike enzyme activation by nitric oxide (NO), which required the presence of enzyme-bound heme, enzyme activation by arachidonic acid was inhibited by heme. Human but not bovine serum albumin in the presence of NaF abolished activation of heme-containing guanylate cyclase by NO and nitroso compounds, whereas enzyme activation by arachidonic acid was markedly enhanced. Addition of heme to enzyme reaction mixtures restored enzyme activation by NO but inhibited enzyme activation by arachidonic acid. Whereas heme-containing guanylate cyclase was activated only 4- to 5-fold by arachidonic or linoleic acid, both heme-deficient and albumin-treated heme-containing enzymes were activated over 20-fold. Spectrophotometric analysis showed that human serum albumin promoted the reversible dissociation of heme from guanylate cyclase. Arachidonic acid appeared to bind to the hydrophobic heme-binding site on guanylate cyclase but the mechanism of enzyme activation was dissimilar to that for NO or protoporphyrin IX. Enzyme activation by arachidonic acid was insensitive to Methylene blue or KCN, was inhibited competitively by metalloporphyrins, and was abolished by lipoxygenase. Whereas NO and protoporphyrin IX lowered the apparent Km and Ki for MgGTP and uncomplexed Mg2+, arachidonic and linoleic acids failed to alter these kinetic parameters. Thus, human serum albumin can promote the reversible dissociation of heme from soluble guanylate cyclase and thereby abolish enzyme activation by NO but markedly enhance activation by polyunsaturated fatty acids. Arachidonic acid activates soluble guanylate cyclase by heme-independent mechanisms that are dissimilar to the mechanism of enzyme activation caused by protoporphyrin IX.
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology*; D001665:Binding Sites; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D006162:Guanylate Cyclase / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D066298:In Vitro Techniques; D008168:Lung / Q000201:enzymology; D009569:Nitric Oxide / Q000494:pharmacology; D012709:Serum Albumin / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D012995:Solubility; D013053:Spectrophotometry
D001095:Arachidonic Acids; D012709:Serum Albumin; D016718:Arachidonic Acid; D009569:Nitric Oxide; D006418:Heme; D012969:Sodium Fluoride; D006162:Guanylate Cyclase