Bernd Mayer

Purification of a CA2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum : cofactor-role of tetrahydrobiopterin

L‐Arginine‐derived nitric oxide acts as an inter‐ and intracellular signal molecule with cytosolic guanylyl cyclase as the effector system. Two NO synthase isoenzymes are postulated: a cytokine‐inducible enzyme in macrophages and a constitutive, Ca2+‐regulated enzyme in various other cells. An NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2,5‐ADP‐Sepharose. The enzyme was identified as an NO synthase with a specific NO‐chemiluminescence method and with purified cytosolic guanylyl cyclase as an NO‐sensitive detection system. The purified NO synthase was, besides Ca2+/calmodulin and NADPH, largely dependent on tetrahydrobiopterin as a cofactor.

Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase

Brain nitric oxide synthase is a Ca2+/calmodulin‐regulated enzyme which converts L‐arginine into NO. Enzymatic activity of this enzyme essentially depends on NADPH and is stimulated by tetrahydrobiopterin (H4biopterin). We found that purified NO synthase contains enzyme‐bound H4 biopterin, explaining the enzymatic activity observed in the absence of added cofactor. Together with the finding that H4 biopterin was effective at substoichiometrical concentrations, these results indicate that NO synthase essentially depends on H4 biopterin as a cofactor which is recycled during enzymatic NO formation. We found that the purified enzyme also contains FAD, FMN and non‐heme iron in equimolar amounts and exhibits striking activities, including a Ca2+/calmodulin‐dependent NADPH oxidase activity, leading to the formation of hydrogen peroxide at suboptimal concentrations of L‐arginine or H4 biopterin.

Biosynthesis of endothelium-derived relaxing factor: A cytosolic enzyme in porcine aortic endothelial cells Ca2+-dependently converts L-arginine into an activator of soluble guanylyl cyclase

In the presence of porcine aortic endothelial cytosol, soluble guanylyl cyclase purified from bovine lung was activated by L-arginine up to 2.5-fold, with an EC50 of about 6 microM. This activation was dependent on NADPH and Ca2+. The EC50 for Ca2+ was about 60 nM. No effect of L-arginine on guanylyl cyclase was observed when the cytosolic proteins were heat-denaturated. The effect of L-arginine was inhibited by NG-monomethyl-L-arginine and hemoglobin. These results indicate that endothelial cells contain a cytosolic enzyme which is directly or indirectly regulated by Ca2+ and converts L-arginine into a compound which in stimulating soluble guanylyl cyclase behaves similar to endothelium-derived relaxing factor.

Ca2+‐dependent formation of an L‐arginine‐derived activator of soluble guanylyl cyclase in bovine lung

Arginine; Ca2+; Guanylyl cyclase; NADPH

Characterization of soluble platelet guanylyl cyclase with peptide antibodies.

SummarySoluble guanylyl cyclase partially purified from bovine and human platelets was characterized with antibodies raised against synthetic peptides corresponding to different sequences of the α1- and β1-subunits of the bovine lung enzyme. On immunoblots, the platelet guanylyl cyclase was recognized by the four antisera used, with the exception of an antiserum against the C-terminus of the β1-subunit which did not react with the human platelet but with the bovine platelet β1-subunit. Furthermore the human platelet β1-subunit exhibited a slightly lower molecular mass than the bovine protein. The C-terminal antibodies precipitated native platelet and lung guanylyl cyclase activity. In contrast an antibody against a peptide out of the putative catalytic domain, which is highly conserved between all guanylyl cyclases sequenced so far, did not precipitate native guanylyl cyclase, although it recognized both subunits on immunoblots, suggesting that the respective amino acid sequence is located in an inner site of the protein.