Eycke Böhme

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.

Formation and release of nitric oxide from human neutrophils and HL-60 cells induced by a chemotactic peptide, platelet activating factor and leukotriene B4

Vascular endothelial cells and neutrophils synthesize and release potent vasodilatatory factors, i.e. endothelium‐derived relaxing factors (EDRF) and neutrophil‐derived relaxing factors (NDRF). One EDRF has been identified as nitric oxide (NO) derived from arginine. We studied the synthesis and release of NO from human neutrophils stimulated with the chemotactic peptide N‐formyl‐L‐methionyl‐L‐leucyl‐L‐phenylalanine, platelet activating factor or leukotriene B4. The formation and release of NO was enhanced several‐fold in the presence of superoxide dismutase, probably by inhibiting superoxide‐induced breakdown of NO. The formation and release of NO but not the formation of superoxide anions was decreased in neutrophils pretreated with L‐canavanine, an inhibitor of arginine‐utilizing enzymes. Our data suggest that at least one NDRF is identical with NO or another labile NO containing compound derived from arginine.

Regulation of neuronal nitric oxide and cyclic GMP formation by Ca2

Abstract: Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration‐dependent manner with half‐maximal effects observed at 0.3 μM and 0.2 μM when its activity was assayed as formation of NO and L‐citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down‐regulation of the signal in NO‐producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3‐isobutyl‐1‐methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin‐dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO‐producing neurons.

Enzymatic formation of nitrogen oxides from L-arginine in bovine brain cytosol

In dialyzed bovine brain cytosol, the enzymatic formation of nitrogen oxides was directly determined. The basal formation of nitrite and nitrate was concentration-dependently enhanced by L-arginine (EC50 about 3.10(-5) M). Both the basal and L-arginine induced formations were inhibited by NG-monomethyl-L-arginine (EC50 about 2.10(-4) M). In the presence of L-arginine, a concomitant formation of citrulline was detected. L-Arginine methyl ester also served as a substrate, but neither D-arginine, D-arginine methyl ester nor N alpha-benzoyl-L-arginine ethyl ester did so. The formation of nitrite and nitrate was time-dependent, increased linearly with the protein concentration of the cytosol and was not observed when the cytosolic proteins were heat-denaturated. Exogenous NADPH (or NADP+) concentration-dependently enhanced the formation of nitrite and nitrate, whereas NADH, NAD+, FAD, Ca2+, Mg2+ and calmodulin were ineffective. These results indicate that bovine brain contains a cytosolic enzyme which uses NADPH or NADP+ as cofactors to form nitrogen oxides from both an endogenous non-dialyzable substrate and from L-arginine.

Guanylyl cyclases, a growing family of signal-transducing enzymes.

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal‐transducing enzymes such as protein tyrosine‐kinases and protein tyrosine‐phosphatases. So far, three isoforms of mammalian membrane‐bound guanylyl cyclases (GC‐A, GC‐B, GC‐C), which are stimulated by either natriuretic peptides (GC‐A, GC‐B) or by the enterotoxin of Escherichia coli (GC‐C), have been identified. These proteins belong to the group of receptor‐linked enzymes, with different NH2‐terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane‐bound enzymes, the heme‐containing soluble guanylyl cyclase is stimulated by NO and NO‐containing compounds and consists of two subunits (α1 and β1). Both subunits contain the putative catalytic domain, which is conserved in the membrane‐bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the α1‐ and β1‐subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated β2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue‐specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.—Koesling, D.; Böhme, E.; Schultz, G. Guanylyl cyclases, a growing family of signal‐transducing enzymes. FASEB J. 5: 2785‐2791; 1991.

Molecular cloning and expression of a new α-subunit of soluble guanylyl cyclase Interchangeability of the α-subunits of the enzyme

A cDNA coding for a new subunit of soluble guanylyl cyclase with a calculated molecular mass of 81.7 kDa was cloned and sequenced. On the basis of sequence homology, the new subunit appears to be an isoform of the α1‐subunit and was designated α2 as the new subunit is very similar to the α1‐subunit in the middle and C‐terminal part: it is quite diverse in the N‐terminal part. Preceding experiments had shown that coexpression of the α1‐ and β1‐subunits is necessary to obtain a catalytically active guanylyl cyclase in COS cells [(1990) FEBS Lett. 272, 221–223]. The finding that the α2‐subunit was able to replace the α1‐ but not the β1‐subunit in expression experiments demonstrates the interchangeability of the α‐subunit isoforms of soluble guanylyl cyclase.

The primary structure of the 70 kDa subunit of bovine soluble guanylate cyclase

The primary structure of the 70 kDa subunit of soluble bovine guanylate cyclase, which catalyzes the formation of cyclic GMP from GTP, has been determined. The alignment of six different clones out of two bovine libraries yielded a total of 3.1 kb with a coding region of 1857 bases. The open reading frame encodes a protein of 619 amino acids and a molecular mass of 70.5 kDa. Antibodies raised against a synthetic peptide, which corresponded to the C‐terminus of the deduced sequence precipitated guanylate cyclase activity from guanylate cyclase‐enriched preparations.

The primary structure of the larger subunit of soluble guanylyl cyclase from bovine lung Homology between the two subunits of the enzyme

The primary structure of the larger subunit of the soluble guanylyl cyclase from bovine lung, which catalyzes the formation of cyclic GMP from GTP, has been determined. Two clones, isolated from two bovine libraries yielded a total of 3261 bp with a coding region of 2073 bp. The open reading frame encodes a protein of 691 amino acids and a molecular mass of 77 500. The deduced amino acid sequence reveals regions which are, to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl cyclases.

Stimulation of soluble guanylate cyclase by endothelium-derived relaxing factor from cultured endothelial cells

Bovine endothelial cells, grown on microcarrier beads and superfused with a saline solution, were stimulated with thimerosal or bradykinin to release endothelium-derived relaxing factor (EDRF). EDRF activity in the effluent was assayed in endothelium-denuded rabbit aorta. The stimulation of purified soluble guanylate cyclase in test tubes by the EDRF-containing effluent amounted to 90-fold of basal activity and its time course correlated with that of the dilator response of the aorta. After preincubation of endothelial cells with gossypol the EDRF-induced dilator response as well as the stimulation of guanylate cyclase was suppressed.