Expression of soluble guanylate cyclase activity requires both enzyme subunits

doi:10.1016/0006-291X(91)90527-E

Biochemical and Biophysical Research Communications

Volume 174, Issue 1, 15 January 1991, Pages 351-357

https://doi.org/10.1016/0006-291X(91)90527-E Get rights and content

Abstract

Soluble guanylate cyclase purified from rat lung exists as a heterodimer of two subunits (70 kDa and 82 kDa). Recent cloning and sequencing of both subunit entities have revealed their primary structures. Transient expression in COS-7 cells by transfection with expression vectors containing the coding regions of the 70 kDa or the 82 kDa subunit cDNA showed no guanylate cyclase activity when cells were transfected with either subunit cDNA alone. However, a marked enzymatic activity was found after transfection with both subunits that was activated by sodium nitroprusside. The combination of separately expressed guanylate cyclase subunits could not reconstitute enzymatic activity in vitro. Furthermore, cotransfection with antisense oligonucleotides against the 70 kDa subunit or the 82 kDa subunit mRNA inhibited the guanylate cyclase activity. These data indicate that both the 70 kDa and the 82 kDa subunits must be present and interactive with each other in order to see basal guanylate cyclase activity and activation with sodium nitroprusside.

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In the present study, we did not analyse the sGC α-subunit. However, although cloning and expression studies indicate that the two subunits have catalytic domains, the enzymatic activity requires the presence of both [31]. Thus, it can be stated that reduced expression of one sGC subunit results in reduced enzymatic activity.
Lead exposure induces hypertension and endothelial dysfunction. However, the effects on the pulmonary vasculature have not been explored. In this study, rats exposed to lead acetate for seven days (4 μg/100 g on the 1st day and 0.05 μg/100 g/day i.m. subsequently) had lead blood level of 3.9 ± 0.7 μg/dL and increased right ventricular pressures. There was an increased Pb deposition and superoxide anions production in the pulmonary arteries, associated with reduced vasoconstriction but unchanged endothelium-dependent vasodilatation to acetylcholine (ACh). In both groups, inhibition of the nitric oxide (NO) synthase with L-NAME blocked the response to ACh, while indomethacin (cycloxygenase inhibitor) had no effect. Incubation with nonspecific potassium channel blocker (tetraethylammonium) reduced the ACh-induced vasodilatation only in the Pb group. Apamin (SK_Ca channel blocker) and 4-aminopyridine (K_v channel blocker), but not iberiotoxin (BK_Ca channel blocker), also inhibited this response in the Pb group. The vasodilatation to exogenous NO was reduced by Pb, while relaxation to the cGMP analogue was similar between groups. Concordantly, the protein level of soluble guanylate cyclase (sGC) was reduced. In conclusion, short-term and low-level exposure to Pb changes pulmonary haemodynamic and increases oxidative stress. The pulmonary vasculature exhibited increased hyperpolarization by the K_v and SK_Ca channels, probably as a compensatory mechanism to the decreased responsiveness to NO.
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Cyclic guanosine-3′,5′-monophosphate (cGMP) signaling in olfactory sensory neurons depends on the balance between cGMP synthesis and degradation, influencing the function of its target molecules, including cyclic nucleotide–gated channels. cGMP is generated by guanylyl cyclases of both the soluble and membrane-bound (particulate) form. Both types, but not all isoforms, exist in olfactory tissue. The distribution of guanylyl cyclases suggests that their expression within an olfactory sensory neuron could cause localized variations in cGMP levels. Similarly, the presence of the cGMP-degrading enzymes, the phosphodiesterases, could differentially influence cGMP concentrations or even affect cAMP levels via complex cGMP-regulated, cAMP-hydrolyzing phosphodiesterases. These various cyclic nucleotide–regulating mechanisms can, in turn, impinge on target molecules like cyclic nucleotide–gated channels to modulate membrane potential. The spatial organization and regulation of cGMP-producing and degrading enzymes may thus be of key importance in shaping the cGMP signal, thus influencing olfactory sensation.
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Evidences have been suggested that phosphodiesterase type 5 (PDE5) inhibition promotes vasculoprotective benefits in patients with cardiovascular diseases.
The aim of this study is to analyze the systemic effect of PDE5 inhibition in type 2 diabetes mellitus patients with erectile dysfunction (ED) determining changes in the expression levels of plasma proteins.
Seventeen patients with controlled type 2 diabetes mellitus and ED were included in the study. Patients received vardenafil hydrochloride 20 mg on demand during 12 weeks. At the beginning and 12 weeks after vardenafil administration, plasma samples were collected and analyzed using proteomics.
International Index of Erectile Function-Erectile Function Domain (IIEF-EFD) and plasma protein expression before and after vardenafil administration. Nitrate/nitrite release, PDE5, and soluble guanylate cyclase (sGC) expression and cyclic guanosine monophosphate (cGMP) content in cultured bovine aortic endothelial cells (BAECs).
The IIEF-EFD score was markedly improved after 12 weeks of vardenafil administration. Plasma levels of alpha 1-antitrypsin isotypes 4 and 6 and β-tropomyosin were decreased, whereas apolipoprotein AI isoype 5 was increased 12 weeks after vardenafil administration. Only β-tropomyosin plasma levels were inversely correlated with IIEF-EFD score. Tropomyosin has been added to cultured BAECs and after 24 hours reduced the protein expression level of sGC-β₁ subunit and decreased the cGMP content. Tropomyosin did not modify PDE5 expression and nitric oxide release in BAECs as compared with control BAECs. Vardenafil (10 μg/mL) did not modify sGC-β₁ subunit expression in tropomyosin + vardenafil-incubated BAECs; however, vardenafil significantly reversed the reduction of cGMP content induced by tropomyosin.
Vardenafil administration improved erectile functionality in controlled type 2 diabetes mellitus patients with ED, which was associated with reduction of circulating plasma β-tropomyosin levels. Tropomyosin affected by itself the cGMP generating system suggesting a possible new mechanism involved in ED. Vardenafil reversed the reduction effect of cGMP content elicited by tropomyosin in BAECs. Zamorano-León JJ, Olivier C, de las Heras N, Mateos-Cáceres PJ, Brime Menéndez R,Rodríguez-Sierra P, Martín Palacios N, Manso LSJ, Modrego J, Segura A, Macaya C, and López-Farré AJ. Vardenafil improves penile erection in type 2 diabetes mellitus patients with erectile dysfunction: Role of tropomyosin. J Sex Med 2013;10:3110–3120.
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Dimerization of N-methyltransferases involved in caffeine biosynthesis
2008, Biochimie
Citation Excerpt :
Dimerization of enzyme proteins has been frequently observed, as exemplified by a human immunodeficiency virus type-1 protease which exhibits enzymatic activity only by homo-dimer formation [18,19]. Guanylyl cyclase α1 forms an active hetero-dimer with the β1 subunits [20–22]. It was thus conceivable that the dimerization of N-methyltransferases involved in the caffeine biosynthetic pathway also positively or negatively affects catalytic properties.
Caffeine is synthesized from the precursor xanthosine through three methylation and one nucleoside removal steps. Methylation is catalyzed by N-methyltransferases, designated as CaXMT1, CaMXMT1 and CaDXMT1, which, respectively, convert xanthosine into 7-methylxanthosine, 7-methylxanthine into 3,7-dimethylxanthine, and 3,7-dimethylxanthine into 1,3,7-trimethylxanthine (caffeine). In the present study, we examined their cytological and biochemical properties using fusion proteins with fluorescent proteins. All three enzymes were found to localize in cytosol as visualized by green fluorescence protein fusions. The possibility of dimer formation among these enzyme proteins was examined in vivo by transient expression of bimolecular fluorescence complementation of yellow fluorescent protein (YFP) using onion epidermal cell layers. Results showed that each enzyme protein formed a homo-dimer in cytosol as seen by a clear reconstituted YFP fluorescence. In addition, each enzyme also formed a hetero-dimer with each of the other two enzymes in cytosol. The biological significance of dimerization among structurally resembling methyltransferases involved in caffeine biosynthesis is discussed.

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Expression of soluble guanylate cyclase activity requires both enzyme subunits

Abstract

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Commun

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Anal. Biochem

J. Biol. Chem

FEBS Lett

J. Cyclic Nucleotide and Protein Phosphorylation Res

Nature