Abstract
Previously characterized mammalian soluble guanylyl cyclases form alpha/beta heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named alpha(1), beta(1), alpha(2), and beta(2). The alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzyme isoforms have been rigorously characterized. The role of the beta(2) subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the beta(2) subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the beta(3) subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mm free Mn(2+). The EC(50) values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the alpha(1)/beta(1) heterodimeric form. In the presence of the detergent Tween, NO sensitivity of beta(2) was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the beta(2) subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.
Highlights
Soluble guanylyl cyclase is a heterodimeric enzyme consisting of one ␣ and one  subunit
We suggest that the 2 subunit is the first mammalian nitric oxide (NO)-sensitive guanylyl cyclase lacking a heterodimeric structure
Co-expression of the human 1 subunit with the human ␣1 or rat ␣2 subunit in Sf9 cells yielded NO and YC-1-sensitive guanylyl cyclase activity in the presence of 3 mM Mg2ϩ [12], analogous co-expression experiments performed with the novel variant 2 subunit together with either of the ␣-subunits did not result in detectable guanylyl cyclase activity (Fig. 2A)
Summary
Materials—YC-1 and ODQ were from Alexis Biochemicals, and equine myoglobin from skeletal muscle was from Serva. A NcoI/BamHI insert from the 2 clone in pcDNA3.1/V5/His-TOPO vector (see above) was ligated together with a HindIII/NcoI fragment from the 338-base pair rapid amplification of cDNA ends product into the HindIII/BamHI cut pcDNA3.1Zeoϩ vector. This clone was checked by DNA sequencing, and sequence information was submitted to GenBankTM (accession number AY004153). Transfection, and Preparation of Cytosolic Fractions of HEK-293 cells—HEK-293 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 1% L-glutamate, 1% (v/v) penicillin/. SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis—Laemmli sample buffer was added to 60 g of cytosolic fractions (for 2 and 2 E596A) of HEK-293 cells transfected with the respective plasmids, and proteins were electrophoretically separated on 8% SDSpolyacrylamide electrophoresis gels. Data represent the means (ϮS.E.) of three independent experiments performed in duplicate using different cytosol preparations in the presence of 4 mM free Mn2ϩ
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