Functional properties of a naturally occurring isoform of soluble guanylyl cyclase

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Abstract
Soluble guanylyl cyclase (sGC), the target enzyme of the signalling molecule NO, contains one prosthetic haem group and consists of an α and a β subunit. So far, only the α1β1 heterodimer has been shown to exist in different cells and tissues, and most biochemical studies of sGC have been performed with the α1β1 heterodimer. Here we demonstrate for the first time the natural occurrence of the α2 subunit on the protein level. The α2 subunit co-precipitated with the β1 subunit from human placenta, showing the existence of the α2β1 isoform in vivo. The new enzyme was expressed in and purified from cells from the Spodoptera frugiperda ovary cell line Sf 9. Spectral analysis showed that the α2β1 heterodimer contains a prosthetic haem group revealing the same characteristics as the haem in the α1β1 form. The kinetic properties of both isoforms and sensitivity towards NO were indistinguishable. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a selective inhibitor of sGC, abolished NO-stimulated activity of both heterodimers. The new NO-independent activator, 3-(5´-hydroxymethyl-2´-furyl)-1-benzyl indazole (YC-1), increased the maximal NO-stimulated activity of the new isoform, caused a leftward-shift in the NO concentration–response curve and turned CO into an effective activator, as it did for the α1β1 heterodimer (200-fold activation). In summary, the differences in primary structure of both α subunits are contrasted by their functional similarity. Further studies will be needed to elucidate the physiological purpose of the new isoform.