Soluble guanylyl cyclase (sGC), the target enzyme of the signalling mo
lecule NO, contains one prosthetic haem group and consists of an alpha
and a beta subunit. So far, only the alpha(1)beta(1) heterodimer has
been shown to exist in different cells and tissues, and most biochemic
al studies of sGC have been performed with the alpha(1)beta(1) heterod
imer. Here we demonstrate for the first time the natural occurrence of
the a, subunit on the protein lever. The alpha(2) subunit co-precipit
ated with the beta(1) subunit from human placenta, showing the existen
ce of the alpha(2)beta(1) isoform in vivo. The new enzyme was expresse
d in and purified from cells from the Spodoptera frugiperda ovary cell
line Sf 9. Spectral analysis showed that the alpha(2)beta(1) heterodi
mer contains a prosthetic haem group revealing the same characteristic
s as the haem in the alpha(1)beta(1) form. The kinetic properties of b
oth isoforms and sensitivity towards NO were indistinguishable. H-1-[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 N
O-independent activator, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazo
le (YC-1), increased the maximal NO-stimulated activity of the new iso
form, caused a leftward-shift in the NO concentration-response curve a
nd turned CO into an effective activator, as it did for the alpha(1)be
ta(1) heterodimer (200-fold activation). In summary, the differences i
n primary structure of both a subunits are contrasted by their functio
nal similarity. Further studies will be needed to elucidate the physio
logical purpose of the new isoform.