Abstract
An insect ovarian cell, Spodoptera frugiperda (Sf9), has been widely used to express recombinant proteins, including adenylyl cyclase, as a host cell in the baculovirus expression system. We report the presence and characterization of a soluble adenylyl cyclase (sAC) distinct from a membrane-bound form of adenylyl cyclase (mAC) that is also present in Sf9 cells. sAC was purified 3,500-fold to near homogeneity; a single band at 25 kDa on SDS-polyacrylamide gel electrophoresis correlated well with adenylyl cyclase catalytic activity. The purified enzyme had a catalytic activity of 0.1 micromol/min.mg and the Km of 0.55 mM for the substrate ATP. In contrast to mAC, sAC was heat-stable. Enzymatic activity of sAC was not stimulated by forskolin and was inhibited by salts at high concentrations. sAC utilized both manganese- and magnesium-ATP as substrate. Di- or triphosphate-containing nucleotides, such as GTP and GDP, as well as pyrophosphate, noncompetitively inhibited sAC. Our data suggest that the physical and biochemical characteristics of sAC are different from those of mAC in Sf9 cells as well as from those of other known forms of adenylyl cyclase in animal cells; sAC in Sf9 cells may constitute a new member of adenylyl cyclase found in animals.
Highlights
An insect ovarian cell, Spodoptera frugiperda (Sf9), has been widely used to express recombinant proteins, including adenylyl cyclase, as a host cell in the baculovirus expression system
We report the presence and characterization of a soluble adenylyl cyclase distinct from a membrane-bound form of adenylyl cyclase that is present in Sf9 cells. sAC was purified 3,500-fold to near homogeneity; a single band at 25 kDa on SDS-polyacrylamide gel electrophoresis correlated well with adenylyl cyclase catalytic activity
What are the physical properties of this soluble adenylyl cyclase in Sf9 cells as compared with those of the membrane-bound form of adenylyl cyclase? Second, how are the biochemical characteristics of this adenylyl cyclase different? Our data suggest that the adenylyl cyclase found in Sf9 cells is similar to the bacterial form of adenylyl cyclase, as well as to the form of adenylyl cyclase found in mammalian germ cells; it may constitute a new member of the adenylyl cyclase family found in animals
Summary
On the other hand, adenylyl cyclase exists in a membrane-bound form to integrate hormonal stimulation This membrane-bound adenylyl cyclase has tandem repeats of a six-transmembrane structure and a cytoplasmic catalytic domain similar to a transporter or ion channel [5]. The germ cells of animals, contain an additional form of adenylyl cyclase that displays physical and functional properties markedly different from those of the membranebound form [7,8,9]. This adenylyl cyclase is not stimulated by G protein or forskolin. What are the physical properties of this soluble adenylyl cyclase in Sf9 cells as compared with those of the membrane-bound form of adenylyl cyclase? Second, how are the biochemical characteristics of this adenylyl cyclase different? Our data suggest that the adenylyl cyclase found in Sf9 cells is similar to the bacterial form of adenylyl cyclase, as well as to the form of adenylyl cyclase found in mammalian germ cells; it may constitute a new member of the adenylyl cyclase family found in animals
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