Soluble adenylyl cyclase from Spodoptera frugiperda (Sf9) cells. Purification and biochemical characterization.
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.
Kawabe
J i
Ji
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Toya
Y
Y
Schwencke
C
C
Oka
N
N
Ebina
T
T
Ishikawa
Y
Y
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D002413:Cations, Divalent / Q000737:chemistry; D005576:Colforsin / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006358:Hot Temperature; D008623:Mercaptoethanol / Q000737:chemistry; D008970:Molecular Weight; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D018411:Spodoptera / Q000201:enzymology*
D000067956:Adenylyl Cyclase Inhibitors; D002413:Cations, Divalent; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008623:Mercaptoethanol; D000262:Adenylyl Cyclases