Nitric oxide synthase (NOS) catalyses the conversion of L-arginine into L-citrulline and nitric oxide. Recently we have developed a method for expression of recombinant rat brain NOS in baculovirus-infected Sf9 cells and purification of the enzymically active enzyme [Harteneck, Klatt, Schmidt and Mayer (1994) Biochem. J. 304, 683–686]. To study how biosynthetic manipulation of the NOS cofactors haem, FAD/FMN, and tetrahydrobiopterin (H4biopterin) affects the properties of the isolated enzyme, Sf9 cells were infected in the absence and presence of haemin chloride (4 μg/ml), riboflavin (0.1 mM), and the inhibitor of H4biopterin biosynthesis 2,4-diamino-6-hydroxypyrimidine (10 mM). In the absence of haemin, NOS was expressed to a very high level but remained predominantly insoluble. Purification of the soluble fraction of the expressed protein showed that it had poor activity (0.35 μmol of citrulline·mg-1·min-1) and was haem-deficient (0.37 equiv. per monomer). Supplementing the culture medium with haemin resulted in pronounced solubilization of the expressed enzyme, which had a specific activity of ~1 μmol of citrulline·mg-1·min-1 and contained 0.95 equiv. of haem per monomer under these conditions. Unexpectedly, the amount of H4biopterin endogenously present in the different NOS preparations positively correlated with the amount of enzyme-bound haem (y = 0.066+0.430x; r = 0.998). Radioligand binding experiments demonstrated that haem-deficient enzyme preparations containing 30–40% of the holoenzyme bound only ~40% of H4biopterin as compared with haem-saturated controls. These results suggest that the prosthetic haem group is essentially involved in the correct folding of NOS that is a requisite for solubilization of the protein and tight binding of H4biopterin.
Overexpression of neuronal nitric oxide synthase in insect cells reveals requirement of haem for tetrahydrobiopterin binding
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Barbara M. LIST, Peter KLATT, Ernst R. WERNER, Kurt SCHMIDT, Bernd MAYER; Overexpression of neuronal nitric oxide synthase in insect cells reveals requirement of haem for tetrahydrobiopterin binding. Biochem J 1 April 1996; 315 (1): 57–63. doi: https://doi.org/10.1042/bj3150057
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