Overexpression of neuronal nitric oxide synthase in insect cells reveals requirement of haem for tetrahydrobiopterin binding

被引:34
作者
List, BM
Klatt, P
Werner, ER
Schmidt, K
Mayer, B
机构
[1] KARL FRANZENS UNIV GRAZ, INST PHARMAKOL & TOXIKOL, A-8010 GRAZ, AUSTRIA
[2] UNIV INNSBRUCK, INST MED CHEM & BIOCHEM, A-6020 INNSBRUCK, AUSTRIA
关键词
D O I
10.1042/bj3150057
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (H(4)biopterin) affects the properties of the isolated enzyme, Sf9 cells were infected in the absence and presence of haemin chloride (4 mu g/ml), riboflavin (0.1 mM), and the inhibitor of H(4)biopterin 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 mu mol 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 similar to 1 mu mol of citrulline . mg(-1). min(-1) and contained 0.95 equiv. of haem per monomer under these conditions. Unexpectedly, the amount of H(4)biopterin 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 similar to 40% of the holoenzyme bound only similar to 40%, of H(4)biopterin 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 H(4)biopterin.
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页码:57 / 63
页数:7
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