Synthase from Rat Liver

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 5361-5365, June 1992 Biochemistry Purification of a distinctive form of endotoxin-induced nitric oxide synthase from rat liver (endothelium-derived relaxing factor/L-arginine/calmodulin) TOM EVANS, ADAM CARPENTER, AND JONATHAN COHEN* Department of Infectious Diseases, Royal Postgraduate Medical School, Du Cane Road, London W12 ONN, United Kingdom Communicated by Robert F. Furchgott, March 6, 1992 (received for review November 18, 1991) ABSTRACT An endotoxin-induced form of nitric oxide We are interested in the hepatic form of the NO synthase, synthase (EC 1.14.23) was purified to homogeneity from rat which is induced by the administration of endotoxin (8). liver by sequential anion-exchange chromatography and afflin- Livers from untreated animals show minimal NO synthase ity chromatography using 2',5'-ADP-Sepharose. The enzyme activity. NO production in the liver seems to suppress protein has a subunit molecular mass of 135 kDa as determined by synthesis and protects the liver against damage (18). We have SDS/PAGE, a maximum specific activity of 462 nmol of purified this enzyme to homogeneity and show that it is citrulline formed from arginine per min per mg, and a K. for strongly stimulated by calmodulin, whereas its activity is arginine of 11 jIM. The enzyme was strongly stimulated by the only affected to a small degree by free calcium concentra- addition of calmodulin with an EC50 of 2 nM, but removal of tions-even in the presence ofcalmodulin. Thus, this hepatic free calcium from the assay medium only reduced activity by endotoxin-induced NO synthase is distinct from that de- 15%. Calmodulin inhibitors significantly reduced the enzyme scribed in endotoxin-treated macrophages, which does not activity. Tetrahydrobiopterin, FAD, and FMN were all re- depend on calmodulin for full enzyme activity (13, 14). quired for full enzyme activity. This form ofendotoxin-induced nitric oxide synthase from liver differs from the inducible MATERIALS AND METHODS enzyme found in macrophages and is unusual in that it is stimulated by calmodulin with little dependence on the calcium L-[2,3,4,5-3H]Arginine (specific activity, 35-70 Ci/mmol; 1 ion concentration. Ci = 37 GBq) was from Amersham. (6R)-5,6,7,8-Terahydro- L-biopterin was from the laboratory of B. Schirks (Jona, Switzerland). NO-Methyl-L-arginine (L-NMA) and the D iSO- Nitric oxide (NO) plays an important role as a physiological mer were a gift of the Wellcome Foundation. All other mediator in a number ofdifferent systems (reviewed in ref. 1). biochemicals were from Sigma. It is responsible for the biological activity of endothelium- Animals and Preparation of Liver Homogenates. Male derived relaxing factor, an important mediator of vascular Wistar rats (250-350 g) were injected intraperitoneally with tone (2), and mediates glutamate-induced stimulation of endotoxin (Klebsiella pneumoniae, phenol extract; Sigma) at guanylate cyclase activity in the brain (3). It is also produced 10 mg/kg and sacrificed by a rising concentration of carbon by activated macrophages (4), where it mediates cytotoxicity dioxide after 6 h. The livers were removed and homogenized against a variety of intracellular pathogens and expression of in 5 vol of ice-cold buffer A [0.32 M sucrose, 50 mM Tris Cl the ability to inhibit tumor target cell metabolism (5, 6). NO (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol (DTT), soybean production has also been demonstrated inter alia in endo- trypsin inhibitor (10 ug/ml), antipain (1 ,ug/ml), pepstatin (1 toxin-treated hepatocytes (7, 8), endotoxin-treated smooth ,ug/ml), aprotonin (1 Ag/ml), 1 mM phenylmethylsulfonyl muscle cells (9), and neutrophils (10). fluoride, 2 AM FAD, 2 ,uM FMN, and 2 AM tetrahydrobio- Characterization of the enzymes responsible for NO pro- pterin] with a Teflon pestle in a glass homogenizer. The duction has broadly classified them into two groups: consti- homogenates were centrifuged at 100,000 x g for 1 h at 40C, tutive NO synthase and inducible NO synthase. A constitu- and the supernatant was used for subsequent purification. tive form ofthe enzyme with a molecular mass of 150 kDa has Assay of NO Synthase. NO synthase activity was assayed been purified from rat cerebellum (11), and its gene has been by the conversion of radiolabeled arginine to citrulline as isolated and sequenced (12). This form of the enzyme is described (11). Citrulline is produced with equimolar strongly dependent on calcium and calmodulin for its activ- amounts ofnitric oxide. The reaction buffer contained 50 mM ity. The inducible form ofthe enzyme from a macrophage cell Tris Cl (pH 7.4), 1 mM NADPH, 1 mM DTT, 4 1uM FAD, 4 line (13) and peritoneal macrophages has also been purified .tM FMN, and 50 ,M tetrahydrobiopterin. An assay mix was (14). This has a subunit molecular mass of 130 kDa in the cell composed of 100 /l of reaction buffer, 25 Al of 100 nM line and 150 kDa in the peritoneal macrophages. In neither radiolabeled arginine, and 10AI of the enzyme fraction to be case was calcium or calmodulin required for full enzyme tested. Reactions were allowed to proceed for 15 min at 370C. activity. An NO synthase that required calcium but not In crude liver preparations, endogenous arginase and other calmodulin for full expression of activity (15) has also been urea cycle enzymes were inhibited by including 50 mM purified from neutrophils. Both of these forms of NO syn- valine, 1 mM ornithine, and 1 mM citrulline in the reaction thase were cytosolic, but in endothelial cells the majority of buffer as described (8). In addition, to validate the enzyme NO-synthesizing capability appears to be membrane bound activity of the purified preparations, these were also assayed (16). This form of the enzyme has also been purified and has for their ability to form nitrite ions, a stable end product of a subunit molecular mass of 135 kDa (17). Taken together, NO in aqueous solutions. The method for nitrite generation these data suggest that there are a number of different is described in ref. 4; 10 /4 of purified enzyme was added to isoforms of the enzyme. 200 /l of reaction buffer containing 1 mM NADPH, 1.25 mM CaCl2, 0.2 mM arginine hydrochloride, 1 mM DTT, 50 mM The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: L-NMA, Nw-methyl-L-arginine; DTT, dithiothreitol. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5361 Downloaded by guest on October 1, 2021 5362 Biochemistry: Evans et al. Proc. Natl. Acad. Sci. USA 89 (1992) Tris Cl (pH 7.4), calmodulin (2.5 ,ug/ml), 50 ,uM tetrahydrobiopterin, 4 gM FAD, and 4 gM FMN. Reactions were allowed to proceed for 3 h at 370C. Nitrite assays were performed as described in ref. 19. Purification of NO Synthase. All procedures were performed at 40C. The supernatant from the liver homogenates E was i10< (two livers) after the ultracentrifugation step pumped at ~~~~~~~~~~~300 2 ml/min onto a 30-ml column of DEAE-cellulose equilibrated with homogenization buffer with no added sucrose. co 1 200 The column was washed with 70 ml of buffer, and then the enzyme was eluted with a 100-ml gradient of0-0.5 M sodium 10- z chloride in the same buffer. Fractions (2 ml) containing the 100 C/) NO synthase activity were pooled and incubated with 2 ml of 2',5'-ADP-Sepharose equilibrated with buffer B [10 mM Tris Cl (pH 7.4), 1 mM EDTA, 1 mM DTT, 2 ILM FAD, 2 ,uM celuls colmn NO Cytaeatvtusshwsterwrdo FMN, and 2 ,M tetrahydrobiopterin] for 30 min. The Seph- 0 10 20 30 40 50 arose was poured into a column, washed with 50 ml of buffer Fraction Number B containing 0.5 M sodium chloride, and then sequentially 2',5'-ADP-Sepharosecolumn.~~~~~~~~~~c washed with 6 ml of buffer B containing 10 mM NADH, 20 FIG. 1. Profile of NO synthase activity eluting from a DEAE- cellulose column. NO synthase activity is shown as the raw radio- ml of buffer B containing 5 mM NADP and 5 mM sodium malate, and 20 ml of buffer B containing 1 mM NADPH. activity of citrulline formed in the assay (o). The concentration of sodium chloride at each point in the gradient is shown on the other was Enzyme activity eluted with 6 ml of buffer B containing y axis (---). 10 mM NADPH. If not used immediately, the NO synthase was stabilized by the addition of bovine serum albumin to 1 tor-requiring enzymes that are specifically bound to the mg/ml and glycerol to 10% (vol/vol); this allowed storage at 2' ,5'-ADP-Sepharose column. -70°C with minimal loss of activity. Protein concentrations The results of a typical purification are shown in Table 1. were determined using the Bio-Rad protein dye binding assay Yields of the NO synthase were between 3% and 5%, with with bovine serum albumin as standard. SDS/PAGE was specific activities ranging from 223 to 462 nmol of citrulline performed according to the method of Laemmli (20). formed per mi per mg. As has been noted in the purification of the constitutive isoform of NO synthase, once the enzyme RESULTS is obtained relatively free of impurities, it is extremely unstable; it loses all of its activity after overnight storage at Previous reports have shown that an hepatic NO synthase is -70dC. This may account for the rather variable specific induced 26-fold by endotoxin treatment in rats, with a peak activities obtained.

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