Identification of inducible calmodulin-dependent nitric oxide synthase ...

Vol. 267, No. 35,Issue of December 15,pp. 25385-25388,1992 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of Inducible Calmodulin-dependentNitric Oxide Synthase in the Liver of Rats* (Received for publication, June 19, 1992)

Sachio IidaS, Hiroshi OhshimaS$YI,Shinobu OguchiS, Toshio Hatall, Hisanori Suzuki$ll**, Hiroshi Kawasaki$$, and Hiroyasu Esurni$@ From the $Biochemistry Division,National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, Japan, the 11Department of Obstetrics and Gynecology, Saitama Medical School,Faculty of Medicine, 38 Morohongo, Iruma-gun, Saitama, Japan, and the $$Department of Molecular Biology, The Tokyo Metropolitan Institute of Medical Science,3-18-22 Honkomagome, Bunkyo-ku, Tokyo, Japan

A calmodulin-dependent nitric oxide synthase was significantly induced in the liver of rats treated intravenously withheat-killed Propionibacterium acnes and 5 days later withEscherichia coli lipopolysaccharide. The apparent calmodulin-dependent and -independent isozymes were separated by Mono Q column chromatography after their partial purification by B’,S‘-ADP-agarose affinity chromatography. Both enzymes had a molecular weight of 125,000 as determined by SDS-polyacrylamide gel electrophoresis and required NADPH, tetrahydrobiopterin, and dithiothreitol as cofactors. Their activities were completely inhibited by the specific nitric oxide synthase inhibitors p-monomethyl-L-arginine and Nu-nitro-L-arginine at 80 and 800 PM, respectively. The peptide maps of these two isozymes with lysylendopeptidase and their reverse-phase column chromatographic profiles were indistinguishable. In the presence of bovine calmodulin, the purified calmodulin-dependent isozyme behaved as a calmodulin-independentisozyme on Mono Q column chromatography. The purified calmodulinindependent isozyme was converted to a calmodulindependent isozyme by EDTA and EGTA. Calmodulin blot analysis using‘251-calmodulinshowed that the two isozymes bound calmodulin equally efficiently.

Nitric oxide (NO)’ has recently been shown to be formed from L-arginine by NO synthase (EC 1.14.23), which is present in various tissues and organs (1-4). This NO appears to be responsible for the cytotoxic effects of macrophages and neutrophils (5), for vasodilatation mediated by endothelial cells (6) and for cell-to-cell communication in the nervous *This study was supported in part by a grant-in-aid from the Ministry of Health and Welfare for a comprehensive 10-year strategy for Cancer Control, Japan, a grant-in-aid for Cancer Research from the Ministry of Health and Welfare, and grants from the Applied Enzyme Foundation and Smoking Science Foundation, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. On leave of absence from International Agency for Research on Cancer, Lyon, France ll Recipients of Foreign Research Fellowships from the Foundation for Promotion of Cancer Research, Tokyo. ** On leave of absence from Istituto di Chimica Biologica, Universita’ di Verona, Verona, Italy. f f To whom all correspondence should be addressed. The abbreviations used are: NO, nitric oxide; LPS, lipopolysaccharide; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis.

system (3). NO activates soluble guanylyl cyclase resulting in increase in cellular cGMP. It has been reported that there are at least two types of NO synthase: a calmodulin-dependent isozyme present constitutively in tissues such as thebrain (7, 8), endothelial cells (9), platelets (lo), and the adrenal gland (11) and a calmodulin-independent isozyme that is induced in the liver of rats by treatment with Escherichia coli lipopolysaccharide (LPS) (4, 12) and in macrophages by their activation with LPS or lymphokines (13, 14).Here, we report that calmodulin-dependent NO synthase is induced in the liver of rats by their treatment with heat-killed Propionibacterium acnesplus LPS andalso provide evidence that atleast a part of the apparent calmodulin-independent NO synthase induced in the liver is a calmodulin-bound form of calmodulindependent NO synthase. EXPERIMENTALPROCEDURES

Materials-~-[2,3-~H]Arginine (specific activity, 55 Ci/mmol; 1 Ci = 37 GBq) and ‘251-calmodulin(specific activity, 80 pCi/pg; 1 Ci = 37 GBq) were obtained from Du Pont-New England Nuclear. flMonomethyl-L-arginine and (6R)-5,6,7,84etrahydro-~-biopterin were from Calbiochem and Dr. B. Schircks Laboratories (Jona, Switzerland), respectively. Other reagents wereof analytical grade and were purchased from Sigma or Wako (Osaka, Japan). Induction of NO Synthase in Viuo-Male Sprague-Dawley rats, weighing between 200 and 300 g, were given heat-killed P. acnes at 75 mg/kg body weightthrough a tail vein, followed 5 days later by an intravenous dose of LPS at 4 mg/kg body weight (12). Livers were removed 5 h after LPS injection and kept at -80 “C until use. Partial Purification of NO Synthase-Frozen rat livers were thawed and homogenized with a Polytron homogenizer in ice-cold buffer A (50 mM Tris-HC1, pH 7.4, containing 0.5 mM EDTA and EGTA, 1 PM leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol) using 3 ml of buffer/g of tissue. All subsequent procedures were carried out at 4 “C. The homogenate was centrifuged at 27,000 X g for 60 min. The resulting supernatant was incubated for 30 min with 2’,5’-ADP-agarose gel (0.75 m1/50 ml supernatant) with gentle agitation and centrifuged at 3000 rpm for 10 min. The precipitated B’,B’-ADP-agarose was transferred to a fritted column. The column was washed with 50 volumes of buffer A containing 0.5 M NaCl and then with 30 volumes of buffer A only. NO synthase was then eluted with 10 mM NADPH in buffer A. The fractions containing the activity were pooled and applied to an FPLC Mono Q anion exchange column HR 5/5 (0.5 X 5 cm, Pharmacia LKBBiotechnology Inc.) equilibrated with buffer A and eluted with a gradient of 0-0.5 M NaCl in buffer A. Assay of NO Synthase-NO synthase activity was measured by determining either the conversion of ~-[2,3-~H]arginine to ~-[2,3-~H] citrulline essentially based on a method of Bredt and Snyder (7, 8). Initial concentration of ~-[2,3-~H]arginine in the reaction mixture was 15 nM. Enzyme activity was also measured by the amount of the formations of nitrite and nitrate, stable oxidized products ofNO, with an automated analyzer (Flow Injector Analyzer, TCI-NOX 1000, Tokyo Kasei) (15). The levels of nitrite and nitrate were determined

25385

Inducible Calmodulin-dependent NO Synthase

25386

after deproteinizing samples by adding 60 ml of 0.5 N NaOH and 40 ml of 0.42 M zinc sulfate. Peptide Mapping-Two types of NO synthase were partially puriX fied by 2’,5’-ADP-agaroseand Mono Q columnchromatographies 0.4 c and were purified further by SDS-PAGE. After brief staining with .-0 Coomassie Brilliant Blue, the bands at 125 kDawere excised from m the gel and digested completely by overnightincubation with lysylen0.3 t C dopeptidase at an enzyme concentration of 2.0 mg/ml. The resulting a solubilized material was subjected to reverse-phase chromatography 0 0.2 5 on an ODS column. Calmodulin Bindings of the Two Enzymes-The partially purified -0 NO synthases were subjected to SDS-PAGE in 6% polyacrylamide 0.1 % gel andtransferred toa nitrocellulose membrane. The membrane was z v) blocked by incubation with 1%bovine serum albumin in TBS/CaMg 0 buffer (50 mM Tris-HC1,pH 7.4, containing 0.2 M NaC1,0.5 mM L CaC12,and 50 mM MgCl2) for 16 h at 4 “C and then incubated with Fraction number 10 pCi/ml of [‘261]-calmodulin in TBS/CaMg bufferfor 3 h at 4 “C. FIG. 1. FPLC Mono Q anion exchange column chromatogIt was then washed three times with TBS/CaMg buffer for 10-min periods and exposed to an imaging plate (Fuji PhotoFilm Co., Tokyo, raphy of NO synthase induced in the liver of rats treated with Japan) for 10 h. Radioactivitywas detected with a Bio-image analyzer P. acnes and LPS. The enzyme activity was measuredin the presence (0)and absence (0)of 1 mM Ca2+and 8 pg/ml calmodulin. (Fuji PhotoFilm Co., Tokyo, Japan). Dashed line. NaCl concentration.

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4-

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RESULTS

Induction of NO Synthase in Liver-NO synthase activity was induced significantly in the liver of rats by treatment with P. acnes and LPS. Itslevel was in the range of 0.15 and 0.35 mmol of NO; formed per mg of protein/h. The activity in theliver of untreated rats was negligible. The nitratelevel in plasma obtained 5 h after LPS injection was also significantly elevated from 25 f 1 mM in control rats to 274 +. 28 mM and was well correlated with the NO synthase activity in t h e liver. Under these conditions, the glutamic-oxaloacetic transaminase andglutamic-pyruvic transaminase levels in the plasma of treated rats also increased about 13- and 6-fold, respectively. FIG. 2. Peptide mapping analysis of calmodulin-dependent Purification of NO Synthase-SolubleNO synthase was and -independent NO synthases from rat liver with lysylenpurifiedfrom the liver of treated rats as described under dopeptidase.A, calmodulin-dependentNO synthase; B , calmodulin“Experimental Procedures.” Most of the NO synthase activity independent NO synthase. in cytosol from the liver of treated ratswas retained on2’,5’of 15 nM ADP-agarose affinity gel and could be eluted from the gel nine or 800 p M Nw-nitro-L-arginine in the presence of L-arginine as substrate. with buffer containing NADPH. SDS-PAGE analysisshowed Peptide Mappingof the Two Isozymes-To characterize the the presence of an inducible protein witha molecular mass of isozymes of NO syn125 kDa,which was not present in theliver of untreated rats calmodulin-dependent and -independent thase, we purified them separately. For this, each isozyme was (15). Material in the eluate from the 2’,5’-ADP-agarose column was purified further by FPLC Mono Q anion exchange purified further by SDS-PAGE andsubjected to peptide mapcolumn chromatography. The enzyme in the first peak re- ping analysis withlysylendopeptidase. As shown in Fig. 2, the quired calmodulin for activity, no activity being detected in peptidemaps of the two isozymes were almost identical, indicating close similarity of the two isozymes, despite their the absence of calmodulin. In contrast, the enzyme in the difference in requirement for calmodulin. second peak showed activity in the absence of calmodulin Calmodulin Bindings of the Two Isozymes-The calmodulin was absolutely (Fig. 1).Although the activity in the first peak binding of these two isozymes after SDS-PAGE and transfer calmodulin-dependent, Ca2+ requirement was not absolute. to a nylon membrane were examined using ‘251-bovinecalEven in the presence of 6 mM EGTA, NO synthase activity modulin. Result showed that the two isozymes had similar wasdetected at one-third of maximal activity whichwas calmodulin binding abilities (Fig. 3), although one isozyme observed at calcium acetate concentration of 1.5-12.5 mM in was calmodulin-independent. The possibility that these two the reaction mixture. A similar calcium requirement was also isozymes are interconvertible, suggested from these data, was observed for calmodulin-independent activity in the second examined directly. After purification of the calmodulin-depeak. Thus the first and second peaks seemed to be due to pendent isozyme, it was rechromatographed on a Mono Q calmodulin-dependentand-independentNOsynthase,recolumn with or without preincubation with bovine calmoduspectively. SDS-PAGEanalysis of theseFPLCfractions lin. As shown in Fig. 4, the calmodulin-dependent isozyme showed that the activity was closely associated with a protein was completely convertedtothecalmodulin-independent in by preincubation withcalmodulinin of 125 kDa (data not shown). The 125-kDa protein present form the absence of these two peak fraction could alsobe detectedby western blot EDTAorEGTA. Conversely, thecalmodulin-independent analysis with the antibody raised against the inducible rat isozyme was partially, although not completely converted to liver NO synthase (15). Neither calmodulin-dependent nor the calmodulin-dependent form by rechromatography on a -independent NO synthase activity was detected in the liver Mono Q column in the presenceof 5 mM EGTA. On rechroof untreated rats (data not shown), indicating that these matography of calmodulin-independent NO synthase, more activities were inducible. Both peaks of NO synthase activity than half the activity was converted to the calmodulin-dewere completely inhibited by 80 p~ NG-monomethyl-L-argi- pendent form and even the activities in fractions containing

Calmodulin-dependent Inducible A

NO Synthase

2,5387

is calmodulinpresentinthebrainandendothelialcells dependent (2, 3), whereas an inducible isozyme in activated NO synthase 205 -C macrophages does not require calmodulin for activity (2, 13, a m (.m -125 kDa 14). We have, however, demonstrated induction of a calmod116.5-C ulin-dependent NO synthase in the liver of rats treated with 80 -C P. acnm a n d LPS. In the present work,we partially purified and characterized 49.5 two induced isozymes from the liver, a calmodulin-dependent a n d a calmodulin-independent enzyme. 1) The peptide maps 1 2 3 4 5 6 7 8 9 1 0 1112 kDa of the two isozymes were almost indistinguishable, strongly indicating the close similarity of the two isozymes. 2) The 205 NO synthase efficient conversion of calmodulin-dependent NO synthase to -125 kDa 116.5the calmodulin-independent form by incubation with calmodulin strongly suggested that at least part of the calmodulin80 -c independent NO synthase was derived from the calmodulin49.5 bound form of calmodulin-dependent NO synthase. 3 ) ""ICalmodulin hound to the calmodulin-dependent and -indeFIG. 3. '2DI-Rovinecalmodulinbinding to calmodulin-dependent and -independentNO synthases. The each enzyme was pendent NO synthesesequallyefficiently.Thesefindings detected using by antiserum against inducihle NO synthase ( A ) and N O synsupport the idea that the calmodulin-independent '"'I-bovine calmodulin ( H ) after transfer to nitrocellulose membrane. thase is derived from the calmodulin-dependent isozyme. ReLanes 1-4, 2',5'-ADP-~garose fractions.Imnes 1 and 2, 1 and 1.6 p g NO of protein from control rat liver, respectively; lanes 3 and 4, 1 and 1.5 cently we cloned and analyzed the cDNA for an inducible synthase from rat liver and found t,hat the putative calmodulin pg of protein from treated rat liver, respectively. h n r s .5-12, fractions after Mono Q column chromatography. Imnes 7 and 8. 400 and 600 binding site of the enzyme is extremely hvdrophobic.2 The ng of protein of peak fraction containing calmodulin-dependent NO binding of calmodulin with the calmodulin-binding sites of synthase of treated rat liver, respectively; lanes I 1 and 12, 400 and many calmodulin-dependent proteins is known to be due to 600 ng of protein of peak fraction containing calmodulin-independent hydrophobic interaction (17). Therefore, it is probable that NO synthase of treated rat liver,respectively. Imnrs ,5 and 6 are rat liver-inducible calmodulin-dependent NO synthase is not fractions from control rat liver corresponding to those in lanes 7 and readily dissociated from calmodulin hy EGTA. This may be 8.Lanes 9 and IO are fractions from control rat liver corresponding the cause of incomplete conversion of calmodulin-independto those in lanes I I and 12. by 5 mM e n t NO synthasetocalmodulin-dependentform EGTA. Although induced NO synthase was dependent on calmodulin, calcium was not required absolutely. This finding was unexpected. Recently, Geiserp t a f . (18) made a similar finding thatamutantcalmodulin,whichdoesnotbindcalcium, present supports yeast strains growth quite normally. The finding is well consistent with this observation. Calcium may he required only for the efficient binding of calmodulin to NO synthase hy only factor 3. This interpretation is also consistent with the results in Fig. 4, in which calmodulin-independent activity was accelerated by the addition of excess calmodulin. Because calmodulin-independent NO synthase is a calmodulin-bound form of calmodulin-dependent NO synthase. gradual release of calmodulin from NO synthase may happen duringMono Q columnchromatography in which 0 5 mM each EDTA and EGTA were present. Calmodulin-independent NO synthase has been reported by others (12) to be induced in the liver of rats treated in the way used in the present study. In the present work, we showed Fracl80n number thatcalmodulin-dependent NO synthase is inducedinthe FIG 4. FPLC Mono Q chromatography of NO synthase. A , liverinthis way. It is noteworthythatthiscalmodulinpattern on first Mono Q chromatographv after 2',5'-ADl'-agarose NO synthasecouldhedemonstratedonlyafter dependent chromatography. H , rechromatography of calmodulin-dependent NO Mono Q column chromatography. There is still a possibility synthase after preincubationwith calmodulin using huffer A without EDTA and EGTA. C , rechromatography of calmodulin-independent that the calmodulin-dependent and -independent forms oft he isozyme using bufferA containing5 mM EGTA. NO synthase activity enzymearedifferent,butwethinkthatafterappropriate was determined by conversion of I.-[2,3-'H]arginine to ~-[2,3-'H] purification,mostof, if notall,theNOsynthaseactivity citrulline in a reaction mixture essentially described under "Experiwould he calmodulin-dependent. mental Procedures" with a minor modification as follows. Calcium Under the present experimental conditions, similar enzyme acetate was added to make a 100 p~ excess over EDTA and EGTA in the reaction mixture. The enzyme activity was measured in the activity was also induced in the lung, spleen, and colon and presence ( 0 )and absence (0)of calmodulin the same as in Fig. 1. the most of activities in these organs were also found to be calmodulin-dependent (19). Recently, calmodulin-independcalmodulin-independent NO synthase were enhanced by ad- e n t NO synthase was purified from 1,PS-stimulated culture (13,14). and we murine macrophages and rat granulocytes dition of calmodulin to the reaction mixture. 1 2 3 4 5 6 7 8 9 1 0

1112

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-

-

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DISCUSSION

There are reports thata constitutive type of NO synthase

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' H. Atlachi. S.lida, S.O ~ w h i1-1. , Ohshima, H. S u z u k i . K, S;I:;Isaki. H. Kawasaki. '1'. Sugimura. a n d H. I