1 submitted may 24, 2002 revised june25, 2002 re-revised july

1

Submitted May 24, 2002Revised June25, 2002Re-revised July 10,2002

BIOCHEMISTRY OF MITOCHONDRIAL NITRIC-OXIDE SYNTHASE*

Elfering, Sarah Liv, Sarkela, Theresa Marie, and Giulivi, Cecilia†

Department of ChemistryUniversity of Minnesota

Duluth, MN 55812

* This work was supported by National Institutes of Health (ES011407-01), MinnesotaMedical Foundation (3048-9227-01), and partially by Grant-In-Aid from University ofMinnesota, Graduate School, United Mitochondrial Disease Foundation (99-1) and CottrellResearch Corporation (CC5675).

SHORT TITLE: mitochondrial nitric-oxide synthase

†To whom correspondence should be addressed:Dr. Cecilia GiuliviDepartment of Chemistry,University of Minnesota,10 University Drive,Duluth, MN 55812e-mail: [email protected]

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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ABSTRACT

We reported that the generation of nitric oxide by mitochondria is catalyzed by a constitutive,

mitochondrial nitric-oxide synthase (mtNOS). Given that this production may establish the

basis for a novel regulatory pathway of energy metabolism, oxygen consumption, and oxygen

free radicals production, it becomes imperative to identify unequivocally and characterize this

enzyme to provide basis for its regulation. The mitochondrial localization of mtNOS was

supported by following the hepatic distribution of mtNOS, immunoblotting submitochondrial

fractions, and immunohistochemistry of liver tissues. mtNOS was identified as bNOSα by

various methods (mass spectrometry of proteolytic fragments, amino acid analysis, MW, pI

and analysis of PCR fragments), excluding the occurrence of a novel isoform or other splice

variants. Distribution of mtNOS transcript indicated its occurrence in liver, brain, heart,

muscle, kidney, lung, testis, and spleen. In contrast to bNOS, mtNOS has two

posttranslational modifications: acylation with myristic acid and phosphorylation at the C-

terminus. The former modification is a reversible and posttranslational process, which may

serve for subcellular targeting or membrane anchoring. The latter modification could be

linked to enzymatic regulation. These results are discussed in terms of the role that nitric

oxide may have in cellular bioenergetics.

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INTRODUCTION

Our (1-5) and other studies (6-10) provided evidence for production of nitric oxide (NO. 1) by

intact, purified mitochondria and by mitochondria-rich fractions, respectively. In our lab the

production of NO. by mitochondria was demonstrated by using two spectroscopic

techniques, i.e., oxidation of oxymyoglobin inhibitable by a nitric oxide synthase (NOS)

inhibitor, and by electron paramagnetic resonance with a spin trapping technique (1-4). In

other labs, the production of NO. has been followed by the formation of L-citrulline from

radiolabeled L-arginine (8-10). These measurements along with the use of purified, intact

mitochondria, provided experimental support for production of NO. by these organelles.

Later, we isolated and purified a protein with nitric oxide synthase activity mainly localized at

the mitochondrial membranes (mtNOS; 2). This mtNOS has characteristics similar to the mac-

isoform (antigenic crossreactivity, and tight-binding of calmodulin). In contrast, mtNOS is

constitutively expressed and membrane-bound (2).

The identification of mtNOS is a required step to assign this protein to one of the known

isoforms (brain NOS, endothelial NOS, or inducible NOS), or to depict it as a novel isoform.

The relevance of this assignment resides on understanding the expression and regulation of

mtNOS, considering the biochemical differences of NOSs. For example, the constitutive

forms, i.e., b-NOS and e-NOS, account for the rapid, transient, Ca2+- dependent production of

NO. (11-13); the inducible form, i.e., mac-NOS, causes the slow onset of Ca2+- independent

NO. synthesis in inflammatory cells (after stimulation by cytokines or lipopolysaccharides; 14-

15).

Considering the relevant role that NO. may have at modulating the O2 consumption (3)

and O2 free radical formation (5, 16) by mitochondria, it is important to gain insight on the

biochemical characterization of this protein. The goals of this study are the following: first, to

identify mtNOS; second, to study the molecular expression of mtNOS in different tissues, and

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third, to investigate putative posttranslational modifications that may target this protein to

mitochondria or may affect enzymatic activity.


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MATERIALS AND METHODS

Biological Materials

Isolation of rat liver mitochondria and Submitochondrial Fractions___ Liver mitochondria were

isolated from adult Wistar rats (180-200 g) by differential centrifugation (1, 17). Purified

mitochondria were obtained by Percoll gradient centrifugation (1, 18), subsequently washed

with 150 mM KCl to dissociate arginase (1, 19) and other nonspecifically bound proteins. The

preparations contained a contamination of non-mitochondrial origin from 1 to 4%, assessed

by evaluating enzymatic markers of liver fractions (20 and references therein). The

respiratory control ratio (RCR) of these mitochondria was 8 ± 1 and the ADP:O ratio was 1.8 ±

0.2 with succinate. Mitoplasts were obtained from Percoll-purified mitochondria subjected to

a controlled digitonin treatment. Mitoplasts, separated from OM and IMS by centrifugation,

were sonicated, and this suspension was separated in two fractions by centrifugation: soluble

(M) and particulate (IM and CS). The IM was purified using a sucrose gradient, sonicated in

water, and washed twice with KCl, to yield SMP (21). The recovery of cytochrome oxidase, a

marker for mitochondrial IM, was 43.5%. The resultant SMP contained less than 0.06% of

the OM marker, monoamine oxidase, and the IM marker, adenylate kinase, and about 2% of

the matrix-specific marker enzyme Mn-superoxide dismutase. The recoveries were based on

the total activity of the mitochondrial markers in mitochondria and SMP.

Purification of mtNOS ___ mtNOS was isolated from purified mitochondria from 6-8 rat livers,

obtained as described before (2).

Measurement of NO. production ___ The reaction medium used to follow NO

. production by

purified mtNOS or crude fractions contained 1 mM L-Arg, 1 mM magnesium acetate, 1 mM

CaCl2, 0.1 mM NADPH, 20 µM tetrahydrobiopterin, 10 mM CHAPS, 1-5 µg/ml calmodulin, in

0.1M Hepes buffer, pH 7.5 (22), and 0.05-0.5 mg of protein. mtNOS activity was followed by

absorption spectrophotometry by following the oxidation of oxymyoglobin. This assay is

based on the oxidation of oxy- to met-myoglobin by NO. (1, 23-24). This oxidation was


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followed at 581-592 nm in a double-beam, dual-wavelength Aminco DW-2C UV-Vis

spectrophotometer at 22˚C in the presence of 1-mM oxymyoglobin (1). Albeit the detection

limit of the oxymyoglobin assay is low (nM concentrations), its specificity could be rather

broad. To overcome this shortcoming, the rate of oxymyoglobin oxidation sensitive to NG-

monomethyl-L-arginine (NMMA), an inhibitor of NOS, was considered representative of an

enzymatic production of NO. (1). The identification of NO

. as a product was performed by

electron paramagnetic resonance with a spin trapping technique as described before (1, 25-

26). Protein was determined by the Lowry assay using bovine serum albumin as a standard

(27).

Two-dimensional electrophoresis of mtNOS and MALDI-ToF analyses___ Immobilized pH

gradient (IPG) strips were rehydrated overnight in Rehydration Buffer (6 M urea, 2 M

thiourea, 2% NP-40, 2% ampholytes, 0.1 M dithiothreitol) using the Immobiline DryStrip

Reswelling Tray. One mg of crude fraction or 50 µg of purified mtNOS was solubilized in

Rehydration Buffer for 30 minutes at room temperature. The samples were then applied to

the strip during the first dimension run. The IEF was performed using the Multiphor II and the

Immobiline DryStrip Kit (Amersham Pharmacia Biotech) for 14-17 kVh (depending on the

length and pH range of the strip being used). Following the first dimension run, the strips

were equilibrated for the second dimension for 10 minutes in 0.05 M Tris, pH 6.8, 6 M urea,

30% glycerol, 1% SDS, and 25 mg/ml of dithiothreitol. Subsequently, the IPG strips were laid

atop a 10% SDS-PAGE gel and layered with 0.5% agarose in 1x Laemmli running buffer.

The electrophoresis was performed using the Hoefer SE 600 at 50 V overnight at 4˚C. After

electrophoresis, one of the gels was electroblotted to PVDF membrane (Immobilon-P,

Millipore Inc., Bedford, MA) and incubated in blocking buffer (1% nonfat dry milk, 10% goat


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serum in Tris-buffered saline with Tween (TBST; 0.3 M NaCl, 40 mM Tris-HCl, pH 7.6, 0.1%

Tween-20) for 1 h. The blot was treated with a 1:2,000 dilution of anti-iNOS monoclonal

antibodies (catalog # N32020; Transduction Laboratories, Lexington, NY) for 1 h in blocking

buffer. The membranes were washed three times with TBST and then anti-mouse IgG,

horseradish peroxidase-conjugate antibody (Upstate Biotechnology, Lake Placid, NY) was

added in a 1:10,000 dilution. The blots were washed 3 times with TBST, and were developed

using ECL reagents. The blots were exposed to chemiluminescent-sensitive Kodak X-Omat

Blue XB-1 film (Eastman Kodak, Co.). The other gels were stained with SYPRO Ruby

(Molecular Probes) and superimposed to the films to identify the spot(s) that crossreacted

with the antibodies to iNOS. Those spots were excised from the gels, and in-gel digestion

was performed with either trypsin (sequencing grade bovine trypsin modified by reductive

methylation, obtained from Promega, Madison, WI) or endoproteinase Glu-C (protease V8,

sequencing grade from Staphylococcus aureus V8; Roche Diagnostics, Germany). The

eluted peptides were analyzed by MALDI-ToF. These analyses were performed at the

University of Minnesota, Mass Spectrometry Facilities (Minneapolis, MN). The mass

fragments were analyzed using two programs which are based on different algorithms: MS-

Fit (ProteinProspector, version 3.4.1.) and ProFound (version 4.10.5).

RT-PCR and PCR conditions ___ RNA was extracted from 1 to 2 g of freshly isolated rat

liver by the guanidium isothiocyanate method (Trizol; Gibco, Grand Island, NY) essentially as

described before (28-29). The enrichment of poly(A)+RNA was performed by using oligodT

resin (MessageMaker, Gibco). The integrity of RNA or poly(A)+-enriched fractions were

assessed by running a minigel and examining ethidium bromide staining of 18S and 28S

rRNA. To prevent the problem created by genomic DNA contamination which may lead to

false-positives (30), we used DNase-treated poly(A)+ RNA for reverse transcription. Half µg


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of rat liver poly(A)+ RNA was treated with RQ1 RNase-Free DNase (Promega, Madison, WI)

at 37˚C for 30 minutes, then heated at 65˚C for 10 minutes to inactivate the DNase. RT-PCR

was performed using Superscript™ One-Step RT-PCR with PLATINUM® Taq (Life

Technologies, Rockville, MD). The reaction contained 1x reaction mix (containing 0.2 mM

dNTP, 1.2 mM MgSO4), 0.5 mg mRNA, 1 µl RT/Taq Mix, and 0.2 mM of both the sense and

anti-sense primers, 40 U RNaseOUT (Gibco BRL, Rockville, MD), and DEPC-treated ddH2O

to bring the final volume to 50 µl. No additional MgSO4 was required owed to the residual salt

carried over from the DNase treatment. The RT-PCR reactions, and other PCR reactions,

were carried out in a Perkin Elmer Gene Amp 2400 PCR System. Reverse transcription was

performed at 50˚C for 60 minutes. PCR amplification followed after a 2 min denaturation at

94˚C. Forty amplification cycles were performed as follows: denaturation at 94˚C for 30s,

annealing at 55˚C for 30s, and elongation at 68˚C for 3 min. A final extension was performed

at 72˚C for 7 min, and then sustained at 4û˚C. Parallel amplification of monocarboxylate

transporter-1 was performed for reference and as a positive control. PCR products were

electrophoresed on a 1.0% agarose gel using TAE buffer containing ethidium bromide and

visualized on a MultiImageFC using FluorChem 2.0 software (AlphaInnotech, San Leandro,

CA). Nested PCR reactions contained 1.25 U AmpliTaq Gold (Applied Biosystems, Foster

City, CA), 1x PCR Buffer II, 1.8 mM MgCl2, 200 mM dNTPs, 0.2 mM of both sense and anti-

sense primers, and 1 µl template from the RT-PCR reaction in a final volume of 50 µl. The

samples were denatured at 94˚C for 5 min, followed by 40 cycles of denaturation at 94˚C for

30s, annealing at 55˚C for 1 min, and extension at 68˚C for 2 min. The final extension was

held at 68˚C for 7min, then set at 4˚C until analyzed by gel electrophoresis as described

above.

PCR reactions for Figure 3 (panel A, lanes 6 and 7, and panels B & C) were performed using

the Rat MTC Panel I (Clontech, Palo Alto, CA) as the template and PCR Master Mix

(Promega, Madison, WI). The PCR conditions for the products obtained in Fig. 3A (lanes 6 &

7) were performed using 1x Master mix (1.25 U Taq DNA polymerase, 200 mM dNTPs, 1.5


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mM MgCl2), 0.5 mM sense and anti-sense primers, and 1 ng template cDNA in a final volume

of 50 µl. Initial denaturation was carried out for 2 min at 95˚C followed by PCR. The PCR

conditions were as follows: 40 cycles of 30s denaturation at 95˚C, 30s annealing at 55˚C, and

3 min elongation at 72˚C. The final extension was performed at 72˚C for 5 min and

maintained at 4˚C until analyzed by gel electrophoresis. Nested PCR reactions were carried

out with the same reaction components and cycling conditions as the parent reactions,

except the cDNA template was substituted for 1 µl of the parent reaction and different sense

and anti-sense primers were used as indicated in the figure legend. Although the PCR

products depicted in Fig. 3B & C were also performed with cDNA from MTC panel, the

experimental conditions were slightly different from those in Fig. 3A with the following

modifications: The parent PCR reactions contained 1.25 U AmpliTaq Gold (Applied

Biosystems, Foster City, CA), 1X PCR Buffer II, 1.8 mM MgCl2, 200 mM dNTPs, 0.2 mM of

both sense and anti-sense primers, 1 ng MTC Panel cDNA in a final volume of 50 µl. The

reactions were amplified under the following conditions: denaturation at 94˚C for 5 min,

followed by 40 cycles of denaturation at 94˚C for 30s, annealing at 55˚C for 1 min, and

extension at 68˚C for 2 min. The final extension was held at 68˚C for 7 min and maintained at

4˚C.

Release and identification of mtNOS-bound fatty acid ___ Purified mtNOS (about 1 mg

protein/ml), obtained following the isolation procedure, was precipitated with 5 V of cold

acetone and kept for 30 min at –20˚C. The solution was centrifuged in a Dupont® centrifuge

in a SS-34 rotor at 10,000 g for 15 min at 4˚C. The pellet was dissolved in 0.5 ml of 2% SDS

W/V and precipitated again with 5 V of cold acetone. The solution was kept at –20˚C for 30

min and centrifuged at 10,000 g for 10 min at 4˚C. This latter procedure (SDS-treatment,

precipitation with acetone, and centrifugation) was repeated twice. The pellet was extracted

with 2 ml Cl3CH-methanol (2/1, v/v), washed with 2 ml of diethyl ether, and dried under a

stream of N2. Deacylation of the protein ___ The delipidated protein pellet was treated with 2


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ml of 0.1 M KOH/anhydrous methanol (less than 0.01% H2O), vortexed, and incubated at

30˚C for 30 min. The solution was centrifuged at 3,000 g for 5 min, and the protein pellet was

saved for acid hydrolysis (See below). The supernatant was acidified with 6 M HCl, and

extracted three times with hexane. These extractions were combined and dried under N2.

The residue, containing the methyl esters of fatty acids originally bound to the protein by oxy-

or thio-ester linkages, was subjected to MS analysis. The protein pellet procured after the

centrifugation at 3,000 g was washed three times with ethanol and dried under N2. This pellet

was resuspended in 1 ml of 3 M HCl in anhydrous methanol and incubated at 105˚C for 20 h

in sealed tubes (earlier purged with a N2 stream) under N2 to release amide-linked fatty acids

from the protein as the corresponding methyl esters. The tubes were cooled and extracted

three times with hexane. The extracts were combined and dried under a stream of N2. The

residue was subjected to MS analysis.

Immunohystochemistry ___ NOS was immunolocalized in paraformaldehyde-fixed rat liver

slices, essentially following the procedure described in ref. 31 with the following

modifications. After fixation, the slices were permeabilized by incubating in ice-cold acetone

for 5 min, then washed 3-times with 0.1% Triton X-100 in phosphate-buffered saline (PBS-T).

The slices were blocked with 10% normal goat serum, 1% bovine serum albumin in PBS-T

for 30 min at room temperature. Primary antibody incubations were performed using a 1:500

dilution of monoclonal antibodies to bNOS (Transduction Labs) and to subunit I of

cytochrome oxidase (Molecular Probes, Eugene, OR) in blocking solution. The bNOS

antibody was labeled with Alexa Fluor™ 594 according to manufacturer's instructions

(Molecular Probes). After labeling the antibody to bNOS, the specificity of these was

confirmed by immunoblotting samples of rat liver and isolated mitochondria, using positive

and negative controls. The antibody to cytochrome oxidase was commercially available as a

conjugate with Alexa Fluor™ 488. Overnight incubation of the slices with primary antibodies

was performed in the dark in moist chambers, at 4˚C. The slices were washed 3-times for 15


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min each with PBS-T. In some experiments, instead of the antibody to cytochrome oxidase,

mitochondria were labeled with MitoTracker Green FM (Molecular Probes) according to

manufacturer's conditions. The slices were washed with PBS-T, the nuclei labeled with 4', 6-

diamidino-2-phenylindole, dihydrochloride (DAPI) according to manufacturer's conditions

(Molecular Probes), air dried, and protected from light. The slides were mounted using

ProLong Antifade medium (Molecular Probes). Rat liver slices were observed with

epifluorescence microscopy using appropriate filters (Nikon E600) and photographed using a

digital camera (Spot II). The digital images were acquired using Metamorph software.

Brightness, contrast and background (the latter defined as areas without cells or with cells

without antibody addition) were adjusted and the images were overlaid using Adobe

Photoshop (Mountain View, CA).


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RESULTS AND DISCUSSION

Distribution Of Nitric Oxide Synthase Activity In Liver

The recovery of rat liver NOS’ activity (followed by either the oxidation of oxymyoglobin or

14C-L-citrulline, inhibitable by an inhibitor of NOS, NG-monomethyl-L-arginine) was 70% in the

mitochondrial fraction and 45 to 50% in Percoll-purified mitochondria, supporting the

mitochondrial localization of this enzyme. The submitochondrial distribution of mtNOS

indicated that inner membrane and contact sites were the only fractions that exhibited the

presence of mtNOS by western blotting. The almost complete recovery (60 to 70%) of

mtNOS activity from mitochondria isolated from purified rat hepatocytes (>98% purity and

>98% viability by trypan blue exclusion) indicated that this enzyme was originated from

parenchymal cells, excluding contribution from other cell types. These observations were

confirmed further by (i) immunoprobing formalin-fixed liver slices for cytochrome oxidase and

NOS, resulting in the colocalization of these proteins in parenchymal mitochondria (Fig. 1;

similar results were obtained by using MitoTracker Green instead of antibodies to cytochrome

oxidase.), and (ii) considering the high contribution of hepatocytes to liver cellular

composition (92.5%; 33) and mitochondrial volume (98.4%; 33).

Identification Of Mitochondrial Nitric-Oxide Synthase

As reported before, isolated rat liver mtNOS shared certain properties with macNOS

(antigenic crossreactivity, Vmax of the purified form), whereas the constitutive expression of

mtNOS, requirement of cofactors, and the main membrane localization suggested the

presence of either a novel isoform, or one of the well-characterized isoforms with co- or post-

translational modifications. To gain insight of the protein sequence, microsequencing of

purified mtNOS from blots was attempted; however, the finding of a blocked N-terminal

residue precluded a direct Edman degradation since the chemistry requires a free NH2 group

(or imino for Pro; ref. 34). Thus, mtNOS was separated by 2D-electrophoresis, followed by

in-gel digestion with either trypsin or endoproteinase V8. MALDI-ToF and/or Q-ToF analyses


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were performed on the eluted fragments. The resulting peptide masses and sequences were

blasted against in-silico trypsin- or V8-digested proteins from the PDB to permit the

identification of mtNOS based on the digest profile (MALDI-ToF) and amino acid sequences

(Q-ToF). The peptidic fragments from both treatments (i.e., trypsin and endoproteinase)

matched sequences of constitutive rat bNOS (Fig. 2). All fragments had a 100% homology to

bNOS, and only between 21 to 78% to mac- and eNOSs precluding these isoforms as

mtNOS. A minor percentage of fragments (5-10%) matched those from mature

carbamoylphosphate synthetase2 and iNOS.

The amino acid composition of mtNOS, obtained after acid hydrolysis of the protein, closely

matched that of bNOS (Table I). The amino acid analysis combined with pI (7.0 ± 0.5) and

MW (130,000 ± 26,000) extracted from 2D-gels, in addition to the peptide mass fingerprinting

and amino acid sequences were used to perform a protein identification search using the

MultiIdent Protein Identification on the ExPASy server. The best integrated score in terms of

amino acid composition, pI, MW, and best-matching set of peptide masses for the species

Rattus resulted in bNOS (Swiss-Prot P29476).

mtNOS And Splice Variants Of bNOS

From these results (vide supra), it could be surmised that mtNOS is the brain isoform.

However, given that four splice variants of full length bNOS (nNOSα) have been identified

(nNOSβ, nNOSγ, nNOSµ, and nNOS-2; 38-40), the question remained whether mtNOS was

one of these products or represented a novel alternative splicing product. Fragments from

amino acids 238 to 242 and from 278 to 290 excluded NOSγ, whereas fragments from amino

acids 512 to 516 and from 548 to 555 precluded NOS-2. Given that the other fragments were

present in all 3 remaining isoforms (NOSα, β, and µ), the identification was not possible by

using data from MALDI or Q-ToF.

To gather more information on what type of isoform is expressed in liver, RT-PCR

experiments were performed on mRNA using primers based on either Q-ToF sequences


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(outline sequences in Fig. 3 and underlined sequences in Table II) or gene-specific (Table II).

To decrease the degeneracy of the primers derived from protein sequences, they were

matched to the corresponding coding oligonucleotide sequences in rat brain cNOS sequence

(Gen EMBL X59949). The other primers used were gene-specific --based on the same

cNOS-- designed using the software Primer3, and selected from 5 alternative sets to best

suit PCR conditions, specificity, and discrimination among the various isoforms.

RT-PCR was performed on an enriched polyA mRNA from rat liver; previously treated with

DNase to avoid genomic DNA contamination. PCR products were separated by gel

electrophoresis and visualized by using ethidium bromide (Fig. 3A). Control experiments

included performing RT-PCR excluding primers from the reaction mixture, followed by PCR

using PS4 (Fig. 3A, lane 2) or PS5 (Fig. 3A, lane 3). The lack of DNA bands when using

these primers (and all the others; not shown) indicates the lack of genomic DNA

contamination which would have lead to false positives. Positive controls included the

amplification of a C-terminal region present in all NOS' isoforms which resulted in a product of

515 bp (Fig. 3A, lane 5). RT-PCR of rat liver polyA mRNA using PS1 followed by a nested

PCR using PS4, resulted in product size similar to that expected for NOSα, NOS-2, and

NOSµ (Fig. 3A, lane 4; 788 bp). This result excluded the possibility of the occurrence of the

transcripts for either NOS-γ or NOS-β (Table III). By performing two consecutive PCR

reactions using PS3 and PS6 on rat liver cDNA the resulting product (206 bp) excluded the

occurrence of the transcript for NOS-2 (Fig. 3A, lane 6). The lack of NOS-2 transcript was

further confirmed by performing an RT-PCR using PS1 followed by two sequential nested

PCR reactions using PS9 and PS8, resulting in a fragment size of 676 bp not present in

NOS-2 (not shown). Combining these results, it was concluded that the transcript amplified

from rat liver was either from NOSα or NOSµ. PCR experiments using PS3 and PS7

performed on rat liver cDNA allowed to obtain a fragment of 81 bp not present in NOSµ

(Table III and Fig. 3A, lane 7). Therefore, our results indicated the presence of the transcript

for NOSα in rat liver, suggesting that the synthesis of NOSα from this transcript is feasible.


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Confirmation of the identity of these PCR products was obtained by purifying the fragments

from the gel, and sequencing them and/or performing restriction maps with Not I (not shown).

The DNA sequences were blasted against the Genome rodent database and the results

matched those from rat brain NOS-1 (Fig. 2, bold sequences).

Distribution Of NOSα Transcript In Other Tissues

Previous work from this lab indicated the presence of a mtNOS activity in a limited number

of tissues (rat liver, rat heart, and pig heart mitochondria; 1-5, 32). Thus, to explore the tissue

distribution of the enzyme, we explore the occurrence of the NOSα transcript. PCR reactions

were performed on rat cDNA from various tissues using PS1 followed by a nested PCR with

PS4. All the tissues exhibited only one band of 788 bp, thus excluding the presence of a

transcript for NOSγ or NOSβ. By performing sequential PCR reactions using PS3 followed by

nested PCR using either PS7 (Fig. 3B, lanes 2-9) or PS6 (Fig. 3C, lanes 11-18) resulted in

the formation of two products (81 bp and 206 bp) consistent with the presence of NOSα

transcript in all these tissues. The only tissue that exhibited another transcript for NOS --in

addition to NOSα-- was skeletal muscle (Fig. 3C, lane 5) resulting in a 183 bp product

consistent with the simultaneous occurrence of NOSµ.

Lastly, our results indicated that the transcript of NOSα was present in liver, brain, heart,

muscle, kidney, lung, testis, and spleen, suggesting that these tissues have the capability to

express NOSα. Since our previous results showed that mtNOS is bNOSα, it could be

surmised that these tissues have the potential of synthesizing mtNOS. However, these

experiments did not provide information on the steady-state concentration of NOSα, nor on

the fraction of NOSα present in mitochondria. To this end, we evaluated the activity of

mtNOS in isolated, purified rat heart mitochondria. This activity resulted 2 to 3-times higher

than that found in liver, but because the content of the enzyme (obtained by

immunoprecipitation of mtNOS from mitochondria) was also lower (about half), the specific

activity of the enzyme remained the same. The results obtained with liver and heart indicated


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that the content of active mtNOS followed that of the transcripts, with the understanding that

this association between activity and content of transcript might not be applicable to other

tissues.

Acylation Of Mitochondrial Nitric-Oxide Synthase

Comparison of the activities of permeabilized mitochondria and SMP indicated that most

NOS activity (30-40%) was detected in the latter fraction, suggesting a localization of the

enzyme at the inner mitochondrial membrane (1-2). The association of mtNOS with

membranes was further supported using a mild detergent to solubilize the enzyme during the

purification procedure (2). The main particulate distribution of mtNOS resembled that of

eNOS (41). The specific distribution of eNOS has been attributed to the presence of an N-

myristoylated terminal and palmitoylated Cys residues (42-43) which may allow a protein-

membrane association. The presence of a blocked N-terminal group in mtNOS, aside from

its membrane distribution, lead us to investigate whether mtNOS was acylated.

To this end, samples of purified mtNOS were precipitated with acetone, washed with organic

solvents (to remove any unspecifically bound lipids), and finally subjected to alkaline

methanolysis to release methyl esters derivatives of the fatty acids. The derivatives were

extracted with hexane, and the fatty acids were identified by mass spectrometry upon

comparison with standards. About 80 to 100% of the fatty acids were recovered as the

methyl ester derivative of myristic acid (Fig. 4) after alkaline methanolysis indicating that this

fatty acid was linked through an oxy- or thio-ester. The starting material had 0.56 nmol of

enzyme and 0.7 nmol of fatty acid was recovered in this procedure indicating that the ratio of

lipid: protein was near unity. The present finding, that mtNOS is acylated with myristic acid

via an ester bond, is consistent with a reversible and posttranslational process, probably

catalyzed by acyltransferases (44-47).

The acylation of mtNOS might explain the discrepancy between the MW of NOSα (160 kDa)

and that obtained from SDS-PAGE and immunoblotting which indicated that mtNOS


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migrated at a slightly faster rate than nNOS. Although this observation has not been studied

for most acylated proteins (for example 48-50), an analogous phenomenon has been

reported for acylreductase (51), acyl carrier protein (52) and some subunits of enzymes

involved in the mitochondrial β-oxidation pathway (53). The difference in the migration of

these latter proteins compared to unacylated ones (12-20%; calculated from 48-50) was

found comparable to that observed with mt- and n-NOS (15-19%).

Phosphorylation Of mtNOS

Agonists promote the phosphorylation of eNOS facilitating its solubilization (54) probably

by decreasing the positive charge of a region that contributed electrostatically to the binding

of the protein to lipid. Other studies provided evidence that all three NOS isoforms are

phosphorylated when immunoprecipitated from host cells (54-55). It has been shown that

kinase- and phosphatase-dependent events occurring in cells modified NOSs activity (56-58).

Recently, it has been reported that regulation of eNOS activity involves coordinated signaling

through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases (59).

To investigate if mtNOS was phosphorylated, this enzyme was purified in the presence of

phosphatase inhibitors, separated by 2D-electrophoresis followed by in-gel digestion with

trypsin. The eluted fragments were treated with either vehicle or phosphatase and the

peptide masses (obtained by MALDI-ToF) were compared (Table IV). The treatment with

phosphatase resulted in the appearance of a new fragment (m/z 1367.70) and the

disappearance of other two (1447.30 and 1588.52). The one of m/z 1367.70 matched

perfectly a C-terminal sequence of bNOS, whereas the other two matched the same

(1447.67) and a partial (1588.52) overlapping sequence containing one phosphorylated site.

This observation was supported by the difference in m/z between two sequences (1367.48

and 1447.30) which corresponded to that (experimental, 79.82 units; theoretical, 80 units)

produced by a loss of phosphate after phosphatase incubation. Although expected, the

fragment of m/z 1508.76, originated from the dephosphorylation of fragment 1588.52, was


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not obtained.

These results indicated that mtNOS isolated from rat liver was phosphorylated, and we

identified the fragment in which this phosphorylation was present. It is evident from our

results that Ser is the phosphorylation site for no Thr or Tyr is present in that fragment (Table

IV). Although our experiments did not indicate which Ser from the 3 possible ones present in

this fragment was phosphorylated, two indications pointed out to Ser-1412: first, two putative

Akt phoshorylation motifs (RXRXXS/T; underlined sequences see below) are present in

eNOS (Ser 635 and 1179 in bovine eNOS; Ser 633 and 1177 in human eNOS) and one motif

is present in nNOS (Ser 1412 in rat, 1415 in human, and 1179 in bovine nNOS), with no

obvious motifs found in iNOS (60); second, by aligning the 3 rat NOS isoforms using

CLUSTALW (see below), the phosphorylated Ser found in bovine (Ser-1179) or human

eNOS (Ser-1177) is homologous to rat bNOS Ser-1412 :

>TQQGKLSEEDAGVFISRLRDDNRYHEDIFGVTLRTYEVTNRLRSESIAFIEESKK---------DADEVFSS nNOS

>ATEGGMELDEAGDVIGVLRDQQRYHEDIFGLTLRTQEVTSRIRTQSFSLQERQLRGAVPWSFDPPGPEIPGS eNOS

>AAKLNLSEEQVEDYFFQLKSQKRYHEDIFGAVFSYGAKKGNTLEEPKGTRL--------------------- iNOS

The phosphorylation of this fragment (in addition to the acylation) might add to the faster

migration of mtNOS compared to nNOS, and provides support for the lower affinity to

monoclonal antibodies to the C-terminal region of nNOS and the crossreactivity to iNOS

earlier observed (1-5).

Conclusions

Our results unequivocally indicate that mitochondria are endowed with a NOS, extending

and confirming our (1-5, 16, 32) and others' (6-10) previous results. mtNOS was identified as

bNOSα with posttranslational modifications. This conclusion is supported by the amino acid

analysis, pI, MW, tryptic- and endoproteinase-peptidic maps, and peptide sequences. The

identification of mtNOS with bNOS has also been suggested through functional studies

performed on isolated cardiac mitochondria of bNOS wild-type and knockout mice (61).


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Given the important role that endogenous NO. has on regulating the O2 consumption and

ATP production of mitochondria (3-5) it is not surprising that the enzyme is localized close to

its target site, cytochrome oxidase. We have showed (3-4) that the production of NO. by

mitochondria modulated the O2 consumption of the organelle by competitive inhibition of

cytochrome oxidase; and, as a consequence, the O2 free radical production (5). In this

regard, the broader implications of our work can help to redefine the way we view regulation

of O2 consumption in vivo. Based on our initial findings, it can be proposed that mitochondrial

production of NO. helps average O2 utilization between cells at different distances from

capillaries. The basic concept is that NO. will slow O2 consumption by cells closest to blood

vessels, allowing O2 to penetrate to cells at the boundary of becoming hypoxic (65). In

addition, NO. might help dilate blood vessels and potentially increase O2 delivery to

borderline hypoxic cells (65).

Regarding the covalent modifications found in mtNOS, i.e., acylation and phosphorylation, it

could be speculated that they are important to target (as with other proteins, e.g., ref. 62)

and regulate mtNOS' activity. Interest in covalent modifications of proteins has been

strengthened by the observations that several viral transforming proteins have covalently

attached lipid. The observations that mutants of the transforming proteins p21 and p60src

that lack acylation are transformation-defective and no longer associated with the plasma

membrane (63-64) serve to underline the importance of the attachment of lipid to these

proteins for full expression of their transforming potential. In the case of mtNOS, and

considering that the acylation pattern of eNOS is required for localization to plasmalemmal

caveolae of endothelial cells (43), it could be speculated that acylation is a required step to

target the protein to mitochondria after its cytosolic synthesis, or its acylation (once inside

mitochondria) facilitates its membrane localization closer to cytochrome oxidase.

The other covalent modification, phosphorylation of Ser, may indicate a more direct pathway

to regulate mtNOS' activity. In fact, phosphorylation of eNOS Ser-1179 enhances the ability

of the enzyme to generate NO. (59-60). Although the mechanism by which this


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phosphorylation increases NO. production has not been elucidated, it has been proposed

that the addition of a negative charge at this site may permit the binding of calmodulin at

resting Ca2+ concentrations (60). If this is the case for mtNOS, then phosphorylation could

represent the main pathway by which this enzyme is regulated without the need for increases

in mitochondrial Ca2+, operating continuously under "resting" Ca2+ concentrations, regulating

the O2 consumption by the [NO. ]/[O2] ratios as it was proposed by our lab before (3-5).


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FOOTNOTES:

1.Abbreviations: NO. , nitric oxide; NOS, nitric-oxide synthase; b- or n-NOS, brain or

neuronal NOS; eNOS, endothelial NOS; iNOS, inducible NOS; mtNOS, mitochondrial NOS;

NMMA, NG-monomethyl-L-arginine; SMP, submitochondrial particles; OM, outer membrane;

IM, inner membrane; CS, contact sites; IMS, intermembrane space; M, matrix; MALDI-ToF;

matrix-assisted laser desorption ionization with time of flight; Q-ToF; quadrupole mass

spectrometry with time of flight; PS, primer set.

2. Although carbamoylphosphate synthetase I is present in the mitochondrial matrix (35),

contamination with this protein is not unexpected given its high concentration (estimated to

represent between 13 to 17% of total mitochondrial protein; 0.4-0.5 mM (36) or 1-1.5 mM

(37)). Assuming that contamination with this enzyme resulted from its entrapment in

membrane vesicles during the sonication procedure, the latter procedure was performed in

water, followed by washes with 0.15 M KCl. The resulting preparation resulted in

undetectable activity of carbamoylphosphate synthetase, without affecting the MS profile of

bNOS.

ACKNOWLEDGEMENTS

We thank Drs. Kent Froberg (Dept. Anatomy), Janet Fitzakerley (Dept. Pharmacology), and

Matthew Ruona with their assistance with the microscopy studies. We appreciate the support

of the staff and the use of imaging software at the Visualization and Digital Imaging

Laboratory, University of Minnesota.


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FIGURE LEGENDS

FIGURE 1 Colocalization Of mtNOS And Cytochrome Oxidase In Liver Slice

Formalin-fixed rat liver slices were probed with fluorescently tagged monoclonal antibodies to

nNOS (panel A) and cytochrome oxidase (panel B). Nuclei were stained with DAPI (panel

C). The superimposition of images from panels A-C was performed with Adobe Photoshop

(panel D). Other experimental details were described under Materials and Methods.

FIGURE 2 Primary Sequence Of Rat mtNOS

Underlined fragments were obtained by MALDI-ToF analysis of trypsin-digested mtNOS;

double-underlined fragments correspond to MALDI-ToF analysis of endoproteinase-digested

mtNOS; outline fragments correspond to those sequenced by Q-ToF; bold sequences

represent sequenced PCR products translated into protein sequences; boxed fragment

indicates the phosphorylated sequence.

FIGURE 3 Gel Electrophoresis Of PCR Products From Rat Liver mRNA or cDNA From

Various Rat Tissues

PANEL A: RT-PCR was performed on rat liver mRNA using PS1 followed by nested PCR

using PS4 (lane 4) or PS2 followed by nested PCR using PS5 (lane 5). To insure that the

resulting PCR products were not amplified from DNA contamination, two "mock" RT-PCR

reactions were performed without primers in the RT-PCR reaction, followed by nested PCR

using PS4 (lane 2) or PS5 (lane 3), respectively. In other set of experiments, PCR was

performed on liver cDNA from MTC panel using PS3 followed by nested PCR using PS6

(lane 6) or using PS3 followed by PS7 (lane 7). Lane 1, corresponds to 50 bp DNA ladder.

Experimental conditions were described in detail under Materials and Methods. PANEL B: The

PCR products were obtained with PS1 on cDNA from various tissues (from MTC panel),

followed by nested PCR using PS4. Lane 1 corresponds to 1 kb DNA ladder, and lanes 2-9

correspond to products resulted from different tissues: 2) liver, 3) brain, 4) heart, 5) muscle,


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6) kidney, 7) lung, 8) testis, and 9) spleen. PANEL C: PCR products obtained using PS3 on

cDNA from the MTC panel followed by nested PCR using PS7 (lanes 2-9) or PS6 (lanes 11-

18). Lanes 1 and 10 correspond to the 50 bp DNA ladder; liver (2 and 11), brain (3 and 12),

heart (4 and 13), muscle (5 and 14), kidney (6 and 15), lung (7 and 16), testis (8 and 17),

spleen (9 and 18).

FIGURE 4 Mass spectra of mtNOS' acylation products

Samples of purified mtNOS were precipitated with acetone, washed with organic solvents to

remove any unspecifically bound lipids, and finally subjected to alkaline methanolysis to

release the fatty acid methyl esters. The derivatives were extracted with hexane, and the

fatty acids were identified by mass spectrometry upon comparison with standards. Generally,

the molecular ion peak of methyl ester of a straight-chain aliphatic is weak, however, the

most characteristic peak is due to the McLafferty rearrangement (m/z= 74). Other

assignments were based on typical cleavage of aliphatic esters and those obtained with a

standard (methyl ester of myristic acid). By comparing the obtained ions to those

theoretically expected, a spectrum match index of 0.9761 was obtained (analysis performed

Mass Spec Calculator Pro™ software, ChemSW).


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Table I: Amino acid analysis of purified mtNOS

Amino acid* Experimental

(%)

Theoretical

(%)

Asx 10.2 10.6

His 5.0 3.0

Ala 8.3 6.5

Val 7.3 7.4

Leu 6.4 9.2

Glx 16.9 12.4

Pro 5.5 6.0

Met 0.9 2.2

Phe 3.2 4.4

Ser 7.2 7.4

Thr 3.3 5.8

Arg 5.9 5.9

Ileu 5.1 5.0

Lys 7.6 6.7

Gly 7.1 7.5

* The amino acids Cys, Tyr, and Trp were not evaluated.


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Table II: Primers used for the PCR reactions

Primer

set

Upstream primer Downstream Primer

Nucleotide

number

Nucleotide sequence Nucleotide

number

Nucleotide sequence

1 356-375 AGA ACA CGT TTG GGG TTC AG 1394-1413 GAA GAG CTG GTC CTT TGT GC

2 3787-3807 GTG GAG GTG CTG GAG GAG TTC 4378-4398 CCC ACA GAC ATA AAT GTG GCC

3 1463-1482 TTT GGC TCC AAG GCC CAC ATG 4378-4398 CCC ACA GAC ATA AAT GTG GCC

4 528-547 TGG AGA CAT CAT TCT CGC AG 1297-1316 TCC AGTGTG CTC TTC AGG TG

5 3751-3770 TAT GAG GAG TGG AAG TGG GG 4247-4266 TCT GAA GAC GCC CTT GTT CT

6 1992-2011 CCC CAA GTT CGA CTG GTT TA 2169-2198 TCC AAA TCC ATC TTC TTG GC

7 2808-2827 ATT CGG CTG TGC TTT AAT GG 2870-2889 GCT GTT GAA TCG GAC CTT GT

8 1522-1541 AGC ACC TAC CAG CTC AAG GA 2169-2198 TCC AAA TCC ATC TTC TTG GC

9 1522-1541 AGC ACC TAC CAG CTC AAG GA 2227-2246 ATC TCC ACC AGT GCT TGG TC

Primers based on Q-ToF sequences were underlined, whereas those based on rat brain cNOS

sequence (Gen EMBL X59949) were indicated in plain text.


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Table III: Expected sizes of PCR products from alternatively spliced bNOS

Primer set NOSα NOS2 NOSµ NOSγ NOSβ

1 1058 1058 1058 ------ ------

2 864 864 864 864 864

3 2955 2640 3057 2955 2955

4 788 788 788 ------ ------

5 515 515 515 515 515

6 206 ------ 206 206 206

7 81 81 183 81 81

8 676 358 676 676 676

A fragment from amino acid 504 to 608 is missing in NOS2, and NOSµ has an insertion at

amino acid 839 with the following sequence KYPEPLRFFPRKGPSLSHVDSEAHSLVAARDSQHR.


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Table IV: MALDI-ToF analyses of phosphorylated peptides

Fragment Expected

m/z*

Experimental

m/z

After phosphatase treatment

SESIAFIEESKK 1367.70 1367.48

LRSESIAFIEESK 1508.76 n.f.

Before phosphatase treatment

SESIAFIEESKK 1447.67 1447.30

LRSESIAFIEESK 1588.76 1588.52

* Expected m/z was calculated by adding (before phosphatase) 80 units to the in-silico trypticdigest of bNOS (after phosphatase). n.f.: Not found.


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A B

C D

Elfering et al.; Figure 1


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1 11 21 31 41 51 61 71

MEENTFGVQQ IQPNVISVRL FKRKVGGLGF LVKERVSKPP VIISDLIRGG AAEQSGLIQA GDIILAVNDR PLVDLSYDSA

81 91 101 111 121 131 141 151

LEVLRGIASE THVVLILRGP EGFTTHLETT FTGDGTPKTI RVTQPLGPPT KAVDLSHQPS ASKDQSLAVD RVTGLGNGPQ161 171 181 191 201 211 221 231

HAQGHGQGAG SVSQANGVAI DPTMKSTKAN LQDIGEHDEL LKEIEPVLSI LNSGSKATNR GGPAKAEMKD TGIQVDRDLD241 251 261 271 281 291 301 311

GKSHKAPPLG GDNDRVFNDL WGKDNVPVIL NNPYSEKEQS PTSGKQSPTK NGSPSRCPRF LKVKNWETDV VLTDTLHLKS

321 331 341 351 361 371 381 391

TLETGCTE H I CMGSIMLPSQ HTRKPE DVRT KDQLFPLAKE FLDQYYSSIK RFGSKAHMDR L EEVN KEIES TSTYQLK DTE

401 411 421 431 441 451 461 471

LIYGAKHAWR NASRCVGRIQ WSKLQVFDAR DCTTAHGMFN YICNHVKYAT NKGNLRSAIT IFPQRTDGKH DFRVWNSQLI

481 491 501 511 521 531 541 551

RYAGYKQPDG STLGDPANVQ FTEICIQQGW KAPRGRFDVL PLLLQANGND PELFQIPPEL VLEVPIRHPK FDWFKDLGLK

561 571 581 591 601 611 621 631

WYGLPAVSNM LLEIGGLEFS ACPFSGWYMG TEIGVRDYCD NSRYNILEEV AKKMDLDMRK TSSLWKDQAL VEINIAVLYS

641 651 661 671 681 691 701 711

FQSDKVTIVD HHSATESFIK HMENEYRCRG GCPADWVWIV PPMSGSITPV FHQEMLNYRL TPSFEYQPDP WNTHVWKGTN

721 731 741 751 761 771 781 791

GTPTKRRAIG FKKLAEAVKF SAKLMGQAMA KRVKATILYA TETGKSQAYA KTLCEIFKHA FDAKAMSMEE YDIVHLEHEA

801 811 821 831 841 851 861 871

LVLVVTSTFG NGDPPENGEK FGCALMEMRH PNSVQEERKS YKVRFNSVSS YSDSRKSSGD GPDLRDNFES TGPLANVRFS

881 891 901 911 921 931 941 951

VFGLGSRAYP HFCAFGHAVD TLLEELGGER ILKMREGDEL CGQEEAF RTW AKKVFK AACD VFCVGDDVNI EKPNNSLISN

961 971 981 991 1001 1011 1021 1031

DRSWKRNKFR LTYVAEAPDL TQGLSNVHKK RVSAARLLSR QNLQSPKFSR STIFVRLHTN GNQELQYQPG DHLGVFPGNH

1041 1051 1061 1071 1081 1091 1101 1111

EDLVNALIER LEDAPPANHV VKVEMLEERN TALGVISNWK DESRLPPCTI FQAFKYYLDI TTPPTPLQLQ QFASLATNEK

1121 1131 1141 1151 1161 1171 1181 1191

EKQRLLVLSK GLQE YEEWKW GKNPTMVE VL EEFPSIQMPA TLLLTQLSLL QPRYYSISSS PDMYPDEVHL TVAIVSYHTR

1201 1211 1221 1231 1241 1251 1261 1271

DGEGPVHHGV CSSWLNRIQA DDVVPCFVRG APSFHLPRNP QVPCILVGPG TGIAPFRSFW QQRQFDIQHK GMNPCPMVLV1281 1291 1301 1311 1321 1331 1341 1351

FGC RQSKIDH IYREETLQAK NKGVFRELYT AYSREPDRPK KYVQDVLQEQ LAESVYRAL K EQGGHIYVCG DVTMAADVLK

1361 1371 1381 1391 1401 1411 1421

A IQRIMTQQG KLSEEDAGVF ISRLRDDNRY HEDIFGVTLR TYEVTNRLRS ESIAFIEESK KDADEVFSS

Elfering et al. Figure 2

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Elfering et al.; FIGURE 3

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A

788 bp515 bp

206 bp81 bp

1 2 3 4 5 6 7 8 9 10 11 1213 14 1516 17 18

183 bp81 bp

206 bp

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788 bp

B

C


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COCH3O

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30O

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Sarah Liv Elfering, Theresa Maria Sarkela and Cecilia GiuliviBiochemistry of mitochondrial nitric-oxide synthase

published online August 1, 2002J. Biol. Chem.

10.1074/jbc.M205256200Access the most updated version of this article at doi:

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Additions and Corrections

Vol. 277 (2002) 47564–47571

Function and solution structure of huwentoxin-IV,a potent neuronal tetrodotoxin (TTX)-sensitive sodiumchannel antagonist from Chinese bird spider Seleno-cosmia huwena.

Kuan Peng, Qin Shu, Zhonghua Liu, and Songping Liang

Page 47567, Fig. 4 legend: The following sentence is missingfrom the figure legend.

“An asterisk indicates that the C-terminal carboxyl group isamidated.”

The figure with the corrected legend is shown below.

Vol. 277 (2002) 38079–38086

Biochemistry of mitochondrial nitric-oxide synthase.

Sarah Liv Elfering, Theresa Maria Sarkela, and CeciliaGiulivi

Page 38081, Fig. 1: Fig. 1 should have printed in color. Thecorrect figure is shown below.

FIG. 4. Comparison of amino acid sequence of HWTX-IV withHWTX-I and previously known conotoxins (CTX) blocking atsite I (O � 4-trans-L-hydroxyproline. The proposed key residuesimportant for their functions are displayed in the frame boxes. Cono-toxin GS (38, 39), �-conotoxin PIIIA (6), �-conotoxin GIIIA (7, 35, 36),and �-conotoxin GIIIB (8, 37) block at site I. HWTX-I was suggested tobe an N-type calcium channel inhibitor (33). Like HWTX-IV and cono-toxin GS, it adopts a 1–4, 2–5, 3–6 disulfide pattern and cystine knotmotif (32). An asterisk indicates that the C-terminal carboxyl group isamidated.

FIG. 1. Colocalization of mtNOS and cytochrome oxidase inliver slice. Formalin-fixed rat liver slices were probed with fluores-cently tagged monoclonal antibodies to nNOS (A) and cytochrome oxi-dase (B). Nuclei were stained with 4,6-diamidino-2-phenylindole (C).The superimposition of images from A–C was performed with AdobePhotoshop (D). Other experimental details are described under “Exper-imental Procedures.”

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 7, Issue of February 14, p. 5489, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriateplaces where the article to be corrected originally appeared. Authors are urged to introduce thesecorrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.

5489

1 submitted may 24, 2002 revised june25, 2002 re-revised july

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