Amphibian peptides that inhibit neuronal nitric oxide synthase : The isolation of lesueurin from the...

Amphibian peptides that inhibit neuronal nitric oxide synthaseThe isolation of lesueurin from the skin secretion of the Australian StonyCreek Frog Litoria lesueuri

Jason Doyle1, Lyndon E. Llewellyn1, Craig S. Brinkworth2, John H. Bowie2, Kate L. Wegener2, Tomas Rozek2,Paul A. Wabnitz2, John C. Wallace3 and Michael J. Tyler4

1Australian Institute of Marine Science, Townsville MC, Queensland, Australia; 2Departments of Chemistry, 3Molecular Biosciences

and 4Environmental Biology, The University of Adelaide, South Australia

Two neuropeptides have been isolated and identi®ed fromthe secretions of the skin glands of the Stony Creek FrogLitoria lesueuri. The ®rst of these, the known neuropeptidecaerulein 1.1, is a common constituent of anuran skinsecretions, and has the sequence pEQY(SO3)TGWMDF-NH2. This neuropeptide is smooth muscle active, an anal-gaesic more potent than morphine and is also thought to bea hormone. The second neuropeptide, a new peptide, hasbeen named lesueurin and has the primary structureGLLDILKKVGKVA-NH2. Lesueurin shows no signi®-cant antibiotic or anticancer activity, but inhibits the for-mation of the ubiquitious chemical messenger nitric oxidefrom neuronal nitric oxide synthase (nNOS) at IC50

(16.2 lM), and is the ®rst amphibian peptide reported toshow inhibition of nNOS. As a consequence of this activity,we have tested other peptides previously isolated fromAustralian amphibians for nNOS inhibition. There are threegroups of peptides that inhibit nNOS (IC50 at lM concen-trations): these are (a) the citropin/aurein type peptides(of which lesueurin is a member), e.g. citropin 1.1

(GLFDVIKKVASVIGGL-NH2) (8.2 lM); (b) the frenatintype peptides, e.g. frenatin 3 (GLMSVLGHAVGNVLGGLFKPK-OH) (6.8 lM); and (c) the caerin 1 peptides, e.g.caerin 1.8 (GLFGVLGSIAKHLLPHVVPVIAEKL-NH2)(1.7 lM). From Lineweaver±Burk plots, the mechanism ofinhibition is revealed as noncompetitive with respect to thenNOS substrate arginine. When the nNOS inhibition testswith the three peptides outlined abovewere carried out in thepresence of increasing concentrations of Ca2+calmodulin,the inhibition dropped by � 50% in each case. In addition,these peptides also inhibit the activity of calcineurin, anotherenzyme that requires the presence of the regulatory proteinCa2+calmodulin. It is proposed that the amphibianpeptidesinhibit nNOS by interacting with Ca2+calmodulin, and as aconsequence, blocks the attachment of this protein to thecalmodulin domain of nNOS.

Keywords: amphibians; Litoria lesueuri; neuropeptides;nNOS inhibition; Ca2+calmodulin interaction.

Amphibians have rich chemical arsenals that form anintegral part of their defence system, and also assist with theregulation of dermal physiological action. In response to avariety of stimuli, host defence compounds are secretedfrom specialized glands onto the dorsal surface and into thegut of the amphibian [1±4]. A number of different types ofbio-active peptides have been identi®ed from the glandularskin secretions of Australian anurans of the Litoria genus,including (a) neuropeptides of the caerulein family [5±8],and (b) wide-spectrum antibiotics, e.g. the caerin peptides

from green tree frogs of the genusLitoria [6±8], the citropinsfrom the tree frogLitoria citropa [9,10], and the aureins fromthe bell frogs Litoria aurea and Litoria raniformis [11].Amongst the most active of these are neuropeptide caeru-lein 1.1, and the antibiotics caerin 1.1, citropin 1.1 andaurein 1.2: caerulein 1.1 pEQGY(SO3)TGWMDF-NH2;caerin 1.1 GLLSVLGSVAKHVLPHVVPVIAEHL-NH2;citropin 1.1 GLFDVIKKVASVIGGL-NH2; aurein 1.2GLFDIIKKIAESF-NH2.

Aurein 1.2 contains only 13 amino-acid residues, and isthe smallest peptide from an anuran reported to havesigni®cant antibiotic activity. The aurein peptides have alsobeen shown to exhibit anticancer activity in tests carried outby the National Cancer Institute (NCI, Washington DC,USA) [12].

The solution structures of the antibiotic (and anticanceractive as appropriate) peptides shown above have beeninvestigated by NMR spectroscopy. In tri¯uoroethanol/water mixtures, caerin 1.1 adopts two well-de®ned helices(Leu2 to Lys11 and from Val17 to His24) separated by ahinge region of less-de®ned helicity and greater ¯exibility,with hydrophilic and hydrophobic residues occupying well-de®ned zones [13]. The central hinge region is necessary foroptimal antibiotic activity [13]. Similar NMR studies of

Correspondence to J. H. Bowie, Department of Chemistry,

The University of Adelaide, South Australia, 5005.

Fax: + 61 08 83034358, Tel.: + 61 08 83035767,

E-mail: [email protected]

Abbreviations: FAD, ¯avin adenine dinucleotide, oxidized form;

FMN, ¯avin mononucleotide; IC50, concentration (of peptide) which

causes 50% inhibition; MS/MS, mass spectroscopy/mass spectro-

scopy; NADPH, nicotinamide adenine nucleotide phosphate, reduced

form; NCI, National Cancer Institute (Washington); nNOS, neuronal

nitric oxide synthase.

(Received 18 June 2001, revised 8 October 2001, accepted 23 October

2001)

Eur. J. Biochem. 269, 100±109 (2002) Ó FEBS 2002

citropin 1.1 [9] and aurein 1.2 [11] show that these peptidesadopt conventional amphipathic ahelical structures, afeature commonly found in membrane-active agents[1±4,8]. Interaction occurs at the membrane surface withthe charged, and normally basic peptide adopting anahelical conformation and attaching itself to charged, andnormally anionic sites on the lipid bilayer. This ultimatelycauses disruption of normal membrane function leading tolysis of the bacterial or cancer cell [14±16].

Many Australian anurans that we have studied conformto the model outlined above in that they have a variety ofhost defence peptides in skin glands including a neuropep-tide that acts on smooth muscle, and at least one powerfulwide-spectrum antibiotic peptide such as those describedabove [8]. However there are some species of anuran thatdivert markedly from this scenario. For example, the RedTree Frog Litoria rubella [17±19], and the related speciesLitoria electrica [20] excrete neither antimicrobial peptidesnor neuropeptides such as caerulein. Instead, they releaselarge quantities of small peptides, called tryptophyllins [21],onto their skin, which are thought to be neurotransmittersor neuromodulators [22], at least partly because they showstructural similarity to the human brain endomorphins (e.g.YPWG-NH2) [23]. The sequences of two tryptophyllins areas follows: tryptophyllin L1, FPWL-NH2; tryptophyllinL2, pEFPWL-NH2.

Even more unusual are the marsh frogs of the Limno-dynastes genus. These produce only minute amounts ofanionic skin peptides, none of which are post-translationallymodi®ed, or show neuropeptide or antimicrobial activity[24,25]. A particular example, dynastin 1 (from Limnodyn-astes interioris) [24] has the sequence GLLSGLGL-OH.

In this paper, we describe the isolation, sequence deter-mination, and activities of two bioactive peptides from theskin glands of the stony creek frog Litoria lesueuri.We havenot studied a member of the L. lesueuri complex of frogspreviously, and we have now found that it does not produceantimicrobial peptides, unlike the majority of the othermembers of the genus Litoria. Instead, it produces aneuropeptide that inhibits the formation of nitric oxide byneuronal nitric oxide synthase (nNOS). We next examinedother peptides isolated earlier from anurans of the genusLitoria. This has led to the discovery of three types ofamphibian peptides that inhibit nNOS.

M A T E R I A L S A N D M E T H O D S

Preparation of skin secretions

L. lesueuri was held by the back legs, the skin moistenedwith deionized water, and the granular dorsal glandssituated on the back were stimulated by means of abipolar electrode of 21G platinum attached to a PalmerStudent Model electrical stimulator. The electrode wasrubbed gently in a circular manner on the particular gland(under study) of the animal, using 10 V and a pulseduration of 3 ms [26]. The resulting secretion was washedfrom the frog with deionized water (50 mL), the mixturediluted with an equal volume of methanol, centrifuged,®ltered through a Millex HV ®lter unit (0.45 lm), andlyophilized. This work conforms with the Code of Practicefor the Care and Use of Animals for Scienti®c Purposes(1990) and the Prevention of Cruelty to Animals Act

(1985), and was approved by the University of AdelaideAnimal Ethics Committee.

HPLC separation of peptide material

HPLC separation of the skin secretion was achieved using aVYDAC C18 HPLC column (5lm, 300 AÊ , 4.6 ´ 250 mm)(Separations Group, Hesperia, CA, USA) equilibrated with10% acetonitrile/aqueous 0.1% tri¯uoroacetic acid. Thelyophilized mixture (0.5 mg) was dissolved in deionizedwater (20 lL) and injected into the column. The elutionpro®le was generated using a linear gradient produced by anICI DP 800 Data Station controlling two LC1100 HPLCpumps, increasing from 10 to 75%acetonitrile over a periodof 60 min at a ¯ow rate of 1 mLámin)1. The eluant wasmonitored by ultraviolet absorbance at 214 nmusing an ICILC-1200 variable wavelength detector (ICI Australia,Melbourne, Australia). The resultant HPLC trace showstwo major peaks (Fig. 1). The two fractions were collected,concentrated and dried in vacuo. The ®rst major fraction(Fig. 1B) (50 lg) contained the known neuropeptide caeru-lein 1.1, identi®ed by HPLC and mass spectrometry [27].The second major fraction (Fig. 1C) contained 10 lg of anew peptide, called lesueurin.

Sequence determination of lesueurin

Electrospray mass spectrometry. Electrospray mass spec-tra were determined using a Finnigan LCQ ion trap massspectrometer. Puri®ed fractions from the HPLC separationwere dissolved in methanol/water (1 : 1, v/v) and infusedinto the electrospray source at 8 lLámin)1. Electrosprayconditions were as follows: needle potential 4.5 kV, tubelens 60 V, heated capillary 200 °Cand 30 V, sheath gas ¯ow30 p.s.i.Mass spectra were acquiredwith the automatic gaincontrol on, a maximum time of 400 ms, and averaging overthree microscans. Mass spectrometric sequencing wascarried by the MS/MS method using B and Y + 2fragmentations [28].

Amino-acid sequencing. Automated Edman sequencing oflesueurin was performed by a standard procedure asdescribed previously [29] using an applied Biosystem 492

Fig. 1. HPLC separation of the glandular secretion of Litoria lesueuri.

B, caerulein 1.1; C, lesueurin. A, nonpeptide material.

Ó FEBS 2002 Amphibian peptides that inhibit nNOS (Eur. J. Biochem. 269) 101

procise sequencer equipped with a 900-A data analysismodule. The best results were obtained using a disc ofimmobilon ®lm treated with bioprene in ethanol, ontowhich the peptide was absorbed from aqueous acetonitrile(90%). The disc was pierced several times with a razor bladeto aid the ¯ow of solvent.

Preparation of synthetic lesueurin

Lesueurin was synthesized (by Mimotopes, Clayton, Vic-toria, Australia) using L-amino acids via the standardN-a-Fmoc method (full details including protecting groupsand deprotection have been reported recently [30]). Syn-thetic lesueurin was shown to be identical to the naturallesueurin by electrospray mass spectrometry, and HPLC.

Bioactivity assays

Antimicrobial testing. Synthetic lesueurin was tested forantibiotic activity by the Microbiology Department of theInstitute of Medical and Veterinary Science (Adelaide,Australia) by a standard method [31]. The method usedinvolved the measurement of inhibition zones (produced bythe applied peptide) on a thin agarose plate containing themicroorganisms under study. The microorganisms used inthis procedure were Bacillus cereus, Escherichia coli, Leuco-nostoc lactis, Listeria innocua, Micrococcus luteus, Pasteu-rella multocida, Staphylococcus aureus, Staphylococcusepidermidis and Streptococcus uberis. Lesueurin showed noactivity at MIC values below 100 lgámL)1 against any ofthese organisms.

Anticancer activity testing. Synthetic lesueurin showed noactivity below 10)4

M in the Ô60-human tumour line testingprogramÕ of the US NCI (Washington) [12].

Neuronal nitric oxide synthase inhibition. Inhibition ofnNOS was measured by monitoring the conversion of[3H]arginine to [3H]citrulline. In brief, this involved incuba-tionof ahomogenateof rat cerebella (whichhadendogenousarginine removed by ion exchange chromatography) in areaction buffer (33 mM Hepes, 0.65 mM EDTA, 0.8 mM

CaCl2,3.5 lgámL)1 calmodulin,670 lMb-NADPH,670 lMdithiothreitol, pH 7.4) containing 20 nM [3H]arginine (NENLife Sciences, Boston, MA, USA). The nNOS inhibitor,Nx-nitro-L-arginine (1 mM) was used to measure back-ground radioactivity. Reactions were terminated after10 min with 50 lL of 0.3 M EGTA. An aliquot (50 lL) ofthis quenched reaction mixture was transferred to 50 lL of500 mM Hepes (pH 5.5). AG50W-X8 (Na+ form) resin(100 lL) was added to separate [3H]arginine from [3H]cit-rulline. After repeated vortexing, this slurry was centrifugedat 1200 g for 10 min, and 70 lLof supernatentwas removedand the [3H]citrulline measured by scintillation counting.Peptides selected for further examination to determine themechanism of inhibition were assayed in the same reactionbuffer as used for initial screening except that it contained30 nM [3H]arginine supplemented with 0.3±13.3 mM argi-nine. Peptide concentrations used are given in the legend toFig. 4.

Data analysis for nNOS studies. Peptide inhibition curveswere ®tted using the curve-®tting routine of SIGMAPLOT

(SPSS, Chicago, IL, USA) with the variation of the Hillequation: fmol [3H]citrulline production � 1/(1 + [inhibi-tor]/IC50n), where IC50 is the concentration at which thepeptide causes 50% inhibition and n is the slope of the curveand can be considered as a pseudo Hill coef®cient [32].Lineweaver±Burk plots [33] were generated using SIGMA-

PLOT (SPSS, Chicago, IL, USA).

Calcineurin assay. This assay was performed following themanufacturer's protocol [34] with minor modi®cations.Calcineurin was diluted to 0.036 UálL)1 in enzyme dilutionbuffer (50 mM Tris, 0.5 mgámL)1 BSA, pH 7.4). Peptides(5 lL) were assayed in duplicate in a reaction mixturecontaining 16 mM p-nitrophenylphosphate, 0.4 mgámL)1

BSA, 0.8 mM NiCl2, 4 lgámL)1 calmodulin in Tris buffer(40 mM, pH 7.4). The reaction was started with the additionof the enzyme (5 lL; 0.1 U). All samples were assayed at30 °Cwith positive controls containing water in the place ofthe test sample and negative controls containing no enzyme.Absorbance (A) readings at 405 nmwere taken after 30 minwith readings averaged, adjusted to the change in A perminute and corrected for background A (negative control).Percent of control was then calculated as (A405 test sample/A405 positive control) ´ 100.

R E S U L T S

L. lesueuri, usually called either Lesueur's Frog or theStony Creek Frog, has varied dorsal colouration rangingfrom yellow to brown, with a black head stripe from thesnout to the tympanum [35]. The animal ranges from 37to 63 mm in length, and is often found in the vicinity ofrocky streams in coastal regions from north of Queens-land to eastern Victoria. It is reported that there are twodistinct populations of this frog, one con®ned to north-eastern Queensland, the other in New South Wales andVictoria. Whether these are two different subspecies ofL. lesueuri or two different species has not yet beendetermined [36].

A single specimen of L. lesueuri, collected at Atherton,Queensland, was used in this study. The electrical stimula-tion method [26] was used to elicit secretion from thegranular skin glands. The animal was not harmed in thisstudy. Less than 0.5 mg of material was obtained followingwork up of the secretion, and this contained less than 100 lgof peptide material. This is an unusually small amount ofpeptide material. It is normal for a species of the genusLitoria to contain 5 mg of peptide material in the glandularsecretion; some frogs, such asLitoria splendida, secrete morethan 100 mg of peptide material [8]. HPLC separation(Fig. 1) revealed two major peptide components. The ®rst,and major component was identi®ed as caerulein 1.1 fromits electrospray mass spectrum and HPLC behaviour [27].This potent smooth muscle agent, analgaesic and hormoneis often a major peptide component of species of the genusLitoria [8].

The minor component is a 13-residue peptide that wehave called lesueurin. Only 10 lg of this material wasavailable for study. The MS/MS of the MH+ ion oflesueurin is shown in Fig. 2. As this mass spectrum does notdifferentiate between isomeric Leu or Ile or isobaric Lys andAsn, we have con®rmed the identity of these four residuesusing the automated Edman procedure [29]. The automated

102 J. H. Bowie et al. (Eur. J. Biochem. 269) Ó FEBS 2002

Edman procedure identi®es all but the last amino acid in thelesueurin sequence. This residue, C-terminal Ala-NH2, isclearly identi®ed by mass spectrometric fragmentation data(Fig. 2). A synthetic sample of lesueurin was prepared, andshown to be identical with natural lesueurin using HPLCand mass spectrometry. The sequence of lesueurin isGLLDILKKVGKVA-NH2.

Lesueurin has no antibiotic activity (at minimuminhibitory concentration values below 100 lgámL)1)against the nine bacteria we use in our test regime. Also,lesueurin exhibited no cytotoxity at concentrations lessthan 10)4

M against any of the 60 human tumour lines inthe NCI test regime. Lesueurin was, however, shown toinhibit nNOS with an IC50 of 16.2 lM (Table 1). This

Fig. 2. Electrospray mass spectrum (MS/MS)

of the MH+ ion of lesueurin.Masses shown in

this spectrum are nominal masses. Data from

B fragmentations give sequence information

from the C-terminal end of lesueurin (see

schematic arrows above the spectrum). Data

from Y + 2 fragmentations give sequence

information from the N-terminal end of the

peptide (see schematic arrows below the

spectrum). For a review of fragmentations of

MH+ ions of peptides see [28]. This method

does not distinguish between isomeric Leu and

Ile (nominal mass 113) and isobaric Lys and

Gln (nominal mass 128). These residues have

been di�erentiated using the automated

Edman procedure [29]. The correct residues

are shown in this ®gure.

Table 1. nNOS Inhibition by amphibian peptides. Amino-acid sequences, source species and naming convention used where a peptide has been

named previously. Peptides with minimal or no e�ect are highlighted with respective concentration used listed in footnotes below. All peptides in

Group 1 are amphipathic ahelices in solution [10,11]. Caerin 1 peptides in solution have ahelices from residues 1±11 and 18±25, with a ¯exible hinge

region including residues 12±17 [13].

Name Source Sequence

IC50

(lM)

Hill

slope

Net

charge

Inactive or weakly active peptides

Rubellidin 3.1a L. rubella [17] IEFFT-NH2 NA NA 0

Trytophyllin L3.1b L. rubella [17] FPWP-NH2 NA NA +1

Electrin 2.1c L. electrica [20] NEEEKVKWEFPDVP-NH2 NA NA )2Caeridin 3d L. caerulea [8] GLFDAIGNLLGGLGL-NH2 NA NA 0

Maculatin 1.3e L. eucnemis f GLLGLLGSVVSHVLPAITQHL-NH2 NA NA +1

Inhibitor Group 1

Lesueurin L. lesueuri g GLLDILKKVGKVA-NH2 16.2 1.5 +3

Aurein 1.1 L. aurea [11] GLFDII KK I AES I-NH2 33.9 2.0 +1

Citropin 1.1 L. citropa [10] GLFDVIKKVASVIGGL-NH2 8.2 1.6 +2

Aurein 2.2 L. aurea [11] GLFDIVKKVVGALGSL-NH2 4.3 2.5 +2

Aurein 2.3 L. aurea [11] GLFDIVKKVVGIAGSL-NH2 1.8 1.7 +2

Aurein 2.4 L. aurea [11] GLFDIVKKVVGTLAGL-NH2 2.1 3.1 +2

Inhibitor Group 2

Not named L. dahlii f GLLGSIGNAIGAFIANKLKP-OH 3.2 2.2 +3

Frenatin 3 L. infrafrenata [8] GLMSVLGHAVGNVLGGLFKPKS-OH 6.8 1.4 +3

Splendipherin L. splendida [67] GLVSSIGKALGGLLADVVKSKGQPA-OH 8.5 1.3 +3

Inhibitor Group 3

Caerin 1.1 L. splendida [6] GLLGVLGSIAKHVLPHVVPVIAEHL-NH2 36.6 1.4 +1

Caerin 1.10 L. chloris [8] GLLSVLGSVAKHVLPHVVPVIAEKL-NH2 41.0 0.6 +2

Caerin 1.6 L. chloris [8] GLFSVLGAVAKHVLPHVVPVIAEKL-NH2 8.5 1.7 +2

Caerin 1.8 L. chloris [8] GLFKVLGSVAKHLLPHVVPVIAEKL-NH2 1.7 3.7 +3

Caerin 1.9 L. chloris [8] GLFGVLGSI AKHVLPHVVPVIAEKL-NH2 6.2 2.2 +2

a Inactive at 13.3 lgámL)1. b Inactive at 33.3 lgámL)1. c Inactive at 66.7 lgámL)1. d Inactive at 133.3 lgámL)1. e Caused 46.4% inhibition at

31.4 lM. Full IC50 determination was not possible because of solubility. f Brinkworth, C., Bowie, J.H., Wallace, J.C. & Tyler, M.J.

unpublished work. g Present paper.


discovery of previously unreported pharmacologicalactivity of an amphibian peptide prompted us to test forsimilar bioactivity of amphibian peptides that we haveinvestigated previously. Table 1 lists the sequences of thenatural peptides tested, both active and inactive towardsnNOS. The solubility of these peptides and the maximumtolerable amount of the peptide solvent in the nNOSassay affected the maximum concentration tested forseveral of the peptides. Those peptides found to inhibitnNOS from the primary screen were then titrated todetermine their IC50 parameters. These results are given inTable 1 together with the slope of the inhibition curvesand various physical properties. Three examples of theinhibition curves, including that generated by lesueurin,are shown in Fig. 3.

Four model peptides were selected from the inhibitorgroups (Table 1) to further examine the mode of action bywhich they were inhibiting nNOS. Lesueurin and citro-pin 1.1 (both from inhibitor group 1), frenatin 3 (inhibitor

group 2) and caerin 1.9 (inhibitor group 3) all producedLineweaver±Burk plots consistent with a noncompetitivemode of inhibition (Fig. 4) with respect to the nNOSsubstrate arginine. Thus inhibition is not mediated by directaction upon the arginine-catalysing site. Rat cerebellarnNOS may retain endogenous calmodulin rendering itunsuitable for a Michaelis±Menten study of enzyme kinet-ics, as was carried out with arginine.

An experimental procedure was used to gauge thepotential for selected peptides to inhibit nNOS by displacingCa2+calmodulin from the calmodulin binding domain ofnNOS. In this procedure, the nNOS inhibition experimentswere carried out with citropin 1.1, frenatin 3, and caerin 1.9with added Ca2+calmodulin to determine the in¯uence ofthe calmodulin on the inhibition of nNOS. These experi-ments measured nNOS activity in the presence of142.9 lgámL)1 of the active peptide (about 10-fold greaterthan the IC50 value of each peptide), with the Ca2+

calmodulin concentration in the assay buffer increased 100-

Fig. 3. Inhibition of nNOS exempli®ed by (A)

lesueurin (circles) and frenatin 3 (squares), and

(B) citropin 1.1 (inverse triangles) and caerin

1.9 (triangles). These peptides represent each

of the groups of peptide inhibitors listed in

Table 1. Curves are drawn to the Hill equa-

tion, and the values for the IC50 and slope of

the curve are given in Table 1.

Fig. 4. Lineweaver±Burk plots derived from

changes in enzyme velocities in the presence of

increasing amounts of the peptides citropin 1.1

(A), lesueurin (B), caerin 1.9 (C) and frenatin 3

(D). Enzyme kinetics data obtained in the

absence of peptide are depicted by ®lled cir-

cles, and represent the uninhibited reaction.

Open circles show the e�ect of the citropin 1.1,

lesueurin, caerin 1.9 and frenatin 3 at 4.1, 24.7,

5.1 and 6.1 lM, respectively. Filled inverse

triangles show the e�ect when the concentra-

tion of citropin 1.1, lesueurin, caerin 1.9 and

frenatin 3 are increased to 8.2, 49.4, 10.3 and

12.2 lM, respectively.


fold to 350 lgámL)1. For citropin 1.1, the inhibitiondecreased from 94 to 58% in the presence of Ca2+calmod-ulin. The results for frenatin 3 and caerin 1.9 are 83±52%,and 84±53%, respectively.

Selected peptides were tested for their ability to inhibitcalcineurin, another calmodulin dependent enzyme.Lesueurin at 74 lM (4.6-fold greater than the IC50 againstnNOS) inhibited calcineurin by 33%. Citropin 1.1 reducedcalcineurin activity by 34% at 31 lM (3.8-fold higher thanthe IC50 against nNOS) but at double this concentration (i.e.7.6-fold greater than its IC50 against nNOS), calcineurin wasinhibited by 96%. Frenatin 3 inhibitied calcineurin by 38%at 46 lM, a concentration that is 6.8-fold greater than theIC50 against nNOS. Finally, caerin 1.9 inhibited calcineurinby 48.1% at 19.3 lM (4.8-fold more concentrated than itsIC50 against nNOS).

D I S C U S S I O N

Lesueurin is the name we have given to a new 13-residuebasic peptide isolated from L. lesueurii. The sequenceshows reasonable homology to that of aurein 1.1, anantimicrobial and anticancer peptide isolated fromL. aurea [11] (Table 2).

There are some critical differences in the hydrophilicresidues of the two peptides with lesueurin having Lys11whereas aurein 1.1 has Glu11. Even so, we predicted thatlesueurin should show similar antimicrobial and anticanceractivity to that of aurein 1.1. Surprisingly, lesueurin neitherexhibits antibiosis below 100 lgámL)1, or cytotoxity below10)4

M in the 60 human tumour lines in the NIC testprogram. Thus, L. lesueuri, as with only a minority ofspecies of the genus Litoria (see introduction), has no hostdefence antibiotic against microbial pathogens. To discoverwhether lesueurin has other biological properties, it wassubjected to the bioactive molecule discovery program ofthe Australian Institute of Marine Science. It effectivelyinhibited nNOS with an IC50 value of 16.2 lM with a slopeof 1.5. This is the ®rst instance of an amphibian peptide thatinhibits the formation of the chemical messenger nitricoxide.

Testing of other amphibian peptides indicated that thereare three well-de®ned groups of basic peptides that inhibitnNOS, and the results are summarized in Table 1. Theseare: (a) the aurein/citropin group of peptides, of whichlesueurin is a member. Most of these (lesueurin is a notableexception) are membrane active peptides, which showpotent antibiotic acitivity, and in the case of the aureins,signi®cant anticancer activity. These peptides are amphi-pathic ahelices, as evidenced by solution NMR studies onaurein 1.2 [11] and citropin 1.1 [9]; (b) the frenatin 3 typepeptides, molecules that are characterized by a C-terminalCO2H group together with two lysine residues near theC-terminus. These peptides show little or no antibiotic and/or anticancer activity; (c) those caerins 1, particularly those

containing Phe3. These molecules are also potent mem-brane-active antibiotics, and NMR studies show they havetwo ahelical regions separated by a central ¯exible hingeregion [13]. How do these three seemingly unrelated groupsof peptides inhibit the formation of nitric oxide by nNOS?

The three nitric oxide synthases, namely neuronal,endothelial and inducible, are highly regulated enzymesresponsible for the synthesis of the signal molecule nitricoxide. They are amongst the most complex enzymes known(for nNOS see [37,38]). By a complex sequence involvingbinding sites for a number of cofactors including heme,tetrahydrobiopterin, FMN, FAD and NADPDH, nNOSconverts arginine to citrulline, releasing the short-lived butreactive radical nitric oxide [39,40]. Nitric oxide synthasesare composed of two domains: (a) the catalytic oxygenasedomain that binds heme, tetrahydrobiopterin and thesubstrate arginine, and (b) the electron-supplying reductasedomain that binds NADPH, FAD and FMN. Communi-cation between the oxygenase and reductase domains isdetermined by the regulatory enzyme calmodulin thatinteracts at a speci®c site between the two domains. In thecases of the nNOS and eNOS isoforms, the calmodulin isregulated by intracellular Ca2+, but not for iNOS [41±44].Dimerization of the oxygenase domain is necessary forcatalytic activity [39,40].

The signi®cant departure of the Hill slope of all of theinhibition curves from unity was the ®rst indication thatthese peptides are not acting directly upon the argininesubstate site of nNOS [33]. A Hill slope � 1 would indicatean interaction between a single active enzyme element with asingle substrate. AsHill slopes>1 are obtained, some otherinteraction must be causing the inhibition of nNOS. Tocon®rm this suspicion and to elucidate the general mech-anism of inhibition, an analysis was conducted of thekinetics of the nNOS inhibition reaction at differentinhibitor concentrations. Lineweaver±Burk plots are shownin Fig. 4 for representatives of each of the inhibitor groupslisted in Table 1, namely, lesueurin as the original nNOSinhibitor found, citropin 1.1, another member of inhibitorgroup 1, frenatin (inhibitor group 2) and caerin 1.9(inhibitor group 3). The fact that the regression lines plottedfor each inhibitory peptide shown in Fig. 4 all intercept at acommon point on the X-axis of these plots is typical ofnoncompetitive inhibition, and so these peptides areunlikely to directly involve the arginine substrate site [33].In its simplest de®nition, noncompetitive inhibition is whenan inhibitor binds at a site other than the active site,changing the enzyme±substrate af®nity.

These four peptides, lesueurin, citropin 1.1, frenatin 3and caerin 1.9, and probably the other active amphibianpeptides of inhibitor groups 1±3, must therefore inhibit theformation of nitric oxide by either blocking one or more ofthe cofactor sites on nNOS or by some chemical modi®ca-tion reaction with nNOS that alters the activity of theenzyme. An obvious example of blocking a cofactor sitewould be if the amphibian peptide reacts with the regulatoryenzyme Ca2+calmodulin, thus changing the three-dimen-sional structure and preventing its attachment to thecalmodulin binding site on nNOS. There are examples ofsmall basic peptides, often ahelices, being captured andenclosed within Ca2+calmodulin, and as a consequencechanging the three-dimensional shape of the calmodulin[45±48], but nNOS deactivation by these peptides has, to

Table 2. Sequence identity of lesueurin and aurein 1.1.

Peptide Sequence

Lesueurin GLLDILKKVGKVA-NH2

Aurein 1.1 GLFDIIKKIAESI-NH2


our knowledge, not been tested. There are also exampleswhere certain small peptides (one of which mirrors part ofthe sequence of the calmodulin binding site of rat nNOS)preferentially interact with Ca2+calmodulin, impeding theinteraction of Ca2+calmodulin with its binding site onnNOS, and, as a consequence, preventing the formation ofnitric oxide (IC50 values at lM concentrations) [49,50].

The possibility that the mechanism of nNOS inhibitionby the amphibian peptides does involve complex formationof peptides with Ca2+calmodulin is supported by thefollowing experiments. The inhibition of nNOS by selectedpeptides (citropin 1.1, frenatin 3 and caerin 1.9) is reducedby the addition of Ca2+calmodulin to the assay buffer. Themaximum reduction of inhibition under the experimentalconditions used is 50%.

The enzyme Ca2+calmodulin regulates not only nNOSbut also a number of other enzymes including calcineurin. Ifthe active amphibian peptides are indeed interacting withCa2+calmodulin, they should also inhibit the activity ofcalcineurin. The four model peptides lesueurin, citropin 1.1,frenatin 3 and caerin 1.9, all inhibit the activity of calcineu-rin, but at concentrations lower than those obtained fornNOS (Table 1). Even so, the fact that all four peptidesinhibit both nNOS and calcineurin enzymes, providescredence to the proposal that the amphibian peptides areaffecting the Ca2+calmodulin interaction with nNOS. Thisis an interesting observation because sequences of theCa2+calmodulin binding sites of nNOS and calcineurin arequite different (see below [51]), even though they have beenclassi®ed as belonging to the same class of enzymes [52].The sequences are nNOS (rat, human) and calcineurin A(rat, human), respectively are as follows:KRRAIGFKKLAEAVKFSAKLMRKEIIRNKIRAIGKMARVFSVLR.

Currently, although three-dimensional structures of cer-tain domains of nitric oxide synthase isoforms are known[53±57], we are not aware of the three-dimensional structureof the calmodulin binding sites of any isoform beingreported. Thus, we cannotmakemeaningful comparisons ofcommon structural motifs that might underlie calmodulinbinding to the enzymes.

Most nNOS inhibitors are small organic molecules thatare either analogues of the arginine substrate such asN-nitro-L-arginine [58] or species that prevent calmodulinconjugation such as tamoxifen [59] and melatonin [60].Protein and peptidic inhibitors of nNOS are few. Apartfrom those we have mentioned above [45,46], caveolin-1, astructural protein component of plasmalemmel caveolae,inhibits endothelial NOS but not neuronal NOS or indu-cible NOS [61]. Residues 82±101 of caveolin-1 in the form ofglutathione S-transferase fusion proteins have been shownto attenuate the activities of all three isoforms of NOS [62].A novel 10-kDa protein inhibitor of nNOS, known as PIN,destabilizes the native dimer form of nNOS preventing itfrom functioning [63±65]. Proteins such as presynapticdensity proteins-93 and -95 and synophin bind to nNOS viaa specialized domain called PDZ domain, which serves ascellular localization signals, but these proteins do notmodulate nNOS activity [66]. The basic amphibian peptidesthat inhibit nNOS show little homology of sequence (a)between the three active groups of peptides shown inTable 1, (b) with the sequence of amino acid residues of theCa2+/calmodulin binding domain of nNOS, (c) with the

sequence of Ca2+/calmodulin itself, and (d) with otherpeptides or proteins which inhibit nNOS. Thus we areunable, at this time, to predict from homology, any featuresof the active amphibian peptides that allow them to inhibitnNOS.

There is, however, a signi®cant homology of sequenceswithin each of the active groups shown in Table 1. Considergroup 1 (Table 1) as an example. All peptides of group 1have a post-translational modi®cation in that theN-terminal group is -CONH2. Lesueurin has high homol-ogy with some of the citropins and aureins. It is severalresidues shorter that aureins 2.2, 2.3, 2.4 and citropin 1.1, allof which have IC50 values more potent than that oflesueurin. Aurein 1.1 has the same number of residues aslesueurin but a lower overall positive charge, and is not aseffective as lesueurin in inhibiting nNOS. The length of thesepeptides seems to be a factor in determining their activity.Another important feature conserved in this group ofpeptides is the central Lys-Lys pair and the basic nature ofthe peptides. It appears that the length of the peptide andthis pair of basic residues are important in determining themagnitude of the nNOS inhibition. All three groups ofnNOS active amphibian peptides (Table 1) are unique newmolecular probes for the functioning of nNOS. We intendto carry out further structural studies on each of the threegroups of active peptides listed in Table 1, in order to probethe relationship between sequence, solution structure(determined by NMR) and nNOS inhibition.

The ®nal question to be addressed is what role do theseactive peptides have in the amphibian integument? Theglandular secretion of the animal is exuded onto the skin(and into the gut) when the animal is stressed, sick, or underattack. Logically, there are two possibilities, either thenNOS inhibitor is playing some regulatory role in theanimal and/or it is acting as part of the primary host defencearsenal against predators.

Consider the ®rst possibility. The nNOS used in thisinvestigation is from rat. Although the sequence of nNOS inanurans has not been determined, it is likely to be verysimilar to that of rat nNOS as the sequences of nNOS so fardetermined (fromman, rat, rabbit and snail) are very similar[51]. We have shown that L. lesueuri secretes two neuro-peptides onto its skin. The ®rst of these, caerulein 1.1 is aknown host defence peptide of many anurans [1±4,8]. It hasa multifaceted role including smooth muscle activity (atngákg)1 body weight), which makes it a powerful toxinagainst predators, it has potent analgaesic properties and itis also thought to be a hormone involved in the hibernationcycle of anurans [67]. The other neuropeptide, lesueurin,inhibits the formation of nitric oxide by nNOS. Nitric oxidehas many roles in animals and plays important roles in thenervous, muscular, cardiovascular and immune systems[68]. In anurans, nitric oxide is already known to be involvedin sight [69], reproduction [70] and modulation of gastricacids [71]. It is possible that together with caerulein 1.1,lesueurin has a role in either stress control and/or temper-ature control.

The other scenario is that nNOS inhibitors are front linedefence compounds. We have now identi®ed nNOS inhib-itors as major skin peptides in 10 out of 12 studied species offrogs of the genusLitoria. These compounds are all active atmicromolar-concentrations. A predator ingesting even asmall amount of the anuran skin secretion could be seriously


affected if even part of its nitric oxidemessenger capability isreduced. The predator could either be large, or small(bacteria have recently been shown to contain NOS[72±75]).

In conclusion, most frogs of the genus Litoria secrete acocktail of bioactive peptides onto their skin, some of whichare cytotoxic and antibiotic, and others which regulate theneuronal isoform of the enzyme NOS.We propose that thiseffect on nNOS is mediated by allosteric modulation ofarginine catalysis, through an effect on Ca2+calmodulinbinding to nNOS. We do not believe this is unexpectedbioactivity. The peptides that inhibit the formation of nitricoxide from nNOS either play a role in the fundamentalphysiology of the animal, and/or are part of the defencearsenal to combat attack by predators, both small and large.

A C K N O W L E D G E M E N T

Amphibian experiments and peptide syntheses were funded by the

Australian Research Council.

R E F E R E N C E S

1. Bevins, C.L. & Zaslo�, M. (1990) Peptides from frog skin.

Ann. Rev. Biochem. 59, 395±414.

2. Lazarus, L.H. & Attila, M. (1993) The toad, ugly and venomous,

wears yet a precious jewel in his skin.Prog. Neurobiol. 41, 473±507.

3. Erspamer, V. (1994) Bioactive secretions of the amphibian integu-

ment. In Amphibian Biology. The Integument. (Heatwole, H., ed.),

pp. 178±350.

4. Barra, D. & Simmaco, M. (1995) Amphibian skin: a promising

resource for antimicrobial peptides. Trends. Biochem. 13, 205±209.

5. Anastasi, A., Erspamer, V. & Endean, R. (1968) The structure of

caerulein. Arch. Biochem. Biophys. 125, 57±63.

6. Stone, D.J.M., Waugh, R.J., Bowie, J.H., Wallace, J.C. & Tyler,

M.J. (1992) The structures of the caerins and caeridin 1 from

Litoria splendida. J. Chem. Soc. Perkin Trans. 1, 3173±3179.

7. Wabnitz, P.A., Bowie, J.H. & Tyler, M.J. (1999) Caerulein like

peptides from the skin glands of the Australian Blue Mountains

tree frog Litoria citropa. Part 1. Sequence determination using

electrospray mass spectrometry. Rapid Commun. Mass Spectrom.

13, 2498±2502.

8. Bowie, J.H., Wegener, K.L., Chia, B.C.S., Wabnitz, P.A., Carver,

J.A., Tyler, M.J. &Wallace, J.C. (1999) Host defence antibacterial

peptides from the skin secretions of Australian amphibians.

Protein Peptide Lett. 6, 259±269.

9. Wegener, K.L.,Wabnitz, P.A., Bowie, J.H., Carver, J.A.,Wallace,

J.C. & Tyler, M.J. (1999) Host defence peptides from the skin

glands of the Australian Blue Mountains tree frog Litoria citropa.

The antibacterial citropin 1 peptides. The solution structure of

citropin 1.1. Eur. J. Biochem. 265, 627±635.

10. Wabnitz, P.A., Bowie, J.H., Wallace, J.C. & Tyler, M.J. (1999)

The antibiotic citropin peptides from the Australian tree frog

Litoria citropa. Structure determination using electrospray

mass spectrometry. Rapid Commun. Mass Spectrom. 13, 1724±

1732.

11. Rozek, T., Wegener, K.L., Bowie, J.H., Olver, I.N., Carver, J.A.,

Wallace, J.C. & Tyler, M.J. (2000) The antibiotic and anticancer

active aurein peptides from the Australian bell frogs Litoria aurea

and Litoria raniformis. The solution structure of aurein 1.2.Eur. J.

Biochem. 267, 5330±5341.

12. Monks, A., Scudiero, D., Skehan, P., Shoemaker, R., Paull, K.,

Vistica, D., Hose, C., Langley, J., Cronise, P. & Vaigro-Wol�, A.

(1991) Feasibility of a high-¯ux anticancer drug screen using a

diverse panel of cultured human tumor cell lines. J. Natl Cancer

Inst 83, 757±766. See also http://dtp.nci.nih.gov

13. Wong, H., Bowie, J.H. & Carver, J.A. (1997) The solution struc-

ture and activity of caerin 1.1, an antibiotic peptide from the

Australian tree frog Litoria splendida. Eur. J. Biochem. 247, 545±

557.

14. Shai, Y.C. (1995) Molecular recognition between membrane-

scanning peptides. Trends Biochem. Sci. 20, 460±464.

15. Bechinger, B. (1997) Structure and function of channel forming

peptides; magainins, cecropins, melittin and alamethicin.

J. Membr. Biol. 156, 197±211.

16. Matsuzaki, K. (1998) Magainins as paradigm for the mode

of pore forming polypeptides. Biochim. Biophys. Acta 1376,

391±400.

17. Steinborner, S.T., Gao, C., Raftery, M.J., Waugh, R.J.,

Blumenthal, T., Bowie, J.H., Wallace, J.C. & Tyler, M.J. (1994)

The structures of four tryptophyllin and three rubellidin peptides

from theAustralian red tree frogLitoria rubella.Aust. J. Chem. 72,

2099±2108.

18. Steinborner, S.T., Wabnitz, P.A., Bowie, J.H. & Tyler, M.J.

(1996) The application of mass spectrometry to the study of evo-

lutionary trends in amphibians. Rapid Commun. Mass Spectrom.

10, 92±97.

19. Steinborner, S.T., Wabnitz, P.A., Waugh, R.J., Bowie, J.H.,

Gao, C., Tyler, M.J. &Wallace, J.C. (1996) The structures of new

peptides from Litoria rubella. Aust. J. Chem. 49, 955±963.

20. Wabnitz, P.A., Bowie, J.H., Tyler, M.J. & Wallace, J.C. (1999)

Peptides from the skin glands of the Australian buzzing tree frog

Litoria electrica. Comparison with the skin peptides of Litoria

rubella. Aust. J. Chem. 52, 639±649.

21. Erspamer, V., Melchiorri, P., Erspamer, G.F., Montecucchi, P.C.

& de Castiglione, R. (1985) Phyllomedusa skin: a large factory

and storehouse of a variety of active peptides. Peptides 6, 7±12.

22. Renda, T.D., -Este, L., Bu�a, R., Usellini, L., Capella, C.,

Vaccaro, R. & Melchiorri, P. (1985) Tryptophyllin-like immu-

noreactivity in rat adenohypophysis. Peptides 6, 197±202.

23. Zadina, J.E., Hackler, L., Ge, L.J. & Kastin, A.J. (1997) A potent

and selective endogenous agonist for the mu-opiate receptor.

Nature 386, 499±500.

24. Raftery, M.J., Bradford, A.M., Bowie, J.H., Wallace, J.C. &

Tyler, M.J. (1993) The structures of the dynastins from the Banjo

frogs Limnodynastes interioris. L. dumerilii and L. terraereginae.

Aust. J. Chem. 46, 833±842.

25. Bradford, A.M., Raftery, M.J., Bowie, J.H., Wallace, J.C. &

Tyler, M.J. (1993) The Structures of the dynastins and ¯etcherin

from Limnodynastes salmini and Limnodynastes ¯etcheri. Aust. J.

Chem. 46, 1235±1244.

26. Tyler, M.J., Stone, D.J.M. & Bowie, J.H. (1992) A novel method

for the release and collection of dermal glandular secretions from

the skin of frogs. J. Pharmacol. Toxicol. Meth 28, 199±200.

27. Wabnitz, P.A., Bowie, J.H. & Tyler, M.J. (1999) Caerulein type

peptides from the skin glands of the Australian Blue Mountains

tree frog Litoria citropa. Part 1. Sequence determination using

electrospray mass spectrometry. Rapid. Commun. Mass Spectrom.

13, 2498±2502.

28. Biemann, K. & Martin. S. (1987) Mass spectrometric determin-

ation of the sequences of peptides and proteins. Mass Spectrom.

Rev. 6, 1±76.

29. Hunkapiller, M.W., Hewick, R.M., Drewer, W.J. & Hood, L.E.

(1983) High sensitivity sequencing with a gas-phase sequencer.

Methods Enzymol. 91, 399±406.

30. Maeji, N.J., Bray, A.M., Valerio, R.M. &Wang,W. (1995) Larger

scale multi-pinpeptide synthesis. Pept. Res. 8, 33±38.

31. Jorgensen, J.H., Cleeland, W.A., Craig, G., Doern, M., Ferraro,

J., Finegold, C.M., Hansen, S.L., Jenkins, S.G., Novick, W.J.,

Pfaller, M.A., Preston, D.A., Reller, L.B. & Swenson, J.M. (1993)

Methods for dilution antimicrobial susceptibility tests for bacteria

that grow aerobically, 3rd approved standard. Natl Committee

Clin. Lab. Stand. 33, 1±12. Document M7±A3.


32. Doyle, D.D., Gua, Y., Lustig, S.L., Satin, J., Rogart, R.B. &

Fozzard, H.A. (1993) Divalent cation competition with [3H]saxi-

toxin binding to tetrodotoxin-resistant and ± sensitive sodium

channels. A two-site structural model of ion/toxin interaction.

J. General Physiol. 101, 153±182.

33. Fersht. A. Enzyme Structure and Mechanism, W.H. Freeman,

New York, NY, USA.

34. Anonymous. (2000) Protein phosphatase-2B. Technical Bulletin

534. Promega, Madison. WI, USA.

35. DumeÂ ril. A.M.J. & Bibron, G. (1841) ErpeÂtologie GeÂneÂrale Ou

Histoire Naturelle CompleÁte des Reptiles, Paris. 8, 792.

36. Barker, J., Grigg, G.C. & Tyler, M.J. (1995) In A Field Guide to

Australian Frogs. p. 68. Surrey Beatty and Sons. ChippingNorton,

New South Wales, Australia.

37. Bredt, D.S., Hwang, P.M., Glatt, C.L., Lowenstein, C., Reed,

R.R. & Snyder, S.H. (1991) Cloned and expressed nitric oxide

synthase structurally resembles cytochrome P-450 reductase.

Nature 351, 714±718.

38. Marletta, M.A. (1993) Nitric oxide synthase and mechanism.

J. Biol. Chem. 268, 12231±12234.

39. Marletta, M.A. (1994) Nitric oxide synthase: aspects concerning

structure and catalysis. Cell 78, 927±930.

40. Pfei�er, S., Mayer, B. & Hemmens, B. (1999) Nitric oxide: a

protective or pathogenic molecule.Angew. Chem. Intl Ed. Engl. 38,

1715±1731.

41. Forsen, S. (1995) Calcium induced structural changes and domain

autonomy in calmodulin. Nat. Struct. Biol. 2, 777±783 and refer-

ences cited therein.

42. Venema, R.C., Sayegh, H.S., Kent, J.D. & Harrison, D.G. (1996)

Identi®cation, characterisation and comparison of the calmodulin-

binding domains of nitric oxide synthases. J.Biol. Chem. 271,

6435±6440.

43. Lee, S.J. & Stull, J.T. (1998) Calmodulin-dependent regulation of

inducible and neuronal nitric oxide synthase. J. Biol. Chem. 273,

27430±27437.

44. Matsuda, H. & Iyanagi, T. (1999) Calmodulin activated intra-

molecular transfer between the two ¯avins of neuronal nitric oxide

synthase. Biochim. Biophys. Acta 1473, 345±355.

45. Malencik, D.A. & Anderson, S.R. (1982) Binding of simple pep-

tides, hormones and neurotransmitters by calmodulin. Biochem-

istry 21, 3480±3486.

46. Ikura, M., Kay, L.E., Krinks, M. & Bax, A. (1991) Triple reso-

nance multidimensional NMR study of calmodulin complexed

with the binding domain of skeletal muscle myosin light-chain

kinase: indication of a comformational change in the central helix.

Biochemistry 30, 5498±5504.

47. Ikura, M., Clore, G.M., Gronenborn, A.M., Zhu, G., Klere, C.B.

& Bax, A. (1992) Solution structure of a calmodulin-target peptide

complex by multidimensional NMR. Science 256, 632±638.

48. Scaloni, A., Miraglia, N., Orru, S., Amodeo, P., Motta, A.,

Marino, G. & Pucci, P. (1998) Topology of the calmodulin-

melittin complex. J. Mol. Biol. 277, 945±958.

49. Matsubara, M., Hayashi, N., Titani, K. & Taniguchi, H. (1997)

Circular dichroism and 1H NMR studies on the structures of

peptides derived from the calmodulin-binding domains of indu-

cible and endothelial NOS in solution and in complex with

calmodulin. Nascent alpha-helical structures are stabilized by

calmodulin both in the presence and absence of Ca2+. J. Biol.

Chem. 272, 23050±23056.

50. Watanabe, Y., Hu, Y. & Hidaka, H. (1997) Identi®cation of a

speci®c amino acid cluster in the calmodulin binding domain of

nNOS. FEBS Lett. 403, 75.

51. For the sequences of various NOS enzymes together with the

positions of the binding domains of the cofactors Ca2+calmodu-

lin. SWISS-PRO http://au.expasy.org/cgi-bin/sprot-search-de

52. Rhoads, A.R. & Frieberg, F. (1997) Sequence motifs for cal-

modulin recognition. FASEB J. 11, 331±342.

53. Crane, B.R., Arvai, A.S., Gachhui, R., Wu, C.O., Ghosh, D.K.,

Getzo�, E.D., Stuehr, D.J. & Tainer, J.A. The structure of nitric

oxide synthase oxygenase domain and inhibitor complexes.

Science 278, 425±431.

54. Raman, C.S., Li, H.Y., Martasek, P., Krai, V., Masters, B.S.S. &

Poulos, T.L. (1998) Crystal structure of constitutive endothelial

oxide synthase; A paradigm for pterin function involving a novel

metal center. Cell 95, 939±950.

55. Crane, B.R., Arvai, A.S., Ghosh, D.K., Wu, C.O., Getzo�, E.D.,

Stuehr, D.J. & Tainer, J.A. (1998) Structure of nitric oxide syn-

thase oxygenase dimer with pterin and substrate. Science 279,

2121±2126.

56. Fischmann, T.O., Hruza, A., Fosseta, J.D., Lunn. C.A., Dolphin.

E., Prongay, A.J., Reichert, P., Lundell, D.J., Naruda, S.K. &

Weber, P.C. (1999) Structural characterisation of nitric oxide

synthase isoforms reveals striking active-site conservation. Nat.

Struct. Biol. 6, 233±242.

57. Tochio, H., Zhang, O., Mandal, O., Li, M. & Zhang, M.J. (1999)

Solution structure of the extended neuronal nitric oxide synthase

PDZ domain complexed with an associated peptide. Nat. Struct.

Biol. 6, 417±421.

58. Moore, P. & Handy, R. (1997) Selective inhibitors of neuronal

nitric oxide synthase ± is noNOS really goodNOS for the nervous

system? Trends Pharmacol. Sci. 272, 15583±15586.

59. Renodon, A., Boucher, J.L., Sari,M.A., Delaforge,M., Ouazzani,

J. & Mansuy, D. (1997) Strong inhibition of nNOS by the cal-

modulin antagonist and anti-estrogen drug tamoxifen. Biochem.

Pharmacol. 54, 1109±1114.

60. Leon, J., Macias, M., Escames, G., Camacho, E., Khaldy, H.,

Martin, M., Espinosa, A., Gallo, M.A. & Acuna-Castroviejo, D.

(2000) Structure related inhibition of calmodulin-dependent

nNOS activity by melatonin and synthetic kynurenines. Mol.

Pharmacol. 58, 967±975.

61. Michel, J.B., Feron, O.D.S. & Michel, T. (1997) Reciprocal reg-

ulation of endothelial NOS by Ca2+calmodulin and caveolin.

J. Biol. Chem. 272, 15583±15586.

62. GarcõÂ a-CardenÄ e, G., Martasek, P., Sue Siler Master, B., Skidd, P.,

Couet, J., Li, S., Lisanti, M. & Sessa, W. (1997) Dissecting the

interaction between NOS and caveolin. J. Biol. Chem. 272, 25437±

25440.

63. Ja�rey, S. & Snyder, S. (1996) PIN: an associated protein inhibitor

of nNOS. Science 274, 774±777.

64. Tochio, H., Ohki, S., Zhang, O., Li, M. & Zhang, M.J. (1998)

Solution structure of a protein inhibitor or neuronal nitric oxide

synthase. Nat. Structure Biol. 5, 965±969.

65. Fan, J.S., Zhang, O.A., Li, M., Tochio, H., Yamazaki, T.,

Shimizu, M. & Zhang, M.J. (1998) Protein inhibitor of

neuronal nitric oxide synthase, PIN, binds to a 17-amino

acid residue fragment of the enzyme. J. Biol. Chem. 273, 33472±

33481.

66. Brenman, E., Chao, S., Gee, S.H., McGee, A.W., Craven, S.E.,

Santillano, D.R., Wu, Z., Huang, F., Xia, H., Peters, M.F.,

Froehner, S.C. & Bredt, D.S. (1996) Interaction of NOS with the

postsynaptic density protein PSD-95 and alpha 1-syntrophin

mediated by PDZ domains. Cell 84, 757±767.

67. Wabnitz, P.A., Bowie, J.H., Tyler, M.J., Wallace, J.C. & Smith,

B.P. (2000) Di�erences in the skin peptides of the male and female

Australian magni®cent tree frog Litoria splendida ± a three year

monitoring survey: the discovery of the male sex pheromone

splendipherin together with phe8caerulein and the antibiotic caerin

1.10. Eur. J. Biochem. 267, 269±275.

68. Ghosh, S.,Wolan, D., Adak, S., Crane, B.R., Kwon, N.S., Tainer,

J.A., Getzo�, E.D. & Stuehr, D.J. (1999) Mutational analysis of

the tetrahydrobiopterin-binding site in inducible nitric oxide syn-

thase. J. Biol. Chem. 274, 24100±24112.

69. Renteria, R.C. & Constantine-Paton, M. (1999) Nitric oxide in

the retinotectal system: a signal but not a retrograde messenger


during map re®nement and segregation. J. Neuroscience 99,

7066±7076.

70. Gobbetti, A. & Zerani, M. (1999) Hormonal and cellular brain

mechanism regulating amplexus of the male and female water frog

Rana esculenta. J. Neuroendocrinol. 11, 589±596.

71. Molero, M., Hernandez, I.M., Lopo, P., Cardenas, P., Romero,

R. & Chacin, J. (1998) Modulation by nitric oxide of gastric acid

secretion in toads. Acta Physiol. Scand. 164, 229±236.

72. Choi, W.S., Chang, M.S., Han, J.W., Hong, S.Y. & Lee, H.W.

(1997) Identi®cation of NOS in Staphylococcus aureus. Biochem.

Biophys. Res. Commun. 237, 554±558.

73. Morita, H., Yoshikawa, H., Sakata, R., Nagata, Y. & Tanaka, H.

(1997) Synthesis of nitric oxide from the two equivalent guanidino

nitrogens of L-arginine by Lactobacillus fermentum. J. Bacteriol.

179, 7812±7815.

74. Choi, W.S., Seo, D.W., Chang, M.S., Han, J.W., Paik, W.K. &

Lee, H.W. (1998) Methylesters of L-arginine and N-nitro-L-argi-

nine induce nitric oxide synthase inStaphyloccoccus aureus. Bio-

chem. Biophys. Res. Commun. 246, 431±435.

75. Er, H., Turkoz, Y., Ozerol, I.H. &Uzmez, E. (1998) E�ect ofNOS

inhibition in experimental Pseudomonas keratitis. rabbits. Eur. J.

Opthalmol 8, 137±141.


Amphibian peptides that inhibit neuronal nitric oxide synthase : The isolation of lesueurin from the...

Documents

Transcript of Amphibian peptides that inhibit neuronal nitric oxide synthase : The isolation of lesueurin from the...