Characterisatn and Localizatn of TMOF

8/3/2019 Characterisatn and Localizatn of TMOF

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Characterizationand localizationof mosquito-gut receptorsfor trypsin modulating oostatic factor using acom plementary peptide and imm unocytochernistry

DOV BOROVSK Y,*.l C . A . PO WELL ,t J. K. N AY AR ,6 J. EDW IN BLALOCK ,1 AND T. K . BAYESS*Institu te of Food and Agricu ltural Sciences, University of Florida, FM EL , Vero Beach Florida 32962, U SA ; tln stitu teof Food and Agricultu ral Scsences, Umversity of Florida, Agricu ltu ral Research and Educational Center, F ort P ie rc e,F lorida 34954, USA , 1Departm ent of Physiology and B iophysics University of Alabam a at B irm ingham Birm ingham ,Alabama 35294 , USA ; 5Department of Entomolog Texas A&M University, College Station, Texas 7 78 43 , U SA

35 0 0 89 2-6 63 8/9 4/0 00 8-0 35 0/$ 0l .5 0. FASEB

ABSTRACT Th e gu t receptor of trypsin -m odulatingoostatic factor (TMOF), a decapeptide hormone thatregulates trypsin biosynthesis in the m osquito gut, hasbeen characterized . The b ind ing of TMOF to mosquitogu t membranes reached m aximum at pH 7.4 and 24#{176}C .No bind ing was observed at pH 2.5 and the b ind ing to themembranes declined rapid ly at pH 8.0 . A t equilibrium ,maxim um bind ing to the receptor was observed at 60 mman d 24#{176}C . synthetic com plem entary decapeptide N H2-lle-Leu-G ly-A rg-G ly-G ly-G ly-G ly-G ly-G ly-COOH (FOMT)for TMOF successfu lly competed w ith the gut receptor,and specifically bound TMOF (K d = 4 M and Kassoc =2.5 x 10 M1). TMOF bind ing to gu t membranes wascharacterized w ith FOMT and a specific ELISA to thehormone at 24 and 72 h after blood feeding. Two classesof binding sites w ere found on the gut membrane; h ighaffinity (Kdl = 4. 6 0.7 x 10 M ; Kassoc = 2.2 X106 M B m a x = 0. 1 pmol/gut) and low affinity(KdI = 4.43 1 x 106 M ; Kassoc = 2. 3 x 10 M1; B m a x= 0. 2 pmollgut) . The tota l binding sites for h igh and lowaffinity classes of TMOF per gut w ere estim ated as6 .3 x 1010 and 1.1 x 1011 sites, respectively . Specificb inding sites on the gut increased after the blood meal andwere visualized by immunocytochem ical sta ining. Theseresults suggest that TM OF regu lates trypsin b iosynthesisby b ind ing to specific receptor sites that are located on themosquito gut, and that th is receptor can be stud ied usinga com plementary peptide approach.- Borovsky, D .,Powell, C . A ., Nayar, J. K ., Blalock, J. E ., Hayes, T . K .Characterization and localization of mosquito-gut recep-tors for trypsin m odulating oostatic factor using a com -p lem entary peptide and im munocytochem istry. FASEBJ.8: 350-355; 1994.Key Words. immunocytochemistry receptor . binding . ELISAp ep tid e h orm on e

ANAUTOGENOUS FEMALE MOSQUITOES LACK reserve proteinsand take a blood meal in order to develop their eggs. Themain digestive enzym e in the m idgut of these females is tryp-sin (1-3). A fter the blood m eal, the m idgut ep ithelial cellsstart to synthesize trypsin and the enzyme is secreted into them idgut (3). A t 24 h, when trypsin biosyn thesis is at theh ighest level, the fo llicular ep ithelium of the ovary beg ins tosynthesize and release TM OF into the hemolymph; m axi-m um synthesis of TMOF is at 36 h and then the synthesisdeclines and stops at 48 h. TMOF binds to the gut and sig-nals the epithelial cells to stop trypsin biosynthesis, which

then rap idly declines and stops at 48 h (4-8). In jectingTMOF immediately after the blood meal in to m osquitos,biting m idges, flies, and fleas inhib ited proteolytic enzymebiosynthesis and blood digestion in the m idgut (5), ind icat-ing that TM OF could be used as a diet pill against blood-sucking insects. [3H ]TMOF injected in to fem ale A ed es ae gy ptior incubated w ith gu t membranes bound specifically to thegut. B ind ing was saturab le, and was reduced by increasingconcentrations of the unlabeled horm one (5 , 7). Theseearlier studies indicated that the m idgut ep ithelial cells haveTM OF-specific b inding sites.

Bost et al. (9) and others (for rev iew , see ref 10) have ob-served the binding of pep tides (termed complem entary) thatare encoded by com plementary nucleotide sequences. Thisapparently resu lts from codons of hydrophobic am ino acidsbeing complem ented by those of hydrophilic am ino acidsand vice versa (11). Thus, b inding of a horm one to a receptormay involve complem entary hydrophobic and hydrophiicdomains that alter the shapes of the molecules and facilita tebinding . Using th is concept, m any laboratories have demon-strated that m any com plementary peptides to hormones in-cluding cortico tropin (ACTH ), y-endorphin , or luteinizinghormone-releasing hormone (LHRH) bound the appropri-ate hormone and that the immune system recognized thesepeptides as an tigenically sim ilar to the respective hormonereceptor binding site (10). Thus, using com plementary pep-tide and immunocytochem ical approaches, TMOF bindingsites on isolated gut membranes were identified and characterized.

M ATERIALS AND M ETHODSInsectsLarval A . a eg yp ti w ere reared at 26#{176}Cn a diet o f brewers yeast and lactal-bum in (1:1), under 16:8 h light:dark cycle. Adults w ere fed on 10% sucroseor on chicken blood. Fem ales were used 3-5 days after em ergence.Synthesis of complementary pep tide (FOMT)A p ep tid e c om ple me nta ry to T MO F (N H2 -Ile -L eu -G ly -A rg-G ly-G ly-G ly -Gly-Gly-Gly-COOH) wa s synthesized in the laboratories ofT K. Hayes at

To whom correspondence should be addressed, at: University ofFlorida-IFAS, Florida Medical Entom ology Laboratory , V eroB each , FL 32962 , USA .

2Abbreviations: ACTH , cortico tropin; LHRH, luteinizinghormone-releasing horm one; TMOF, trypsin-m odulating oostaticfactor; TSTB, Tris saline Triton buffer; FOMT, TMOF com -p lem entary p eptid e.


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TIME (mm )

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MOSQUITO TMOF GUT RECEPTOR 35 1

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Figure 1. T ime cou rse of specific bind ing of TMOF to gut mem-branes. Each incubation well was incubated at 24#{176}Cnd conta inedgu t membranes from seven female A. aegypti that w ere fed blood 24h earlier and TM OF (2 .0 tg ). Nonspec ific b ind ing (17-26% of tota lbinding) was subtracted from each poin t and was determined by in -cubating FOM T (8.0 tg) in parallel w ells. E ach point is th e a ve ra geof t wo d e te rm i na ti on s.

Texas A&M University an d J. E . B la lo ck , University of A labama at Birming-ham. Th e p ep tid e p ur ity a nd h om o ge ne ity w as confirm ed by reversed phaseC1 8 HPLC an d by fa st a to m bombardmen t mass spectrometry in which th epeptide exhib ited an abundant ion at m/ z 799.7.Preparation of gut m em branesG uts w ere dissected from fem ale A . a eg yp ti 24 and 72 h after the b lood m eal.Th e guts (100 pe r group) were hom ogenized in 0.1 m l 20 mM T ri s- ma le icacid buffer, pH 6.5, containing 500 mM NaC1, 1 mM EDTA , 10 ig b ac itra -cm , 1 0 s g a pr oto nin , 50 sg BSA, an d 1 mM PM SF at 5#{176}Cith a Teflonhom ogenizer. The hom ogenate w as then centrifuged at 14,000 x g a t 5 #{ 17 6} Ca nd th e s up ern ata nt w as re mo ve d. T he p elle t w as re ho mo ge niz ed , s on ic ate dfo r 60 s, and recentri fuged fo r 10 mm a t 5 #{1 76 }C .h e p elle t was thenr es us pe nd ed a t 5 #{1 76 }Cn 0.1 m l TBS (20 mM Tris -HC I, 500 mM NaC l, pH

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0. 4

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Figure 2 . Effect of FOMT on the binding of TMOF to gut mem -branes. Gut mem branes from seven female mosquitoes were in-cubated at 24#{176}Cor I h with TMOF (2.0 tg ) and increasingamounts of FOMT (0-40 ig). Each point represen ts an average oft wo d et erm in a tio ns .

C

z0U-02I-

pHFigure 3. Relationship between pH and bind ing of TMOF to gutmembranes. Each well conta ined gut membranes from seven fe-male A . ae gy pti and TMOF (2.0 tg) and was incubated for 1 h at24#{176}Cn 20 mM G lycine-HC I buffer for pH 2.6 , 20 mM T ris m ale-ate buffer for pH 4.0 to 6.5, and 20 mM Tris HC I buffer for pH 7.0to 8.5. Each point represents an average of two determ inations.

7.5) in such a way that 1 l o f m em brane preparation was equivalent to onegut. O nly fres hly prepared g ut m em branes w ere used fo r th e b in din g e xp er i-ments.Binding of TMOF to gut membranesB in din g assays w ere carried O ut in C ostars n itro cellu lo se filter strip system(Cam bridge, M ass.). M embranes in wells w ere wetted w ith 10 0 sl TBS, pH7 .5 , fo r 5 mm, and the b uffer w as rem oved from th e w ells by g entle vacuum .G ut m em bra ne s (5-10 per w ell) or d iffe rent am ounts o f T MO F com plem en-tary peptide (FOMT) were added and allowed to incubate for 30 mm inTBS (100 tl) a t room tem pera ture. The solution was then rem oved underg en tle v a cu um , an d the w ells w ere w ashed three tim es w ith TB S. The w ellsw ere b lo ck ed w ith T BS , 1% Tween 2 0 ( Si gm a , St . Louis, M o.), and 2% BSA(Sigma), pH 7.5 (100 il), for 30 m m at room tem pera ture. The blocking so-lu tion was removed from the wells by vacuum and diffe rent amounts ofTM OF were added an d allow ed to incubate in the w ells for 1 h at room tem -p era tu re . T o d ete rm in e n on sp ec ific binding, TMOF complementary pep-tide (FO MT) w as added at concentrations fourfo ld h igher than TM OF. A f-ter incubations, the solutions were rem oved from the wells w ith a gentlevacuum and the membranes were washed th ree times w ith TBS , 0.05%Tween 20, pH 7.5, and 1% rabbit anti-TMOF serum (100 s1) (in TBS, pH7.5) was added and the filter s trip s w ere in cu ba te d a t 4 #{1 76 }Cor 18 h . Str ipsw ere th en d ev elo pe d w ith g oa t a nti-ra bb it im m un og lo bu lin c ov ale ntly lin ke dwith a lka line phosphatase and p-n itropheny l phospha te (12). The absor-bances of contro l w ells con ta in ing gut m em branes w ere subtracted fromw ells tha t con ta ined T MO F a nd g ut rece ptor. E ach as say w as repeate d th reet imes.

Immunocytochem istry of TM OF bind ing to mosquito gu tM idguts of sugar-fed and blood-fed fem ale A. aegypti w ere d iss ec ted inphos phate-sa line buffer and fixed in B ouin H ollandes s ublim ate fix ativ e fo r24 h (13). A fter fixation, the tissues w ere w ashed in d istilled w ater for 12 h,dehy drated in 75% , 95 %, and 100% ethanol, treated w ith xy lo lle th anol, a nde m be dd ed in P ar ap la st. S er ia l s ec tio ns (7 sm ) of m idguts were cut using arotary m icrotom e and attache d to glass s lid es w ith egg albu min. Tiss ue s ec-tio ns w ere im m un oc he mic ally s ta in ed u sin g a m od ifie d te ch niq ue d es crib edby V andesande (14). B rie fly, the tissue sections w ere depara ffinated andhydrated in 1 00% x ylo l (tw o c hanges of 5 m m), 100% e thanol (thre e c hange sof 3 mm ), and 1 mm in distilled water. The sublimate was removed by in-cubating tissue sections in Lugol (two times, for 2 mm ) and in 5% sod iumth iosulfate. The tissue sections were washed w ith distilled water andequilibrated for 5 m m in Tns saline Triton buffer (TSTB) conta in ing 10mM Tris-HCI, 150 mM NaC I, 1 mM NaN3, pH 7.6, 0.1% Triton X-lO O. T is-sue sections were then incubated for 45 m m with pre im mune goat serum


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1000 2000 3000 4000 5000S

1500T MO F (h g)

TMOF

2000 3000TM OF (ng)

RES EARCH COMMUNICAT IONS

352 Vol. 8 March 1994 T he F AS EB J ou rn al BORO VSKY ET AL.

Figure 4. B ind ing of TMOF to its complementary peptide(FOMT). Increasing amounts of TMOF (0-2,800 ng ) were in -cuba ted w ith FOMT bound to nitroce llu lose wells for 1 h at 24#{176}C .Scatchard analysis of the data is shown in the inset. The data area typ ica l experiment of two independent determ inations. A dissoci-a tion constant of 4 x 10-6 M was calcu la ted from these data.

(S igm a, S t. Louis , M o.), d ilu ted 1:5 in TSTB , an d rin se d s ev era l tim es inT ST B to rem ov e unbo und pro te ins . E ach slide w ith tis sue se ctio ns w as thenincubated w ith TM OF (0.2 m l, 130 sg) in TSTB for 1 h, washed in TSTB,an d incubate d ov ern ight at ro om tem perature w ith ra bbit antiserum againstTM OF (1:300 dilu tion in TSTB). A fter incubation , s lides w ith tissue sec-tions were washed, incubated for 25 m m w ith goat anti-rabbit antibody(S igm a), d ilu ted 1:30 in TSTB , washed an d incubated for 25 mm w ithp ero xid as e-a ntig oa t-p ero dix as e-c om ple x d ilu te d 1 :3 00 in T ST B, w as he d inTSTB, and developed in 3-3-d iam inobenz id ine. The tissue sections w erethen dehydrated in increas ing concentrations of e thanol and clarified in100% xylo l and m ounted w ith Perm ount.

F igu re 5 . Specific b ind ing of TMOF to gut membranes of femaleA . a eg yp ti that were fed blood 24 h earlier. Each well contained sevengut membranes and various amounts of TMOF (0-5,000 ng).N onspecific b inding was determ ined by incubating TMOF in th epresence of FOM T an d represented about 26% of tota l b ind ing atthe maximum . Scatchard analysis of the data is shown in the inset.The data are a typ ica l experiment of three independent determ ina-tions . H igh (Kdl 5.7 x 10 M) an d low (Kd2 = 2.2 x 10-6 M)dissoc ia tion constants were calcu la ted from these data.

5000

Figure 6. Spec ific b ind ing of TMOF to gut membranes of fem aleA . a e gy p tithat were fed blood 72 h earlier. Each we ll c o nt ai ne d sevengut mem branes and different am ounts of TMOF (0-4000 ng).Other conditions are as in F ig. 4. The data are a typical experimentof three independent determ inations. H igh (Kdl = 3.6 x 10 M)and low (Kd2 = 6.2 x 10-6 M) dissocia tions constants w ere calcu-la ted from these da ta.

RESULTS AND DISCUSSION

Preliminary data have indicated that mosquito gu t has asaturable receptor for TM OF (5, 7). To characterize theTMOF receptors, gu t membranes w ere iso lated 24 h afterthe blood m eal from seven fem ale A . a eg ypti, homogenized ,sonicated , and bound to nitrocellu lose membranes innitrocellulose strips (7 gu t membranes per w ell) and in -cubated for different periods (0-180 mm) w ith TMOF in thepresence and absence of its complem entary peptide(FOM T). The specific b inding of TM OF (2.0 g) to the gu tmembranes reached an equiibrium after incubation for 60mm at room tem perature (F ig. 1), and all subsequent incu-bations were carried out at 60 mm and room tem perature.The binding of TM OF (2.0 &g) to the gut membranes wasreversed in the presence of FOMT Maxim um displacementof TM OF from the gut membrane by FOMT was achievedw ith am ounts tw ofold higher than TM OF; increasing theamount of FOMT up to 40 eg did not d isp lace m ore of thehorm one (F ig . 2). Thus, about 26% of the binding of thehormone to th e gut m em brane is nonspecific. No inhibitionof TMOF binding to gu t membranes w as observed when re-nm i nh ib it or ( NH2P ro -H i s- Pr o- Ph e-H is -P h e- Ph e-V al -T y r-Lys-COOH) (4 ftg) w as substituted w ith FOM T (results notshown) indicating that the binding of TMOF to FMOT isspecif ic .

Incubation of the horm one at different pHs indicated thatoptimum binding w as between pH of 5 .5 an d 7.4 , w ith maxi-mum binding at pH 7.4 (Fig . 3). A s the pH values for thehemolymph of three mosquito species Aedes ta en io rh yn ch us, C u-lexpipiens, an d A ed es d or sa lis are between 7.5 and 7.6 (15), pH7.4 was used to characterize the binding of th e hormone toi ts re ce pt or.To de term in e th e affin ity of TM OF for its com plementarypeptide, nitrocellulose wells w ere incubated w ith FOMT (7eg), and the wells were w ash ed to re mo ve unbound peptide,blocked , and incubated w ith increasing concentrations of


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Figure 7. A superimposed stereov iew of TMOF and its complem en-tary peptide FOMT represented by a stick model. TMOFhydrophilic am ino acids Tyr and Asp2 are colored red (bottom ),whe reas the hydrophobic am ino acids (P ro3, A la4 , P ro5#{176})reco lo red green. The NH2 term inus of both peptides is at the bottom ,the COOH term inus is on top. FOMT backbone atoms are: whitecarbons, red oxygens, and b lue nitrogens.

TMOF (0-2 .8 tg). A fter equilibrium had been reached (1h), each well was analyzed for binding of TMOF usingELISA (12). A nalysis of the binding data using Scatchardanalysis (16) confirmed that the binding w as l inear with asingle class of b inding sites and an appa ren t d is soc ia ti onconstant (Kd) of 4 x 10-6 M and an affinity (K assoc) of2.5 x 10 M1 (F ig. 4). A single class of binding sites is ex-pected from complementary peptide studies (9) w ith a theo-retical b inding ratio of 1 :1 if al l the FOMT (7 peg) bound eachwell and if each TM OF molecule binds one FOM Tmolecule. M aximum binding ( Bm a e ) that was calculatedfrom the Scatchard analysis (Fig . 4) indicated that 5300 ngof TM OF bound 7000 ng of FOM T (1:0 .76 binding ratio) if100% of FOM T bound each w ell. If , on the other hand, only76% of FOMT bound each well then the ratio of b inding ofTM OF to FOMT is 1:1, suggesting a m onomolecular inter-action. Complem entary peptides to ribonuclease S p ep tid e(2 0 am ino acids) and substance P (9 am ino acids) also ex-hib ited sim ilar d issociation constants (1 .2 x 10-6 M an d6 x 10-6 M , respectively) (10). The ability of FOM T to com -pete specifically w ith the binding of the horm one to its gu treceptor enabled us to characterize the binding of TMOF tothe gut receptor.

Im portan t physio logical changes in the female mosquitooccur after the blood meal; trypsin is synthesized in them idgut, and the egg yolk protein vitellogenin is synthesizedby the fat body (3 , 17). To characterize the binding of TMOFto its receptor, m idgut m embranes w ere removed from fe-m ale m osquitoes 24 and 72 h after the blood meal and as-sayed for specific TMOF binding. B ecause binding reachedequilibrium in 60 mm at room tem perature w ith seven gutm embrane equivalents (Fig . 1), all incubations were fo llowedfor 60 mm using membranes from seven guts. The resultsare expressed as specific b inding per gut rather than peram ount of pro tein , because after the blood meal free am ino

acids and peptides released from the blood meal m ake quan-titation difficu lt, e.g., a membrane preparation of sugar-fedfem ale has I ig pro tein pe r gut, whereas a m embrane prepa-ration at 24 h after b lood digestion has 4 ig protein per gut.W hen gut m embranes were prepared 24 h and 72 h after theblood m eal and analyzed for specific b inding of TM OF bythe m ethod of Scatchard (16), tw o classes of binding sitesw ere found (Fig. 5 and F ig. 6). The apparent high affin ityconstants (Kdj ) for m embrane preparations at 24 and 72 hw ere sim ilar (5 .7 x 10 M and 3.6 x 10 M , respectively) .The apparent low affin ity constants (Kd2) for m em branepreparations at 24 and 72 h w ere 2 .2 x 106 M an d6.2 x 10-6 M , respectively (F ig. 5 and F ig. 6). B ecause thebinding constants at 24 h and 72 h w ere sim ilar, we ex-pressed the results as the average SE M (n = 6) of threedeterm inations at 24 h and three determ inations at 72 h.The apparent average high-affinity dissociation constant(Kdl) is 4 .6 0.7 x 10 M and the association constant(Kassec) is on the order of 2 .2 x 106 M , which is 10-fo ldhigher than for the com plementary peptide, FOM T (Fig . 4).The m aximum amount of TM OF bound per gut membranepreparation (Bmax) to the high affin ity receptor is 0.1pmol/gut which is equivalent to 6.3 x 1010 binding sites pergut. The average low affin ity dissociation and associationconstants w ere 10-fold lower (Kd2 4.43 I x 10-6 M an dKassoc = 2.3 x 10 M-1). The maxim um amount of TM OFbound per low affin ity sites (Bmas) was calculated as 0.2pm ol/gut, w hich is the equivalent of 1 .1 x 10 binding sites.Because the to tal amount of TMOF in one pair of ovaries ofa fem ale m osquito is about 112 pmol or 117 ng (12), there isa sufficient am ount of hormone in one female to bind boththe high and low affin ity sites when the biosynthesis of tryp-sin is rap idly term inated 24 to 48 h after the blood m eal.H igh and low affin ity binding sites of insect hormones havebeen reported for the juvenile horm one receptors of the cock-roach Leuc o ph a ea mad er ae and for the ecdystero id receptors offemale D ro so ph il a m el an og as te r. A 10-fo ld difference betweenthe high and low affin ity constants w as observed (18, 19).

A molecular m odel of the com plem entary peptide su-perim posed onto TM OF was developed using SYBYLmolecular m odeling softw are version 5.2 (T ripos A ssociatesInc., S t. Louis, M o.). The sim ilitaries between the TMOFlow affin ity binding constants for the gut receptors and theTM OF binding constants for FOM T suggested that a com -parison of the two peptides m ight y ield additional structuralrelationships. M olecular m odels for the tw o peptides were in-itially constructed independent of one another. S im pleenergy m inim izations for each peptide resulted in a helicalstrucutre for the COOH -term inal polyammno acid tail ofeach compound. NM R studies have recently confirm ed thatthe range of so lution conformations for TMOF are dom i-nated by this feature (5 , 6 , 20). Use of the F it command inSYBYL aligns the superim posed structures to disp lay thesim ilarities (Fig . 7). The negative charge of the Asp2 sidechain on TMOF and the positive charge of the A rg4 sidechain on FOMT are in sim ilar positions in the two struc-tures. That predicts one logical site among many for the tw opeptides to in teract. H owever, com puter modeling alonecannot prove the conform ation of TM OF in the gut receptoror where is is bound to FOMT. However, the prediction ofsuch sim ilar s tructures by simple computational chem istrym ight account for som e aspects of the in teraction of TMOFw ith its com plem entary peptide, and that prediction cer-tain ly can be used to initiate a structural hypothesis to p laninvestigations of the TMOF-FOMT complex. Sim ilar com -parative com putational models have been used to pred icteffective inhibito rs of cysteine and serine pro teases from th e


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FOMT was preincubated w ith TM OF (results not shown).The specificity of the binding to certain regions on them osquito gut w as demonstrated when three consecutive gutsections (7 jm in thickness) w ere cut, fixed , and incubatedw ith TMOF. Only the m iddle gu t section bound Ti OF(Fig. 8b ) whereas the other sections apparently did not haveTM OF receptors and no staining was observed (F ig. 8a, c).Sim ilar results were observed when guts w ere removed 24and 48 h after the blood m eal and analyzed by immunocyto-chem istry (resu lts not shown). W hen whole mosquitoes weresectioned longitudinally and the sections w ere incubatedw ith TM OF and stained using the immunocytochem icalm ethod, the horm one bound only to the m idgut and to thefo llicu lar epithelium 48 h and 72 h after the blood m eal.P revious studies had shown that the follicu lar epitheliumsynthesizes an d stores the hormone (8). Thus, we concludethat TMOF specific binding sites are located on themosquito m idgut, and upon bind ing of the hormone to itsrecep tor, a signal is transm itted that initiates the term inationof trypsin b iosynthesis in the m idgut.

This work was partially supported by the Lady Davis Foundationv isiting professorsh ip award to D . B ., by FDACS contract 1415 toD . B ., and by PHS gran ts DK 38024 and NS 29719 toJ. E . B . Thispaper is part of University of F lorida-IFAS Experim ental S tationJournal Series No. R -03448 .

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c hem . P h ys io L 1 7, 1 15 -1 272. Briegel, H. , and Lea, 0 . A . (1975) R elationship be tween prote in andproteo lytic activity in the m idgut of m osquitoes. j I ns e ct P h ys io l. 21 ,1597-16043. B orov sk y, D ., and S chle in , Y . (1988 ) Q uantita tiv e dete rm ination of t ryp-s inlik e an d c hym otr yp sin lik e e nz ym e s i n i ns ec ts . Arch. I ns ec t B io ch em .Physiol. 8 , 2 49 -2 604. Borovsky, D. (1985) Iso la tion and cha racterization of h igh ly purif iedmosqui to oostat ic hormone. A rc h. I ns ec t B io ch en s. P hy sz ol . 2 , 3 33 -3 495. B orov sk y, D ., C arlson, D . A ., G riffin , P . R ., S ha banow itz , J. , a nd H un t,D . F. (1990) M os qu ito o os ta tic fa cto r: a n ov el d ec ap ep tid e m o du la tin gtrypsin -like enzym e biosynthesis in t he m id g ut . FA S E B J 4, 3015-30206. Borovsky , D ., C arls on , D . A ., G riffin , P . R ., S ha ba now itz , J. , a nd H un t,D . F. (1993) Mass spectrometry a nd cha rac teriza tion of Aedes aegyptitrypsin m odulating O ostatic facto r (T MO F) an d i ts a n al og s . Insect B i o-che,n. Mo le c. B io l. 2 3, 7 03 -7 12

7. B oro vs ky , D ., C arls on , D . A ., G riffin , P . R . , S ha ba no witz , J. , a nd H un t,D . F. ( 1 993 ) Sequen c in g an d characterization of t ry ps in mo d ula ti ngo os ta tic fa cto r. In Ho st R egu la te d Dev el opment al M ech an ism s in V ecto r A rih ro-p od s, P ro ce ed in gs o f t he T hir d S ym po si um , Vero Beach , Florida (Borovsky,D. , an d Spielman, A ., eds) pp. 36-47, U niv ersity of F lorida-IF AS ,F lo rid a M ed ic al E nto mo lo gy L ab ora to ry , V ero B ea ch8. B orov sk y, D ., S ong, Q.,Ma, M ., and C arlso n, D . A . (1993) B io8y nthe-sis, secretion and imm unocytochem istry of trypsin modulating oostaticfactor of A ed es a eg yp ti. A rc h. I ns ec t B io ch em . P hy sio l. (I n p re ss )9. Bost, K . L., Sm ith , E . M ., an d Blalock, J. E . (1985) S im ilarity betw eenthe corticotrop in (ACTH) recepto r and a peptide encoded by an RNAthat is com plem entary to ACTH m RNA. Pr oc. Na il . A ca# {1 2 8}d. US A 82 ,1372-137510. Jarpe, M . A ., and B la lock, J. E . (1 99 3) C om ple me nta ry p ep tid es : a pp li-cat ions of the m olecular recogn ition theory to peptide and prote inpurif ication an d design. In P e pt id es : D e si gn , S ynt he s is , a n d B i ol og ic a l A c ti vi ty(B us ava, C ., and A nanthram aia h, G . M ., eds ) B irk h#{228}user,oston (Inpress)1 1. B la lo c k, J. E ., and Sm ith, E . M . (1984) Hydropath ic anti-complementar i ly of a mino acids base d on th e g en e tic c o de . B io ch em . B lo -p hy s. R e s. Commun . 1 21 , 2 0 3- 20 712. B orovsky, D ., Pow ell, C . A ., and C arlson, D . A . (1992) D evelopm ent ofspecific RI A and ELISA to study t rypsin m odulating oo static factor inmosqui toes. A rc h. I ns ec t B io ch em . P hy sio f 21 , 13-2113. S te rnbe rger, L . A . (1979) Immunocytochemistry. W iley M edica l P ublic a-tion, New York14. Vandesand; P . (1983) Immunocytochemical double sta in ing tech-n iq ue s. In Immunocytochemistry (Cuello, A . C ., ed) pp. 257-272, Wiley ,N ew York15. C lem ents , A . N . (1992 ) Th e B io log y of Mosquitoes. Chapman & Hall , Lon-don, 509 pp.16. S catc hard, G . (19 49) T he attra ctions of prote ins fo r s mall m olec ules andions. A n n. N Y A ca d. S ci. 31 , 660-67217. Bo rovsky, D ., Thom as, B . R., C arlson, D . A ., W hisenton, L . R. , an dFuchs, M . S. (1985) Juvenile hormone and 2 0-h yd ro xy ec dy so ne a sprimary a nd s ec on da ry s tim uli o f v ite ilo ge ne sis in Aedes aegypti. A rc h. In -sect B i och em . Ph ys io l. 2 , 75-9018. E ngelm ann , F ., M ala, J. , and To be, S . S . (1 987) C yt os ol ic a nd n uc le arr ec ep to rs fo r ju ve nile h or mo ne in fa t bodies of L euco ph a ea ma d er ae . InsectBiochcn. 17 , 1045-105219 . Handler, A. M ., and Maroy, P . (1989) E cd ys te ro id r ec ep to rs inDrosophila melanogaster a du lt fe m ale s. M ol. C ell. Endocrinol . 63 , 103-109

20 . Curto, E. V ., Jarpe, M . A ., B la loc k, J. E ., B orov sk y, D ., a nd K rish na,N . R . (1 99 3) S olu tio n s tru ctu re of tryps in m od ulating oos tatic fac tor isl ef t- ha nd ed h e lix . B ioc hem . B io ph ys . R es. C om mun . 193, 688-69321. R ing, C . S ., Sun, E ., M cKerrow , J. H. , L ee, C . K. , R os en th al, P . J. ,

Kuntz, I. D ., and C ohen, F . E . (1993) S truc ture-b ased inh ib itor des ignby using pro te in m odels for the developm ent of antiparasitic agents .Pr oc. N aIl. Acad. Sci. U SA 9 0, 3 58 3-3 58 7

Rece ived fo r publication O ctober 14, 1993.Accep te d f or p u bl ic a ti on November 30, 1993

ERRATUMThe authors w ish to correct a paragraph that appeared in aresearch communications article on page 115 in the January1994 issue of The FASEB Journal [Chinsky, J. M., Bohlen ,L. M ., and Costeas, P . A . (1994) Noncoordinated responsesof branched-chain a-ketoacid dehydrogenase subunit genesto dietary pro tein . FASEB j 8 , 114-120]. Changes are inboldface.

METHODSAnimalsA ll m ic e [C 57 5L /6 J, ob/ob a nd le an (oh/i., +1+) litte rm ate sl w ere obtainedfrom the Jackson Laboratory , Ba r H arbor, M e. The diets used w ere ob -ta ined from Purina Tests D iets , R ichm ond, Ind., a nd in clu de d standardLaboratory R odent D iet (#5001) c onta in ing 23.4% protein, 5.5% fat, and49.0% carbohydrate; Prote in F ree Purified D iet (#5765C ) conta in ing 0%

pro te in , 10.0% fa t, and 81.70% carbohydrate; and 50% ProteinPurifiedD iet (#5786C ) conta in ing 50.5% prote in , 13.50 % f at , a nd 2 3. 35% ca rb o hy -d ra te . T he la tte r tw o diets are iso caloric, provid ing 4.17 Kcal/g of calcu-la te d digestib le energy (phys io log ic fue l v alue). Standard lab orato ry ro -dent d ie t has a potentia l energy content (gross ene rgy) of 4.0 Kcal/g bu tprovides on ly 3.3 Kcal/g according to its ca lcu la ted physio log ic fue lvalue, as dete rm ined by th e manufacturer. A ll t hr ee d ie ts c on ta in similarm inera l and fiber content. The m ice w ere fed these diets an d water on anad lib itum basis. In general, no significant differences in the volumes of foodor water consumed were noted in the groups o f m ic e fed t he se t hr e e d if fe r en tis oc alo ric d ie ts . Experimental groups, conta in ing at least four m ice pe rg ro up , w er e a ge -m a tc he d, s ex -m a tc he d, an d when po ss ib le , l it te r -ma t ch ed.A ll experim ents w ere repeate d at least tw ice w ith d ifferent groups of m ic e.O nly fem ale m ice were used because their weight gain du ring th is age(6.5-8.5 w eek s) is m in im al com pared to m ale m ice an d s tandard w eight ga inc urves av ailab le from the J ac kso n Laboratories . R esu lts are show n fo r thoseex perim enta l grou ps of m ic e w ho cons um ed e quiv alent vo lum es o f food anda s a g ro up d em on stra te d similar weight changes during the experimentalpe riods (1 -2 weeks). The weight changes observed in C57BL/6J m ice ons tandard laboratory cho w (23 % prote in) d uring the expe rim enta l periods(6 .5-8.5 w eek s of age) averaged 3.97% ( 2.25 for 3 expe rim ents invo lvingn = 1 2 m i ce ) pe r w ee k, s im ila r to th os e re po rte d by th e su pp lie r, th e JacksonLaboratories.3

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