Change in phenoloxidase and its precursor during silkworm (a80

」． Fac． Agr．， Kynshu Univ．， 45 （2）， 487一一493 （2eOl）
Change in phenoloxidase and its prec皿sor d曲g s皿㎞orm
（a80 strain） development
Kollji Yamamotoi・‘）， Hiroshi Fr“’i’2）， Takahiro Kusakabe3）， Katsurni Koga3），
Yoichi A304），｛md Masats㎜e lshig皿o＊4）
i） The University of Texas Healtth Center at ’13iler， 11937 ww 271 Tyler TX 75708， U． S． A．
2） Laboratory of Silkworm Genetics， 3） Laboratory of Sericultural Science， and
4）Laboratory of Protein Che如stry and Eng加eerj皿g， Graduate School of
Biorescurce and BioenvironmeAtal Sciences， Kyushu Uulversity，
6−lg−1 Hakozaki， Higashi−ku， ｝ikikuoka 812−8581， japan．
（Rθcei警θd o蕊。わθゲ 3iン20θo『ζ善鴛ζオesc6eg）ted N（）y8m∼｝酬jO，200θ）
The ehaxxges iR pheBoloxidase and its pyecursor， so−cal｝ed propheno｝oxidase， of the
silkworm， Bo励yx mori（a8◎stra加）were exam漉d鷺sing detect圭。照6say職h or wiもhouも
detergent to distinguish PO and pro一一PO in heiytolymph （￥araainoto， et ag．， 1999）． Little or Ro
phe鍛0玉◎XidaSe aCtiv董ty waS deteCおed溢helnOly難ph aも撫e S君kwOm≧deve玉◎P搬ent dur類g the
inal laival instar and pupal stage． On the other hand， prophenoloxtdase wa＄ detected for same
t㎞e丘ame． In males， after prophenolo）ddase血creaSed S蓑ghtly at day 4， it deαeaSed to a m血一
mum level at ＄pinning and increased to a maximum level on the day of pupation and decreased
graduaHy to day 5 after pupation． In females， prophenoloxidase decreased graduany to a m血i−
mum level on day of spinning artd a slight increase was observed at day 8 and it ，increased to
maxtrnum level between day 2 and 3 after pupation and decreased thereafter． Prophenoloxidase
mRNA expression was preceded a day or two before， when cgmparing the change of phe−
noloxidase mRNA with its product．
INTRODUCTION
Phenoloxidase （PO： monophenol， dihydroxyphenylalaRkrie； oxygen oxidoreductase；
EC 1． 14． 18． 1） catalyzes two ＄equential reactions； the hydroxy｝ation ef monopheRol te
o−dlphenel and the oxidatioR of o−dipheRoke o−qtmelte （Ashida and Yamazaki， 1990）．
In insects，．PO is generally believed to be crucia｝ for the cuticular melanization and sele−
rotization（S6derhaU，1982；Hir㎜a and Rid曲rd，1988；Sug㎜aranθ弼，，1992）．
The existence of a precuesor protein，．called prophenoloxidase （pro−PO）， was
reported from many insects： e．g．， the fruit fly DTosophila melanogaster （Fujtmoto et al．，’
1993）， the sillrvvorm Bombygv mori （Yasuhara， et al．， 1995； Yamamoto， et al．， 1999）， the
tabacco homworm Mesndzeca sexta （Hall， et al．， 1995）， the cockroach BtabeTzes dis−
oo掘α鰯3（Durra蕊， et a9．，1993），もhe wax m◎th Ga99eriαme990ne99α（K◎pacek，θ彦α1．，
1995）， aRd a colecpteraR iRsect ffogetrichin diomphggta （KwoR， et ffl．， 1997）． The
activatiek of pro−PO h3ve beeR showi｝ by tke propheRglgxtdase−activating factcr in vitre
（Lee， et ag．， 1998； JiaRg， et esl．， 1998； Satoh， et ag．， 1999） aRd by organie cempoands snch
as sbdiura dodecy｝ sulfate （Funatsu aRd IRaba， 1962； IRaba and ｝F’llkatsu， 1964），
ceもy玉pyri（泣薮疑m d瞭◎ri（ie（Ha11， eS al。，1995），2−propariol（Asada，θ孟編．，1993）a戯d（㎞eもyL
benzy㎞罪is融m：m◎船囲． c撮◎r圭d￠（DBMA）αa搬a搬ot◎， et a9．，ユ999）． Fuもhe］㎜◎re，もhe
＊ Corresponding author． FAX number： 092−642−3051， E−mail： ishiguro＠agr．kymshu−u．ac．jp．
487
488
K Yamamoto et al．
cDNA ekcodiRg pro−PO have beeA clolled aRd sequenced recently frera the raosquite
Amaigeres szebalbatzes （Cho， et al．， 1998）， the malaria vector Anophetes gambiae （Jiang，
etα乙．，1997），乞he油日webw◎r搬吻猶α窺短αo％暢θα，（Pa意， etαs9、，1997），もhe c◎1e◎pteran
insect Tenebrio molitor （Lee， et aZ．， 1999）， the frult fiy D． melanogaster （Fujimoto， et
ae．， 1995）， the tabacco hornworm M． sexta （HaR， et al．， 1995）， and the silkworm B． mowi
（Kawaめ厩a，θ孟α霧。，1995；Ya難alm（）t◎婁θ脇ε．，2◎◎◎）．
Although irtaRy knowleges about pro−PO have been accumniated as described above，
the blgchemical aRd geketicai aspeets ef the regulation ef PO activity during deve｝opmekt
is st皿poorly understood． In house且y，」VzLscα（Zomesticα， the change of PO activity was
examiRed from larvae・to pupae （Fimatsu and IRaba， 1962）． The authers feuxd that the
noticeable change in PO activity occurred at the stage（）f pupati◎n． A㎞ost the highe就
PO actwity iR the homogenate of the fiRal instar larvae disappeared suddeniy iR the
homcgenate of the prepupae and thell it is appeared again in the homegenate ef aged
pupae． However， the disappearErnce of PO activity in the prepupae did not imply that the
e轟zy蟄e v畿賊shed becauseもhe h（）m（）ge蓑aもe〈）fもhe prep糠pae exhibiもed PO acも鯖ty up◎簑
the addition of an anionic detergent such as sodium oleic acid or an extract血om the aged
pupae． lt’meaRs ・that pheRoloxidase is present as inactive form in prepupae aRd there is
natural ac伽ator， which converts inactive fbrm． to active form， in 3ged pupae．
The伽ctuaもi◎n of PO activ靭d．顧蓑g meもam◎rph◎sis 1嚢the s三三kworm has頚ever
investigated． IR erder to get a better understaRdmg of the regulatory events inve｝ved in
the metarnorphosis of the silkworm， we analyzed the change in PO activity and pro−PO
during developmeRtal st3ges． The compariso1i cf these fiuetuations with that for pre−PO
mRNA was aiso included．
MATERIALS and METHODS
夏山sects a灘d preparatio難of蓋e受理0璽y斑ph a無d hemo￠ytes
The a80 strain of B， moTi was reared as described previously （Yamamoto， et al．，
i999）．・ Females aRd ma｝es were distiRguished by the irrtaginal bud oR the abdeiniRa｝
surface． The hemolymph specimens collected into liquid nitrogen， were lyophilized and
the p◎wder◎bもa圭Red was used fbr癒e deもer漁ati◎R◎fも◎認PO ac慧嘘y。
Measurement of PO activity in the crude extract
PO ac糠v並y w’as・m年asured spectr〈）phoも◎搬etrically厩th or witho登もac翫ti◎Ric
detergent， DBMA， as an activator （］Yamamoto， et al．， 1999）． The lyophilized hemolymph
（5 mg） frgm various days was dissolved in 1 ml of I O ；r［M pctassium phosphate buffer （pH
7．0）． Fifty 2Ll of this mmure was added to the reactioft mixture （1 mi） containing O．es M
potasslum phosphate buffer （pH 6．5）， 5 mM L−3一（3，4−dihydroxyphenyl） alanine （ydopa）
and with 6r witheut O．4mM DBMA． While incubating at 3e aC for O．5min， the absorbaAce
at 475 nm was monitored with a Hitachi U−3210 spectrophotometer （Tokyo， Japan）． One
uRit of PO was defined as the araggRt of active PO capable ef predueikg i psmole of
dopachrome per Min．
Measurement of pro−PO mRNA titer
Northern hybridization was done according to previous paper （Yamamoto， et al．，
Chgnge in ph（？？zoloxUtase activ吻（）f3igkwoma
489
2000）． Level ef pro−PO mRNA was assessed by scanRing of the electrophoregram by
dexxsitoraetry gsing NIH twage sgftware akd replo￡ted．
RESULTS and DISCUSSION
C数a灘ge i盤PO aRd prO−rPO at deve豆OP斑￠鍛ta監stages
The fuctuatioft of Pe activity during metamorphesis in the silkworm has never inves−
tigated． Therefore， we measured the fiuctuation of PO or pro−PO and we made better use
of detection assay with or without DBMA to disttnguish PO．and pro−PO血hemolymph．
By using this method， little or Ro PO was detected， even if the metaraorphosis frora
｝arva to pllpar was eccurred （Fig． 1）． IR housefiy， hewever， the eRdggeReeits PO aetwity
was detected in the homogenate of the final instar larvae， and then it was odsappeared
suddenly血the homogenate of the prepupae（Funatsu and Inaba，1962）． The presence of
active PO in herRolymph is thought to be harmfUl to the insects health aRd it is Reccesary
to activate it duriRg a Reed． ［1］heyefore， insects Reed te coRtro｝ active PO． Receptly，
endogeneous PO inhibiters were identified in the housefiy， M． aemestiea （Daqtmag， et
al．， 1995） and in the tabacco hornworm， M． ＄exta （Sugumaran and Nellaiappan， 2000）
a嚢dbo撫were sh◎wnも。盛bits（i薩recも1y the ac慧viもy of PO。 The PO姫biもor◎f h◎use盤y
co濾d i磁biもPO acも紘y◎f s江kworm Crama瓢伽，θ脇ゑ，20◎◎，膿pubhshed results）．丁海s
fact suggests that there might be the strong㎞漁bitor in the s皿kworm that involves in the
regulation system of PO activity， because actjve PO cannot be detected．
When the ’changes in pro−PO was determined in hemolymph at developmental stages
frem fifth larva｝ instar to pupa， pro−PO was detected for thirteeR days from larva to pupa
（Kg． 1）． IR males， after pro−PO increased stightly at day 4， it decrea＄ed to a rmum
level （1．39×10−3 unit／mg lyophilized hemolymph） at spirming （day 6） and increased to a
maximum level （7．54×10−3 unit／mg lyophilized hernolymph） on the day of pupation and
deereased gradually to day 5 after．pupation． IR feraa｝es， pro−PO decreased gradually to a
無論m慧搬level◎R day（）f spiRRiRg（2．41×ヱぴ3 uRiも／難91y◎phi蓋zed hemdy斑ph）and a
slight increase was observed at day 8 and it increased to maximum level （9．80−9．74×10−3
unit／mg lyophilized hemolymph） between day 2 and 3 after pupation and decreased
thereafter． Pro−PO is always preseRt in this perlod， whereas Ro active PO was detected．
As ＄hown in Fig． 1， the large ameunt of pro−PO is stored during a few days after pupation．
It is postulated that pro−PO in hemolymph was thought to be stored in case of emer−
gency． Xiao−Qiang et al． demonstrated that after inj ection of bacteria， novel lectin mRNA
apPeared and lead to sthaulate the activ就ion of pr◎一PO（Yu， etαZ．，1999）．
There is a pessibi｝ity that sgme chymetryp＄in iRhibitors had the ability to inhibit
pro−PO activating faetor of the same species・（Aso， et at．， 1994）． lt was reported some
inhibitors found from the locust Locustα migγ伽riα（Brehe㎞， et al．，1991）and M． se漁
（Saul， et al．， 1986） could control pro−PO activation． Pro−PO is always present from larva
to pupa， whereas no PO activity is detected （Fig． 1）． ［1］his result iRdicates that chy−
raotrypsin lll｝rgbitor is lil〈ely to coRtro｝ prc−PO activaticf｛．
We have recently investigated the developmental changes of mRNA titer in the
silkworm， B moTi （Yamamoto， et al．， 2000）． The strong signal of rRRNA was detected on
d翫y30f the f蟄；h蟄sもar a蓑d◎嚢e（iay be致）re p慧pa雛◎難鎗Ptales， w圭聖ereas f6males exhibited
those on day 4 of the fifth instar and oR the day ef pupatieR． Smi｝ar resiilts weye obtained
K， Yamamoto et al．
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F圭9。LDeve韮・P搬e貧もa璽。蓋a捻ges細・◎一PO c幡e鯉va三uated Wi嶽PO
acも重v重毛y aτ芝く匪Pro−PO搬RNA
PO activity of erude extract was measured after activation
with DBMA as described in Materials and Methods． Closed cir−
cle＄ represent the a＄say conducted in the presense of DBMA．
Open circles represent the assay conducted in the absense of
DBMA． Closed squares represent the level of mRNA． S and P
即rese勲乞spi麟鍛g a轟d p脳も織r￠sρec乞ively・Baぞs紬ca乞e
the staRdard deviatiofi （n＝3）．
Chα％gθ￠銘 phe％oto謬乞（オα3θ㏄麗砂客惣（∼プ8ilkworm
491
when the pro−PO concentrations were measured by enzyme−linked immunosorbent
assay．
It is interestirtg that the fiuctuatioR of pre−PO 一mRNA of the silkworm was 〈Mfferent
frem that of the faR webworm， H． c2snea （Park， et at．， 1997）． They deraomstrated that the
highest level of pre−PO iriRNA was detected in the inid−instay larvae aRd there was Rg
sigRa1蟄pupae， adu駕a嚢d eg9． There is a｝S◎もhe（圭廷fく）rence beもweenもhe f隻UCtuati◎R ofもhe
silkwomi PO aRd that of heusefiy PO （Daqtmag， et al．， 1995）． in the housefiy， M aome＄一
tica， by a Western blot detectien method using polyclomal’ antibodies raised agaiRst
housefiy PO， no significant change in PO protein content wa＄ observed in the
homogenates of the pharate adult compared with that of the homogenates of other stages
of aged pupae． They suggested that phenoloxidase inhibitor functions as a regulator of
the necessary ｝eve｝ of PO．
As shgwn in Fig． 1， pre−PO i￥［RNA expressieR was preceded a day or twe before，
whek cevapariRg the chakge cf pheRelcxidase i￥｝RNA with its preduct． This imdiRg
suggests that the expre＄sieR of pre−PO ce￡｛｝d be ceRtroged at traRscriptienal step． insect
deve｝epment is kRown to be regulated by ecdystereids and juvemie hormokes （」｝1）．
Po＄sible roles of these hermoRes in the ccntrol ef pro−PO ’expressioR during development
w皿be studied血deta黛． The effects of J：H on the synthesis of PO were reported． The
granular PO・that is responsible for cuticular melanization in M． seafta larva was controlled
by JH （Hiruma， K． and Riddiford， L． M．， 1984， 1985 and 1988）． Recently， the transcription
of PO in A． gambiae was regniated by 20−hydroxyecdysone （Martin， et al．， 1999）． lt is
alse possib｝e for PO ＄ymthesis in s“kworm to be coRtrolled by 2e−hydroxyecdyseRe at
traftscript重◎薙至evel． Takξ｝R t◎geもher，もhere areもhree p◎ssib粗品ieS（）fもhe feg譲at圭◎R瓢ech−
a通s瓢of PO acもivity，廊aもis，綬a繋scripti◎聾al cokも翌◎1， act重7ati◎R coRtr（）｝a難d the i血ibiも沁蓑
◎facもive PO、 In， f就uどe rese鼠rch， iもis㎞p（）r捻蕊f◎r us to lo（）k fbr PO辻戯ibiもor a嚢d we厩経
endeaver to uaderstaRd how this hermeRe regniates the expression of PO．
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