Purification And Characterization Of A Protein Associated With Genetically-determined Longevity Differece In Drosophilq Melanogaster

Her`滋 ′ αsゴ ′ア_15ノ ー258`ノ 992)

PurincatiOh and characterization of a protein associated with genetically口 deterllllined longevity difFerence ln ″ D″ θsθ ′力liJa“ ιJα οgα s′ ι

“

AKIRA OKAN01,ISAMU YONEMURA2,YOSHIHARU SHIMIZU3, YASUNORI YANAGIDAIRA4,HAYATO HASEKURA2 and BARRY BOETTCHER5 12)9,α

rrzη θ ″′

Me務

2Dυ`772θ α″

,Mα

`“ `″

」￠′ακ

ιう οノ グ Bノ οε力θ777お ′ r″ ′ θ り ,771s′ ′ 7710`0,ヨ フθJψ α″

`s″ ぽ ιttα ′″

Jψ α77 3D9,α ″777θ ″ Ifθ

`√

εЙ

`,Lり

ο

f/t‐

cα ″ ″ουαSε ン/rrr Dお θαs′ ,だヌ カあ り s力 ν び′ わ′ぉノ rl Sι 力οο′て ノ

α助 ルα 磁 わ′rs′ ヶ

SC力 οο ′げ

rブ

`グ

4Dψ α″ ′′ ″

`″

`√

P夕 ろ′ た 〃θ α′ ,S`力 οο′″ ′θ″ε777θ ,及 ,た αJこ ルわ `力

`rs′

″

`′

ιレ ,3ン ″り ο,動 り ο ,

rl,3ο s`′ αα′ ,お

勲υ ′ ′ ο ″ ″′ ″ ノP/111s′ ο あ″,ル s′ ル ′ ″ο υ α S`ク ルr θグ Cα ″

`力

Dノ s′ α s′ ,`'カ

α″ α,κ α″

lllα

`=α

,259-ノ ′

ノ ″s力 trび ″ノ υ′rs′ rl

Sι /7ο ο ノ

`グ `α “ ,コ “ ′α οrο ,39θ J″ α″ グ ルセ″ `777′ `s″ “ 5 Dψ α7r“ ′″′げ B′ ο′ οgた αノS`た ″ε′s,7/P′ び″tl′ ぉJヶ て αルs,2Jθ θ Иνs″ α′ カ √ A● ″εαSrた ,Ⅳ ιll Sο ″Й Иイ

OKANO,A,YoNEMURA,I,SHIMIzu,Y,YANACIDAIRA,Y,HASEKURA,H and BoETTCHER,B 1992 Purincation and charactcrization of a protcin associatcd with genctically― Drο sο′/2ブ ル 717`力 ″ Ogα s′ ′ ″――Herθ ′ ∵ ′ αs yゴ 7:251-258 Lund,Swcdcn ISSN

dctcrmincd longcvity difFcrcncc in

0018‐ 0661 Rcccivcd January 30,

1992 Accepted July 3, 1992

Earlier studics havc shown a cOrrclatiOn bctwccn thc prcsence of a 77 kDa protein in the proteins extractcd

iom yOung adult D″ ο,0ゅ みブ gα s″ r(D″ )and thc autosomallongevity allclc ル 771′ 滋″ο

И2 at thc J″ ′ 10cus

ln this study, a77 kDa protcin has bccn isolatcd from pupac of D 772 0f a long livcd strain of genotypc

/2/2,and was purined by DEAE chromatography,ConA column chrOmatography,and two cyclcs of gcl nltration The purincd protcin has a molccular wcight of 76,600(by SDS‐

PAGE),an iSOCICctric point of pH

65,and molar cxtinction cocmcicnt A(1絡 )=18 3 1t is a glycoprotcin containing 3 3%hcxosc Supplc menting the foOd of D 777 With thc purincd protcin at 5 x 10 4

μg/ml,beginning at day 5 aftcr cmcrgcncc,

rcsultcd in an incrcase in the survival ratc and maximal lifc span Of bOth short―

livcd and long― lived strains

Of D 777

おα 777″ b716771ν rα ,D″ α r`777θ ″ ′げ ι α′″ι ″ε ttσ ,動 /cl οルαS力 清αυ ′′ υ ′ ″ ざ り γ″ s力 ″α ″ ,Bッ ″ りο,動 り ο,″ ψα “ `洗

Sc力 ο ο ′〆

ν

`滋

σ ″a,′ タイ5

is undcr polygcnic control, thc lifc span of

or longcvity potcntial,bcforc cmcrgcncc(YoNE‐ MURA Ct a1 1991a)ThC ncxt stcp was thc inding

D″ οsρ ρカメ あ

of a corrclation bct、 ′ een thc cxtcndcd prcscncc of a

Contrary to thc carly gcncral conccpt that lifc span 777′

ル″Ogα s`ε ″(D771)haS bCCn shown to

bc grcatly innucnccd by a fcw maJor genes(YoNE ccntly,thc modc ofinhcritancc and the function of

77 kDa protcin in cxtracts from young adult iD 727 and thc prcscncc of thc autosoma1 longevity allclc И2at thc 3mИ locus lt has been concludcd that this

thc nl■ or gcnCS(J711 gcncs)haVC bCcn dcscribcd in

protcin is involvcd in thc intra― strain dittcrcncc of

, havc bccn ablc to bc undcrtakcn from an cntircly ncw aspcct

lifc span in D″

(YoNEMURA ct al 1989,1990,1991a and b)

cation of thc 77 kDa protcin and somc of its

Thc basis for thc ncw way ofstudying thc control Of lifcspan in D″ , is thc nnding that thc J42 gcncs in■ ucncc dcvclopmcntal spccd in thc larval stagcs of D″ 7, 、 vith long― livcd ■ies emcrging carlicr than short― livcd nics,and adult lifespan bcing ablc to bc

chcnlical and bi01ogical charactcristics

MURA ct al 1986;LucKINBILL Ct al 1988)Re―

dctail,and thc studics on lifcspan of D″

prcdictcd at thc tilnc of cmergence Thcsc rcsults suggcst that thc function of thc J770 genes is to scgrcgatc thc gcnctically dctcrnlincd adult lifc span,

(YoNEMURA Ct al 1992)

Thc prcscnt rcpOrt dcscribcs dctails of thc purin‐

NIlatcrials and mcthods

D“ ′″″οgα srar Two inbrcd strains of D 771 WCrC uscd,as in the prcvious rcports: a short‐ livcd strain (S Strain)

252

A OKANO ET AL

〃θ″′ α s′ /7`′ 992) rθ

cstablishcd through sib‐ nlating rcpcatcd for morc

than 20 gcncrations from a wild typc stock,and a

was kcpt rccycling ovcrnight to assurc colmplctc adsorption

long-livcd strain (L strain)silllilarly cstablishcd

Thc column was clutcd irst with 100111l of

through morc than 13 gcncrations fronl thc Orc‐ gon R strain Lifc spans of thcse strains havc bccn

buffcr D and thcn with 50 1121 of buffcr E(buffCr D plus 02M α‐mcthyl― D‐ mannoddc)ThC SCcond

sho、 vn

cluatc(Fraction E,FrE)was dialyscd against PBS

to bc dctcrmincd by nuclcar longcvity gcncs ´

at thc SmИ and J727Й 10Ci,and an cxtranuclear gcne

at thc J“ E

locus(YoNEMURA ct al 1989,1990, 1991b)Thc nics wcrc brcd at 27° C and wcre

providcd with cookcd food containing 8シ 6 dry ycast, 5シ 6 sugar, 1シ 6 agar, 、 vith prcscrvativcs of

05シ 6

n‐ butyl p‐

hydroxybcnzoatc and 0 5ツ 6

pro―

pionic acid

PM-10 membrane to 3 ml G′ ′ メ ο″ ′/r″ α′

――Thc conccntratcd FrE was applied

to a Scphadcx G-200 column(1.5× 98 cm)pre‐ cqulibratcd with PBS, and was elutcd with thc

samc bufFcr(PBS)Absorbancc at 278 nm was mcasurcd for cvcry 3 m1/tubC Of thc cluatc. Tubcs numbcr 28-36, 、 vhich includcd thc pcak

P“ ″ ′εαrブ οκ ぽ

r/2′

of optical dcnsity, wcrc harvcstcd for thc nnal

77た Dα ′″ οたlll

All proccdurcs 、 vcrC pcrformcd at 4CC P″

(pH 78)and waS COnccntratcd on an Amicon

αrα ″0″

θθ χ α

Q′

―-23 5 g of L strain

`r7/α `′ `″ `ρ pupac,gcnotypc И2A2 obtaincd soon aftcr pupa一 tion, wcrc homogcnizcd in icc‐ cold phosphatc bu∬ crcd salinc(PBS,pH 7.8)and thC hOmogcnatc

was cxtractcd ovcrnight

purincation, which was pcrformcd by rc― matography、 vith the samc column

chro―

s―

Dθ ′ ″αr′ ο′ ぽ ρ′ bο ″ 77・ α′ ′ ιr″ ノ ο ″s α″グ ια″

Protein was quantitated`′by thc incthod of Lo、

vRY

et al(1951)using bovine serum albunlin as thc

standard Carbohydrates wcrc mcasurcd by thc mcthod of DuBols Ct al(1951)with glucosc as thc

Thc homogcnatc was ccntrifugcd at 100,000g for 2 hours; thc supcrnatant(thc nrst crudc cx― tract,about 100 ml)was rCtaincd:thc scdimcnt was again cxtractcd with 100 1nl of PBS ovcrnight,and

ccntrifugcd, to yicld thc sccond cxtract(about

standard. Pο ケαε ― Sodium dO― り ″777ノ 滋 gイ リ カο″ `″ `″ `sお dccyl sulphatc polyacrylanlidc gcl clcctrophorcsis

(SDS― PAGE)was

pCrfOrmcd using a 10%slab gcl

100 1nl) Thc nrst and thc sccond crudc cxtracts

(10%SDS― PAGE)according to LAEMMLI(1970)

wcrc poolcd (PBS cXtract)and COnccntratcd to

Protcins in thc gcl

20 ml using Amicon PM-10 mcmbranc(W R ison,MA 0195,USA); Gracc Corp,AmicOn D

water,10ノ 10/80v≠ v/V)

thc conccntratcd samplc was thcn dialyscd against 50 mM tris― HCl,pH

DEИ E―

7ル ツ 0-グ ′ ″,′7PS′ 0″ αノ′″ιr″ 9′ 力ο″ ιsお

――Thc

mcthod of

O'FARRELL(1975)was uSCd fOr thc nrst dimcn―

″ ο α′ ンル θχ― slon of isoclcctric focussing(IEF)and thC SCCond ∝″ ψ リ グ r力 θ ε′ “ dimcnsion of SDS― PAGE Standard IEF markcrs vas applied to a Thc abovc dialysatc 、 wcrc obtaincd fl・ om Bio― Rad Laboratorics(Ca, Scphaccl(Pharmacia Fine Chemicals AB, 力αε ′ノ

`17ρ ′ ″ αε′――

DEAE‐

82(buttr A)

、 vcrc staincd 、 vith Coomassic bluc R-250(0005,も w/vin 2‐ propano1/aCCtic acid/

`力

Uppsala,Sweden)C01umn(16× 25 cm)whiCh had bccn prc‐

USA)

cquilibratcd with buttcr A Thc column

was washcd 、 vith 200 nll of buttcr ´、 , 200 1nl of bumcr B(450 mM Tris― HCl,pH 82),and thCn with 200 ml of buttr C(500 mM Tris― HCl,pH 82,containing 0 5 M NaCl)ThC fl・ actions clutcd by thc rcspcctivc bu∬ crs 、 vcrc tcrmcd Fraction A

(FrA), Fraction B(FrB)and Fraction C (FrC), rcspcctivcly FrC was dialyscd against bu∬ cr D(20 mM Tris― HCl,pH 72,containing 0 5 M NaCl)

И7711Й ノ s″ α

rブ

0″

οr′ ノ ′′ οD 7271 ぽ 77た Dα ′″

Thc purincd 77 kDa protcin and othcr prOtcins (FrA)wcrc SCrially dilutcd to makc four protcin conccntrations,20 μg/ml,2 μg/ml,02μ g‐ ml,and 0 02

μg/mlin 1/4 dilutc PBS(1/4 PBS,pH 72)

Thcsc prOtcin solutions wcrc stcrilizcd by 0 22

μm

Milliporc nltration and wcrc givcn to D 721 by adding 25 μl to onc llll of food Thc protcins(77

FrC, aftcr dialysis,was applicd to a Concanavalin A collJu‐

kDa protein or FrA)concCntrations wcrc thcn rcspcctivcly,05,005,0 005 or 0 0005 μg/ml ThC

gatcd Scpharose 4B (Pharmacia Finc Chcmicals AB,Uppsala,Swcdcn)column(12× 5cm)prC‐ cquilibratcd 、 vith bumcr D. Thc samplc solution

adult lifc span of thc S and thc L strains provided 、 vith thc dittcrcnt food lnixturcs、 vcrc obscrvcd and

Cο κ И Sep力 αrο s′

ε 力″ ο″α′ Ogr`ψ /71

-―

rccordcd

″′ ″″′ αs′ 77(,992)

A PROTEIN ASSOCIATED WITH LONGEVITY DIFFERENCE

As controls, two grOups wcrc cstablished: onc

1

was id ordinary food(contr01)and thC Other was fcd 25μ l of l′ 4 PBS addcd tO onc ml of thc ordinary food to providc thc samc PBS conccntra‐ tion as in thc cxpcrimcntal bod(1′ 4 PBS control)

D″ .werc kcpt at 27° C and werc givcn thcir respcctivc food, accOrding to thc grOup, from day 5 of cmcrgcncc Thc dcad wcrc countcd evcry day,

whcn old food was changed、 vith ncw; thc culturc

2

3

kDa 770 662 450

攣

勃 ￨■

.―

‐ ‐

Purincation and chemical characteristics of the 77 kI)a protein

Fig l shows thc 10シ 6 SDS― PAGE pattcrns of protcins extractcd from D 777′ ′ α72ο gα s′ θr pupac and in thc fractiOns obtained at cach stcp in thc purin―

cation of thc 77 kDa protcin Lanc 2 shou/s thc protcins in thc salinc cxtract frOm thc pupac Lancs 3-5 show thc pattcrns of thc protcins in thc

fractiOns Obtaincd from I)EAE chromatOgraphy. Thc 77 kDa protcin was not dctectcd in FrA(lanc

3); a nlinOr prOportion of it was fOund in FrB (lanc 4)among many othcr protcin、 it was a mttOr prOtein component of FrC(lanC 5),about 45シ 6

FrC was subnlittcd to COnA Scpharosc chro―

matOr辱車井→曇lding

FrIΣ ttanc 6),whiCh COn_ taincd a low cOnccntration Of thc 77 kDa protcin among othcr protcins,and FrE(lanc 7),conSiSting largcly of the 77 kDa protcin、 vith a small amount (about 5シ 6)of COntallaination,appcaring as bands of 3 1arger and 5 smallcr polypeptidcs Thc 77 kDa protcin in FrE、 vas further purined by gci nltration

with Scphadex G‐ 200 using PBS as thc cluant Aftcr two cyclcs of gcl■ ltration,the 77 kDa pro‐ tcin was isolatcd with abOut 98 9そ purity(lanc 8)

Thc molccular wcight Of thc prOtcin isolated from D.″, pupac was dctcrlllincd as 76.6kDa by

10%SDS―PAGE. Tablc l shows prOtcin yiclds at cach stcp of the

purincation Thc isoclectric point(IEP)ofthC 77 kDa protcin

摯 鸞

警鸞

257

172 123

暉

笏呵 隆覇嬢琴… 1

攀

・

‐

Rcsults

4

1 111■ ■ ″= ‐

tubc、vas rcnewcd cvcry、 veek Othcr details of thc mcthod of culturing nics 、 vcrc as in thc prcvious

rcport(YoNEMURA Ct al 1989)

253

■ .■ ■■ 攀

‐

_鰺

.

_

Fig. 1. SDS― PAGE protein pattcrns of thc fractions at cach step in thc purincation of the 77 kDa protcin from pupac Of the long lived strain of D″

,′

ノ α770gα s′ ′ ′

Lanc l: Molccular wcight standards: ovOtransferrin

ne scrum albumin(66 2 kDa),ovalbu_ min(45 0 kDa),chymOtrypsinogcn A(25 7 kDa),myO― (77 0 kDa),bo

globin(17 2 kDa),cytOChromc(12 3 kDa) Lanc 2:PBS cxtract from pupac

Lanc 3-5:DEAE chromatography,Lanc 3:Fraction A (50 mM Tris― HCl eluatc)Lane 4: Fraction B (450 mM Tris‐ HC l eluatc)Lane 5:Fraction C(eluate of 500 mM Tris― HCl contalning 0 5 M NaCl)

Lanc 6-7:ConA chromatography Lane 6:Fraction D (eluatC Of 20 mM Tris― HCl cOntaining 0 5 M NaCl) Lanc 7:Fraction E(cluate Of o 2 M α― methyl― D― man―

nosidc in 20 mM Tris HCl cOntaining 0 5 M NaCl) Lane 8:thc 77 kDa protein purincd on gel■

ltratiOn

Fig 3 shows the ultraviolet spcctrum of thc purincd 77 kDa protcin lt has a maxilnum ab― sorbancc at 278 5 nm 2ヽ s it lacks a shOuldcr at 290 nm,thc prOtcin、vas assumcd to contain no,or vcry fc、 v, tryptOphan residucs. The molccular ex‐

tinction cocmcient was A(3る )=18.3,as calculated

from thc protcin contcnt(■leaSurcd by thc Lowry

mcthod), as WCll as by thc optical dcnsity at 280 nm Only PBS was used for thc cxtraction of thc 77 kDa protcin, avOiding thc usc of prOtcasc

was pH 6 5 as dcternlincd by isoclcctric focussing

inhibitors Or protcin dcnaturants and dclipidatiOn by organic solvcnts,sO that it cOuld bc subnlittcd

clcctrophorcsis(Fig 2, uppcr) Two― dimcnsional

to further tcsts for bi01ogical activities

clectrophorcsis rcvcalcd 2 minor componcnts in thc

WVc found that the 77 kDa protcin has nO, Or

rangc of pH 3 5 to 5 2 and molccular wcight of

cxtremely fc、 v,tryptOphan rcsidues,but has a largc

65,000 to 75,000(Fig 2,lowcr).

molecular cxtinction coemcicnt,18 3 Thcse results

254

A OKANO ET AL

rr7ゎ /c2

′ Protcin yicld at each step of the proccdurc uscd in thc purincation of the 77 kDa

″θ ″ θ ″′ α s′ ′ 7r'994・ 9

protcin

Stcp

TOtal

Yicld

protcln

(°

o)

Starting material(pupaC):WCt WCight 23 5 g

39g

l PBS Extract

2 DEAE chromatOgraphy Elution(1)50 mM Tris HCl bufFcr pH8 2 HCi bufFcr pH8 2 (2)450 mM T (3)500 mM Tris‐ HCi bumcr pH8 2 s―

containing 0 5ヽ イNaCI

21 mg

054

H mg

028

5 mg

013

3 Con‐ A chrOmatOgraphy

Elution(1)20 mM Tris‐ HCi bullcr pH7 2 containing 0 5 M NaCl

(2)02 M Mcthy卜 mannoddc in

20 mM Tris HCi buf「 er pH7 2 Containing 0 5 M NaCI

4 Gcl■ ltration on scphadex G-200 5 Rc―

chromatography

い 一 ︲︱︱︱︱

い い  ︱︱︱︱︱

F号

￨￨

い ヽ  ︲ ︱︱︱︱

肥艦Ⅷ

￨￨

2 0

in 一¨ ︶一 一´ や一 一︵ ︶′ニイ ・

77kDa ゆ← ∽∪ 摯 ﹁＞〇 ロ

278 5 l

1 ()

1→ IEF Fig.2.Isoelectric focussing(IEF)pattern and two―

di―

mcnsional clcctrophorcsis pattern of thc purincd 77 kDa protcin

Uppcr:IEF pattcrn Lowcr:T、 vo― dilncnsional clcctrophorcsis pattern

indicatc that JP has abundant tyrosinc(Tyr)and phcnylalaninc (Phe) reSiducs Our prclinlinary anlino acid analysis revcalcd high Phc and Tyr con―

tents of 8%cach(16%in total,data nOt shown) The hcxosc cOntent was 3 3 0/O as mcasurcd by thc phcnol sulfuric acid mcthod

22o

270 いave lel,gth(1ヽ m)

Erect on lit spans of adult D.″ ι力″ρgα s″ ι″

Fig. 3. Ultraviolet spcctrum of thc puriflcd 77 kDa protein 550 μg of the 77 kDa protein was dissolved in

Table 2 shows thc adult life span of D″

one ml of PBS(pH 78)Absorbancc was measurcd in a

groups

fcd with a supplcmcnt of the 77 kDa protcin

onc cm light path,with PBS as the rcfcrcnce

320

A PROTEIN ASSOCIATED WITH LONGEVITY DIFFERENCE

″cr″ r/1′ αざ′77(′ 992)

rα わ 々2

255

Lifc spans of lies fed on f00d supplemented with thc 77 kDa protein,FractiOn A or 1/4 PBS

Group

ヽlalc

No

Fcmalc

Lilc span

M_Hχ

No

2

Life span

(dayS)

Max

M_Hχ 2

(dayS)

Max

Modc

235 235

39 36

32

2162ホ

31

58 88

35

31

140 279

35

31

ヽIodc

S strain

77 kDa

234

41

2976*

Fr A

235 60 86

34 35 35

004 069

77 kDa Fr A

235 237

3286*

245

58 87

003 025

236

l14PBS Control

60 52 54 52

1/4PBS Control L strain

49 46 46 48

55 91

2205* 140

093

77 kDa1 77 kDa protcin,Fr A:flactiOn A 2: Chi‐ M― Hχ squarc valuc by Mantcl‐ Hacnszcl prOccdurc * Signilcantly longcr than control(pく 005)

「

αわ′

`θ

Lifc spans of flics fcd On food supplcmcntcd、 vith di∬ crcnt amounts of thc 77 kDa protcin

Conccntration of 77 kDa

ヽ4alc

protein(μ g,ml)

NO

Fcmalc M_Hχ

Life span

2

NO

(dayS)

M_Hχ 2

Lifc span (dayS)

N(ax

Mode

58 58 60 58 86

41

34

36 34 35 35

31

56 57 60 62 87

60 57 55 54 52

Max

ヽlodc

60 57 59 59 88

39 37 36 36 35

36 33 32 32

64 59 60 62

60 57 57 54 53

55 49 49 47 48

S strain

00005

0005 005

05 0(COntr01)

31

3294* 2745* 1010*

30 29

423*

55

4204* 1669* 1406* 1224*

3554* 853* 166

012

31

L strain

00005

0005 005

05 0(Contr01)

51

50 50 46

91

2447* 1355* 1012* 553*

ヽ4-Hχ 21 chi_squarc valuc by Mantel Hacnszcl proccdure ネSignincantly 10ngcr than cOntrol(pく 005)

(p001Cd data), FrA (poolCd data), 1/4 PBS, and ordinary food(contr01) Lifc span of cithcr S Or I」

strain was signincantly

prolongcd in thc 77 kDa protcin groups by com― parison with thc cOntr01(p<0005)by thC Mantcl― Hacnszcl's chi― squarc

tcst(MANTEL 1963)

Lifc spans of FrA groups and l′ 4 PBS groups

Tablc 3 sho、 vs thc adult lifc spans Of nics fcd

with diffcrc,t amounts of thc 77 kDa protcin Prolongation of lifc span in thc 77 kDa groups 、 vas rclatcd tO protcin conccntration;lifc span、 vas

longcr with thc morc dilutc protcin supplcmcnt to thc fOOd, within thc rangc of this cxpcrilncnt.

Thc grOups fcd with thc mOst dilutc prOtcin

μg′ ml)ShOWCd mcan and ma

wcrc ncarly thc same as that of the control

(0 0005

(p>005)

spans prolongcd about 15ツ t in both strains Sur―

mal lifc

256

A OKANO ET AL

rrarθ ″′ α∫′′ア(1992)

J″ 乙 and an cxtranuclcar locus J“

E(YoNEMURA

ct a1 1989,1990,1991b;LucKINBILL ct al 1988).

ヽ ベ

is an autosomal longcvity genc systen■ with two known alleles,И r and Иス giVing fllndamental Jη 2И

環 ¶一

iS an X chromosomallongevity gene systcm with two allelcs,χF and χ2 adding more

Cont_― 一 tt

lifc span J771χ

lifc span onto fundamcntal lifc span;」 rF shOws a dosagc cffcct and causcs scx difFcrcnccs y74」

g is an

cxtranuclcar gcnc systcm with two gcncs, Er and E2; E2supprcsscs thC CXprcssion of thc allclcs at the Jη ,И and Jη r loci, giving a short lifc Thc longevity potcntial of D″ 2 controlled by nuclear

キ

7」

J791 gcncs is assumed to dimerentiatc bcforc emcr‐

gcncc(YoNEMURA ct al 1991a) Wc havc found a 77 kDa protein band isolated by onc‐ dilllcnsional SDS―

PAGE that appcars in

young adults soon aftcr cmcrgcncc This is also inhcritcd in association 、 vlth 5mA allclcs This 77 kDa protcin 、 vas fOund to bc thc only such

Cont_― ハ

protcin relatcd to thc diffcrcntiation of 10ngcvity potcntial.Wc havc rcpOrtcd this protcin as a prob― ablc genc product of J727И

allClcs(YoNEMURA ct

al 1992) Thc present study rcports thc mcasured molccu‐ of this extractcd protein and thc cmects of its usc as a nutritional supplemcnt, that is, it signincantly pr。 longed thc adult lifc of D″ っ 、 vhcn fcd at lo、 v conccntration to D.“ .adults lts molcc‐ ular、 vcight agrecs with that of thc 77 kDa protcin lar v′ cight

o

Fig。

10

20

30

40

50

4. Survival curvcs for thc short livcd strain of D 771

JP:GrOup fcd with thc 77 kDa protcin(0 0005 μg/1nl) as a supplcment

PBS:Group fcd with 1/4 dlutcd PBS as a supplcmcnt ContI Control group fcd、 vith ordinary food

lowcr band rcportcd in thc prcvious papcr(YoNE‐

MURA ct al 1992) On thc othcr hand, FrA, containing thc othcr protcins in the pupal cxtract, which was dcvoid

of thc 77 kDa protcin, did not show any life― prolonging cfFccts when fcd to adult

■ies Somc matcrials arc known to pro10ng lifc of D“

whcn administcrcd in thc adult stagc, for cxam― plc, improvcd nutrition, antioxidants, or othcr

vival curvcs of the mOst dilutc 77 kDa protein group(0 0005 μg/1111)and Of thc contr01 arc sho、 vn in Fig 4(S strain)and Fig 5(L strain) Arnong thc FrA groups,lifc span was shortcr in

chcmicals(LINTS and SoLIMAN 1988).How― cvcr, it is cvidcnt that thc cmcct of thc 77 kDa protcin prolongation of lifc span was not siinply

by supplying more prOtcins on thcir cOmpo―

groups fcd with highcr conccntration of FrA L

ncnt anlino acids, becausc no prolongation of lifc

strain malcs fcd with thc highcst conccntration of

was observed in thc FrA group supplied with thc samc conccntration of protcins in the food sup―

FrA had signincantly shortcr mcan lifc span (p<005)and slightly shortcr ma

mal lifc span

than thc cOntrols(dctailCd data arc onlittcd)

plcmcnt Orally adnlinistcrcd protcins of thc prcscnt cx‐

pcrilllcnt may bc cxpcctcd to bc digcstcd mostly

intO thcir amino acids.LINTS and SoLIMAN(1988) fOund that only mcthiOninc was cmcctivc in pro‐

E)iscusslon

longing lifc span among thc many anlino acids

Lifc span of D 771 haS bccn found to be grcatly inaucnccd by gencs at thc nuclcar loci, J″

,И

and

they tcstcd;0 01 M conccntration of rncthionine in

food incrcascd life span by l1 4シ 6 Wc found a

A PROTEIN ASSOCIATED WITH LONGEVITY DIFFERENCE

″crご ″′ ぉ′ J7″ 992)

257

0

Days after ellergence

Fig.5. Survival curvcs for the long-lived straln of D″

.

JP:GrOup fcd with thc 77 kDa protein(0 0005 μg/ml)as a supplement PBS:Group fcd with 1/4 dilutcd PBS as a supplcmcnt Cont:Control group fcd with ordinary food

prolongation of life span of about 15 0/O with a

soon after emergence(LINTS and SoLIMAN 1988)

supplcmcnt of 5× 10 4μ g/m1 0f the 77kDa

The pattcrn of protcins of D.″ ,. on day S aftcr cmcrgcncc, as sccn in onc¨ diincnsional SDS‐

protcin. Thc incrcasc in lifc span is silnilar in thc t、

vo studics Thc 77 kDa protcin has a mcthioninc

PAC}Iち iS Similar to that of a scnilc dcad lly,whcrc

contcnt of 3 0/。 (data nOt shown). ConSequently,

the 77 kDa protcin lowcr band has disappcarcd

thc mcthionine supplement in the present study is

(YoNEMURA ct al 1992).ConSCqucntly,fccding

only l× 10 8 。f the supplement provided by LINTS and SoLIMAN (1988). TherCfOre, thc in‐ crease in life span observed here is highly unlikely

to be due to a supplement of methionine The

the 77 kDa protcin from day 5 onwards,providcs a protcin which is norlnally undetcctablc in the ny, but is present at carlicr stagcs

Generaly, protcins are bclicvcd to bc absorbcd

77 kDa protcin incrcascd lifc span at a conccntra‐

fron■ thc intcstinc after being digested into anlino―

tion lowcr than that known for any othcr sub‐

acids.However, WARSHAW et al(1974)have

stancc. Additionally, it was ettective when given

from day 5 aftcr cmcrging, unlike Vitanlin E,

shown in rats that polypcptidcs 、 vith molccular weight of 50,000 or morc arc rcadily absorbcd

which is known to prolong life only if it is givcn

from alimcntary canals. Thc orally ad■ linistcrcd

258

A OKANOET AL

″gra″ ′ ω′ ′ 7α 99η

77 kDa protcin may givc a life‐ prolonging cnbct through absorption in the form of high molccular 、 vcight polypcptidcs aftcr incomplctc digcstion. Inhcritancc of thc 77 kDa protcin coincidcs with that ofthc longevity gcnc J“ И allclcs(YoNEMURA

ct al.1992).Thc 77 kDa protcin cxtractcd from L strain individuals、 vas found to prolong not only the

References DuBols,M,GILLES,K,HAMILTON,J K,REBERS,P A and SMITH, F 1951 A colorilnctric mcthod for thc dctcrmination of sugars ―― Nα′ ″re J68: 167 LAEヽ lMLl, U K 1970 Clcavagc of structural protcins during thc asscmbly of the head of bactcriOphagc T4 -― Nα′ ″r′ 227:

680-685

LINTS,F A and SoLIMAN,M H 1988 D″ οs″ みブル as a mOdcl organism for ageing studies

――Bル

た αれ′ Sο ″, Gた sgο lt α77r/

lifc span of S strain(whiCh nOrmally haslcss amount of this protcin)but also that of the L Strain This mcans that this protcin does not merely supplement

LowRY,O H,RosEBROUGH,N J,FARR A L and RANDALL,

a genetic defect in thc S strain

" LucKlNBILL, L S, GRAVES, J L, REED, A H and KoEST―

Thc modal day(mode)Of thC 10ngcvity distribu‐ tion curvc of D 711 corrcspOnds to thc valucs for

spccinc longcvity gcnotypcs(YoNEMURA ct al

`た

L072″077

R J

1951

Protcin mcasurcmcnt with thc Folin Phcnol B,ο ′C力 ′ ′ 93:265-275

rcagcnt― ―工

AWANG, S 1988 Localizing gcncs that dcrcr scncsccncc in ル″οgα ∫′ ′″ ― ″′r′ ″ r16θ :367-374 squarc tcst with onc‐ dcgrcc of frccdonl: Extcnsions of thc Mantcl‐ Hacnszcl proccdurc一 工 И S′α ` “ И5,ο ε ∫θ1 690-700 D/ο S9ρ み′ 物

771′

MANTEL, N 1963 Chi‐

1989,1990)It waS incrcascd with supplcmcntation ofthc 77 kDa protcin in food(Tablc 2,Tablc 3).On

NILSSON,B,NoRDEN,N E and SvENSSON,S 1979 Structural

thc basis ofthc abovc nndingS,we consider that thc 77 kDa protcin is a protein produced by J72,И gencs which g c a basic longc ty;it can modi夕 (thrOugh

0'FARRELL, P

an unknown mcchanism)a gCnctically dctcrrnincd

WARSHAヽ V, A L, WALKER, W A and lssELBACHER, K J

lifc span of adult nics aftcr thcir longcvity potcntial

has alrcady dimcrcntiatcd Wc nov′ tcrm this protcin

Ju-lnyo Protcin(JP),mCaning a protcin produccd by 5憔 (Ju‐ myO)gCncs JP is a glycoprotcin with 3.3シ 6 hcxosci and,with a molccular mass of 76,600,it is calculatcd to havc

13-15 hcxosc rcsiducs pcr molcculc. It combincs arl121yヽ

Vith ConA,and is assumcd to posscss polysac― 、 vith mannosc corcs, 1lkc fctuin

studics on thc carbohydrate protcin of Fctuin Cた′772 254:4545-4553

H

1975

――J B,ο ′

High rcsolution two‐

clcctrOphorcsis Of prOtcins 4021

dimcnsional

β′ ο′ C/1′″ 25θ :

― 工

4007-

1974 Protcin uptakc by thc intcstinc: cvidcncc for absorp‐

tion of intact macromolcculcs ― 992

Gα s″ ο′″′ ′″ 0′Ogy

`θ 987-

YoNEMURA,I,MOTclYAMA,T and HASEKURA,H 1986 Evi‐ dence of ma」 or genes controlling longe θ″″οgα s′ ′r― ―Prο εJc′ α″ Иσα″

ty in D″ οs9′ 力′ 物

62 Sar B:41-44

“ YoNEMURA,I,MOToYAMA,T and HASEKURA,H 1989

ヽlode

of inhcritancc of lllaJor gcncs controling lifc span diffcrcnccs between two inbrcd strains of Drο s9′ み′ 滋 121`滋 ″οgas′ ′″ ― Her`′ ′ αsII′ :207-214

YoNEMURA, I, ABE, M, ISHIDATE, R, IsHIYAMA, T, MO―

charidc chains

TOYAMA, T, HASEKURA, H and BoETTCHER, B 1990

(NILSSON Ct al 1979) Wc havc produccd an antibody against JP (un―

lifc span in D″

publishcd data)ThC ncxt stcp in our studics will bc thc dctcrnlination ofthc anlino acid scqucncc in thc activc part of JP which clongatcs lifc span,followcd by dctcrnlination ofthc basc scqucncc of3綸 Aallclcs and localization ofthc Jη

2И

gcnclocus on a chromo―

somc Thc rcsults would hclp in dctcrnlining thc mcchanism of gcnctic control of longcvity Иcた 71011々 r/gο ″´ ″お ― This study was supported by the Smoking Rcscarch FOundation(Japan)

1nflucncc

of tcmpcraturc

on

thc

inhcritancc

ο s″ 力 ′ /r7″ ′ 力″Ogα s`′ ″― /′ ―〃θ

r/7′

of aoult

α s fr2: H7-

127

YoNEMURA, I, MOToYAMA, T, HASEKURA, H BOETTCHER, B longcvity

gcncs

and

1991a Relationship betwecn genotypcs of and

dcvclopmcntal

spccd

in

″ ″θ′ α″οgα s′ θr ―― Hcr`沈 ヶ 66: 143-149 YoNEMURA, I, ｀4oToYAMA, T, HASEKURA,

Drο

H

sο ρ/1ア ル

and

BOETTCHER, B 1991b Cytoplasnlic in■ ucncc on thc cxprcs‐ sion of nuclcar gcncs a∬ ccting lifc span in D″ οs)pЙ jル ″′′ α72ο gα s′ ′″ ―― fferθ ″ r1 66: 259-261

YoNEMURA,I,OKANO,A,HASEKURA,H and BoETTCHER,B 1992 A di∬ erence in the proteins found in young adults of inbrcd strains of Drο s9′ 力,ル ο麟″οgα s″ ″whiCh Corrclatcs with gcnctically‐ dctcnnincd,10ng “ or short lifc span ――Herθ ′ ‐ ′ αs ′′7:241-250