αsゴ ′ア_15ノ ー258`ノ 992)

Her`滋 ′

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″′ ιう
οノグ Bノ οε力θ777お ′
り ,771s′ ′
r″ ′
θ 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
`√ `√
Me務 ,Mα 7710`0,ヨ フθJψ α″
2Dυ`772θ
α″ `s″
ιレ ,3ン ″り ο,動 り ο
ぽ ιttα ′″ εЙ ο α助 ルα 磁 わ′rs′ ヶ SC力 οο′げ ″
`,Lり
f/t‐ ,

`“ `″ `″ `′
Ifθ Jψ α77
3D9,α ″777θ ″ た 〃θ
P夕 ろ′
rブ
α′ ,S`力 οο′″ ′θ″ε777θ ,及 ,た αJこ ルわ rl,3ο s`′ αα′ α″
α,κ α″
,お lllα ,259-ノ ′
」￠′ακ `グ `力 `rs′ `力 `=α

4Dψ α″
′′″ 勲υ
′′ο
″″′
″ ノP/111s′ ο
あ″,ル s′ ル ′
θグ Cα ″
″ου
αS`ク ルr Dノ s′ αs′ ,`'カ ノ
″s力 trび ″ノ
υ′rs′ rl Sι /7ο οノ
“ ,コ `グ `α
J″ α″ “
グ ルセ″ ′α οrο ,39θ
5 Dψ α7r“
`777′ `s″ “
′″′げ B′ ο′ √ A● ″εαSrた ,Ⅳ ιll Sο ″Й Иイ
οgた αノS`た ″ε′s,7/P′ び″tl′ ぉJヶ て αルs,2Jθ θ Иνs″ α′
カ

OKANO,A,YoNEMURA,I,SHIMIzu,Y,YANACIDAIRA,Y,HASEKURA,H and BoETTCHER,B 1992

Purincation and charactcrization of a protcin associatcd with genctically― dctcrmincd longcvity difFcrcncc in
Drο sο′/2ブ ル 717`力 ″ ″――Herθ ′
Ogα s′ ′ ∵′αs yゴ 7:251-258 Lund,Swcdcn ISSN 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″ αr`777θ ″′げ ι α′″ι
″εttσ ,動 /cl οルαS力 清αυ′′
υ′″
ざり Sc力 οο
′〆 ν σ
″a,′ タイ5
`滋
γ″
s力 ″α ,Bッ ″ りο,動 り ο,″ ψα″
“
`洗

Contrary to thc carly gcncral conccpt that lifc span
is undcr polygcnic control, thc lifc span of
D″ οsρ ρカメ
あ 777′ ル″Ogα s`ε ″(D771)haS bCCn shown to
bc grcatly innucnccd by a fcw maJor genes(YoNE
MURA ct al 1986;LucKINBILL Ct al 1988)Re―
ccntly,thc modc ofinhcritancc and the function of
thc nl■ or gcnCS(J711 gcncs)haVC bCcn dcscribcd in
, havc bccn
dctail,and thc studics on lifcspan of D″
ablc to bc undcrtakcn from an cntircly ncw aspcct
(YoNEMURA ct al 1989,1990,1991a and b)
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
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,
or longcvity potcntial,bcforc cmcrgcncc(YoNE‐
MURA Ct a1 1991a)ThC ncxt stcp was thc inding
of a corrclation bct、 ′
een thc cxtcndcd prcscncc of a
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
protcin is involvcd in thc intra― strain dittcrcncc of
lifc span in D″ (YoNEMURA Ct al 1992)
Thc prcscnt rcpOrt dcscribcs dctails of thc purin‐
cation of thc 77 kDa protcin and somc of its
chcnlical and bi01ogical charactcristics
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
rθs′ /7`′ 992)

cstablishcd through sib‐ nlating rcpcatcd for morc was kcpt rccycling ovcrnight to assurc colmplctc
than 20 gcncrations from a wild typc stock,and a adsorption
long-livcd strain (L strain)silllilarly cstablishcd Thc column was clutcd irst with 100111l of
through morc than 13 gcncrations fronl thc Orc‐ buffcr D and thcn with 50 1121 of buffcr E(buffCr D
gon R strain Lifc spans of thcse strains havc bccn plus 02M α‐mcthyl― D‐ mannoddc)ThC SCcond
sho、 vn to bc dctcrmincd by nuclcar longcvity gcncs cluatc(Fraction E,FrE)was dialyscd against PBS
´
at thc SmИ and J727Й 10Ci,and an cxtranuclear gcne (pH 78)and waS COnccntratcd on an Amicon
at thc J“ Elocus(YoNEMURA ct al 1989,1990, PM-10 membrane to 3 ml
1991b)Thc nics wcrc brcd at 27° C and wcre
G′ ′
′/r″ α′
メο″ ――Thc conccntratcd FrE was applied
providcd with cookcd food containing 8シ 6 dry
to a Scphadcx G-200 column(1.5× 98 cm)pre‐
ycast, 5シ 6 sugar, 1シ 6 agar, 、
vith prcscrvativcs of
cqulibratcd with PBS, and was elutcd with thc
05シ 6 n‐ butyl p‐ hydroxybcnzoatc and 0 5ツ 6 pro―
samc bufFcr(PBS)Absorbancc at 278 nm was
pionic acid
mcasurcd for cvcry 3 m1/tubC Of thc cluatc.
Tubcs numbcr 28-36, 、 vhich includcd thc pcak
P“ ″ 77た Dα ′″ of optical dcnsity, wcrc harvcstcd for thc nnal
′εαrブ οκ ぽ r/2′ οたlll
purincation, which was pcrformcd by rc― chro―
All proccdurcs 、
vcrC pcrformcd at 4CC matography、 vith the samc column
P″ αrα ″0″ Q′
`r7/α `′
θθ
χ α ―-23 5 g of L strain ιr″ ノ
Dθ ′ ο ″s α″グ ια″
″αr′ ο′ ぽ ρ′ bο ″ 77・ α′
′ s―
pupac,gcnotypc
`ρ И2A2 obtaincd
`″
soon aftcr pupa一 Protein was quantitated`′by thc incthod of Lo、 vRY
tion, wcrc homogcnizcd in icc‐ cold phosphatc et al(1951)using bovine serum albunlin as thc
bu∬ crcd salinc(PBS,pH 7.8)and thC hOmogcnatc standard Carbohydrates wcrc mcasurcd by thc
was cxtractcd ovcrnight mcthod of DuBols Ct al(1951)with glucosc as thc
Thc homogcnatc was ccntrifugcd at 100,000g standard.
for 2 hours; thc supcrnatant(thc nrst crudc cx―
tract,about 100 ml)was rCtaincd:thc scdimcnt was Pο ケαε り ″777ノ 滋 gイ リ カο″ ― Sodium dO―
`″ `″ `sお
again cxtractcd with 100 1nl of PBS ovcrnight,and dccyl sulphatc polyacrylanlidc gcl clcctrophorcsis
ccntrifugcd, to yicld thc sccond cxtract(about (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
、
vcrc staincd 、 vith Coomassic
20 ml using Amicon PM-10 mcmbranc(W R bluc R-250(0005,も w/vin 2‐ propano1/aCCtic acid/
Gracc Corp,AmicOn D ison,MA 0195,USA); water,10ノ 10/80v≠ v/V)
thc conccntratcd samplc was thcn dialyscd against 7ル ツ
0-グ ′
″,′7PS′ 0″ αノ′″ιr″ 9′ 力ο″
ιsお ――Thc mcthod of
50 mM tris― HCl,pH 82(buttr A) O'FARRELL(1975)was uSCd fOr thc nrst dimcn―
DEИ E― 力αε
′ノ ″
ο α′ ∝″ ψ リ グ r力 θ ε′ ンル θχ― slon of isoclcctric focussing(IEF)and thC SCCond
′
″αε′――
`17ρ “`力
Thc abovc dialysatc 、 vas applied to a dimcnsion of SDS― PAGE Standard IEF markcrs
DEAE‐ Scphaccl(Pharmacia Fine Chemicals AB, wcrc obtaincd fl・ om Bio― Rad Laboratorics(Ca,
Uppsala,Sweden)C01umn(16× 25 cm)whiCh had USA)
bccn prc‐ cquilibratcd with buttcr A Thc column
was washcd 、vith 200 nll of buttcr ´、
, 200 1nl of И7711Й ノ
s″ α rブ 0″ ぽ 77た Dα ′″
οr′ ノ
′′οD 7271
bumcr B(450 mM Tris― HCl,pH 82),and thCn
Thc purincd 77 kDa protcin and othcr prOtcins
with 200 ml of buttr C(500 mM Tris― HCl,pH
82,containing 0 5 M NaCl)ThC fl・ actions clutcd (FrA)wcrc SCrially dilutcd to makc four protcin
conccntrations,20 μg/ml,2 μg/ml,02μ g‐ ml,and
vcrc tcrmcd Fraction A
by thc rcspcctivc bu∬ crs 、
0 02 μg/mlin 1/4 dilutc PBS(1/4 PBS,pH 72)
(FrA), Fraction B(FrB)and Fraction C (FrC),
rcspcctivcly FrC was dialyscd against bu∬ cr D(20
Thcsc prOtcin solutions wcrc stcrilizcd by 0 22 μm
Milliporc nltration and wcrc givcn to D 721 by
mM Tris― HCl,pH 72,containing 0 5 M NaCl)
adding 25 μl to onc llll of food Thc protcins(77
Cο κ И Sep力 αrο s′ ε
力″ο″α′ FrC, aftcr
Ogr`ψ /71 -― kDa protein or FrA)concCntrations wcrc thcn
dialysis,was applicd to a Concanavalin A collJu‐ rcspcctivcly,05,005,0 005 or 0 0005 μg/ml ThC
gatcd Scpharose 4B (Pharmacia Finc Chcmicals adult lifc span of thc S and thc L strains provided
AB,Uppsala,Swcdcn)column(12× 5cm)prC‐ 、vith thc dittcrcnt food lnixturcs、 vcrc obscrvcd and
vith bumcr D. Thc samplc solution
cquilibratcd 、 rccordcd
″′ αs′ 77(,992)
″″′
As controls, two grOups wcrc cstablished: onc
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
tubc、vas rcnewcd cvcry、 veek Othcr details of thc
mcthod of culturing nics 、vcrc as in thc prcvious
rcport(YoNEMURA Ct al 1989)
Rcsults
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
was pH 6 5 as dcternlincd by isoclcctric focussing
clcctrophorcsis(Fig 2, uppcr) Two― dimcnsional
clectrophorcsis rcvcalcd 2 minor componcnts in thc
rangc of pH 3 5 to 5 2 and molccular wcight of
65,000 to 75,000(Fig 2,lowcr).
A PROTEIN ASSOCIATED WITH LONGEVITY DIFFERENCE 253
1 2 3 4
1 111■ ■ ″= ‐
kDa 勃 ￨■ 攣 暉
笏呵 隆覇嬢琴…
770
662
1
450 .― ‐ ‐
警鸞 摯 鸞
257 攀 ・
‐
‐ _鰺
172 ■■■
.■ .
123 攀 _
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
(77 0 kDa),bo ne scrum albumin(66 2 kDa),ovalbu_
min(45 0 kDa),chymOtrypsinogcn A(25 7 kDa),myO―
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
inhibitors Or protcin dcnaturants and dclipidatiOn
by organic solvcnts,sO that it cOuld bc subnlittcd
to further tcsts for bi01ogical activities
WVc found that the 77 kDa protcin has nO, Or
cxtremely fc、 v,tryptOphan rcsidues,but has a largc
molecular cxtinction coemcicnt,18 3 Thcse results
254 A OKANO ET AL ″θ
″θ″′
αs′ ′
7r'994・ 9

rr7ゎ /c2 ′ Protcin yicld at each step of the proccdurc uscd in thc purincation of the 77 kDa
protcin

Stcp TOtal Yicld

protcln (° o)

Starting material(pupaC):WCt WCight 23 5 g

l PBS Extract 39g
2 DEAE chromatOgraphy
Elution(1)50 mM Tris HCl bufFcr pH8 2
(2)450 mM T HCi bufFcr pH8 2
s―

(3)500 mM Tris‐ HCi bumcr pH8 2

containing 0 5ヽ イNaCI 21 mg 054
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 H mg 028
4 Gcl■ ltration on scphadex G-200
5 Rc― chromatography 5 mg 013
い ヽ  ︲
い い  ︱︱︱︱︱

い 一 ︲︱︱︱︱
︱︱︱︱

2 0

￨￨
肥艦ⅧF号
in
￨￨
・
一¨︶一
一´や一 一︵

77kDa
︶′ニイ
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 22o 270 320
thc phcnol sulfuric acid mcthod
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″ groups one ml of PBS(pH 78)Absorbancc was measurcd in a
fcd with a supplcmcnt of the 77 kDa protcin onc cm light path,with PBS as the rcfcrcnce
″cr″ r/1′ αざ′77(′ 992) A PROTEIN ASSOCIATED WITH LONGEVITY DIFFERENCE 255

rα わ々2 Lifc spans of lies fed on f00d supplemented with thc 77 kDa protein,FractiOn A or 1/4 PBS

Group ヽlalc Fcmalc

No Lilc span M_Hχ 2

No Life span M_Hχ 2
(dayS) (dayS)

Max ヽIodc Max Modc

S strain
77 kDa 234 41 2976* 235 39 32 2162ホ
Fr A 235 34 004 235 36 31 140
1/4PBS 60 35 069 58 35 31 279
Control 86 35 88 35 31

L strain
77 kDa 235 60 3286* 245 49 2205*
Fr A 237 52 003 236 46 140
l14PBS 58 54 025 55 46 093
Control 87 52 91 48

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

Conccntration ヽ4alc Fcmalc

of 77 kDa
protein(μ g,ml) NO Life span M_Hχ 2
NO Lifc span M_Hχ 2
(dayS) (dayS)

N(ax Mode Max ヽlodc

S strain
00005 58 41 34 3294* 60 39 36 3554*
0005 58 36 31 2745* 57 37 33 853*
005 60 34 31 1010* 59 36 32 166
05 58 35 30 423* 59 36 32 012
0(COntr01) 86 35 29 88 35 31

L strain
00005 56 60 55 4204* 64 60 55 2447*
0005 57 57 51 1669* 59 57 49 1355*
005 60 55 50 1406* 60 57 49 1012*
05 62 54 50 1224* 62 54 47 553*
0(Contr01) 87 52 46 91 53 48

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 Tablc 3 sho、 vs thc adult lifc spans Of nics fcd
ordinary food(contr01) with diffcrc,t amounts of thc 77 kDa protcin
Lifc span of cithcr S Or I」 strain was signincantly Prolongation of lifc span in thc 77 kDa groups
prolongcd in thc 77 kDa protcin groups by com― 、
vas rclatcd tO protcin conccntration;lifc span、 vas
parison with thc cOntr01(p<0005)by thC Mantcl― longcr with thc morc dilutc protcin supplcmcnt
Hacnszcl's chi― squarc tcst(MANTEL 1963) to thc fOOd, within thc rangc of this cxpcrilncnt.
Lifc spans of FrA groups and l′ 4 PBS groups Thc grOups fcd with thc mOst dilutc prOtcin
wcrc ncarly thc same as that of the control (0 0005 μg′ ml)ShOWCd mcan and ma mal lifc
(p>005) spans prolongcd about 15ツ t in both strains Sur―
256 A OKANO ET AL
ヽ Jη 2И is an autosomal longcvity genc systen■ with
ベ two known alleles,И r and Иス giVing fllndamental
¶一
Cont_― 一 tt
環 systcm with two allelcs,χF and χ2 adding more
キ E2; E2supprcsscs thC CXprcssion of thc allclcs at
Cont_― ハ
o 10 20 30 40
Fig。 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
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
groups fcd with highcr conccntration of FrA L
strain malcs fcd with thc highcst conccntration of
FrA had signincantly shortcr mcan lifc span
(p<005)and slightly shortcr ma mal lifc span
than thc cOntrols(dctailCd data arc onlittcd)
E)iscusslon
Lifc span of D 771 haS bccn found to be grcatly
inaucnccd by gencs at thc nuclcar loci, J″ ,И
rrarθ ″′
α∫′′ア(1992)
J″ 乙 and an cxtranuclcar locus J“ E(YoNEMURA
ct a1 1989,1990,1991b;LucKINBILL ct al 1988).
lifc span J771χiS an X chromosomallongevity gene
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
the Jη ,И and Jη r loci, giving a short lifc Thc
7」
longevity potcntial of D″ 2 controlled by nuclear
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
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‐
lar v′ cight 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
50
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
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―
ncnt anlino acids, becausc no prolongation of lifc
was observed in thc FrA group supplied with thc
samc conccntration of protcins in the food sup―
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‐
longing lifc span among thc many anlino acids
they tcstcd;0 01 M conccntration of rncthionine in
and food incrcascd life span by l1 4シ 6 Wc found a
″crご ″′
ぉ′J7″ 992)
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
supplcmcnt of 5× 10 4μ g/m1 0f the 77kDa
protcin. Thc incrcasc in lifc span is silnilar in thc
t、 vo studics Thc 77 kDa protcin has a mcthioninc
contcnt of 3 0/。 (data nOt shown). ConSequently,
thc mcthionine supplement in the present study is
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
77 kDa protcin incrcascd lifc span at a conccntra‐
tion lowcr than that known for any othcr sub‐
stancc. Additionally, it was ettective when given
from day 5 aftcr cmcrging, unlike Vitanlin E,
which is known to prolong life only if it is givcn
A PROTEIN ASSOCIATED WITH LONGEVITY DIFFERENCE 257
.
soon after emergence(LINTS and SoLIMAN 1988)
The pattcrn of protcins of D.″ ,. on day S aftcr
cmcrgcncc, as sccn in onc¨ diincnsional SDS‐
PAC}Iち iS Similar to that of a scnilc dcad lly,whcrc
the 77 kDa protcin lowcr band has disappcarcd
(YoNEMURA ct al 1992).ConSCqucntly,fccding
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
fron■ thc intcstinc after being digested into anlino―
acids.However, WARSHAW et al(1974)have
shown in rats that polypcptidcs 、vith molccular
weight of 50,000 or morc arc rcadily absorbcd
from alimcntary canals. Thc orally ad■ linistcrcd
258 A OKANOET AL ″gra″ ′
ω′′
7α 99η

77 kDa protcin may givc a life‐ prolonging cnbct References

through absorption in the form of high molccular DuBols,M,GILLES,K,HAMILTON,J K,REBERS,P A and
、vcight polypcptidcs aftcr incomplctc digcstion. SMITH, F 1951 A colorilnctric mcthod for thc dctcrmination
Inhcritancc of thc 77 kDa protcin coincidcs with of sugars ―― Nα′ ″re J68: 167
LAEヽ lMLl, U K 1970 Clcavagc of structural protcins during
that ofthc longevity gcnc J“ И allclcs(YoNEMURA thc asscmbly of the head of bactcriOphagc T4 -― ″r′ 227:
Nα′
ct al.1992).Thc 77 kDa protcin cxtractcd from L 680-685
strain individuals、 vas found to prolong not only the 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 L072″077
`た

of this protcin)but also that of the L Strain This LowRY,O H,RosEBROUGH,N J,FARR A L and RANDALL,
mcans that this protcin does not merely supplement R J 1951 Protcin mcasurcmcnt with thc Folin Phcnol
rcagcnt― ―工 B,ο ′C力 ′ ′93:265-275
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‐ AWANG, S 1988 Localizing gcncs that dcrcr scncsccncc in
D/ο S9ρ み′
物 ル″οgα ∫′′″ ― ″′r′ ″ r16θ :367-374
tion curvc of D 711 corrcspOnds to thc valucs for 771′

MANTEL, N 1963 Chi‐ squarc tcst with onc‐ dcgrcc of frccdonl:

spccinc longcvity gcnotypcs(YoNEMURA ct al Extcnsions of thc Mantcl‐ Hacnszcl proccdurc一 工 И S′α
1989,1990)It waS incrcascd with supplcmcntation И5,ο ε ∫θ1 690-700 “ `

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

ofthc 77 kDa protcin in food(Tablc 2,Tablc 3).On
studics on thc carbohydrate protcin of Fctuin ――J B,ο ′
thc basis ofthc abovc nndingS,we consider that thc Cた′772 254:4545-4553
77 kDa protcin is a protein produced by J72,И gencs 0'FARRELL, P H 1975 High rcsolution two‐ dimcnsional
clcctrOphorcsis Of prOtcins ― 工 ο′ C/1′″ 25θ :
β′ 4007-
which g c a basic longc ty;it can modi夕 (thrOugh 4021
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 1974 Protcin uptakc by thc intcstinc: cvidcncc for absorp‐
tion of intact macromolcculcs ― ′″
Gα s″ ο′″′ 0′Ogy `θ 987-
has alrcady dimcrcntiatcd Wc nov′ tcrm this protcin 992
Ju-lnyo Protcin(JP),mCaning a protcin produccd by YoNEMURA,I,MOTclYAMA,T and HASEKURA,H 1986 Evi‐
5憔 (Ju‐ myO)gCncs dence of ma」 or genes controlling longe ty in D″ οs9′ 力′
物
θ″″οgα s′ ′r― ―Prο εJc′ α″ Иσα″ 62 Sar B:41-44
JP is a glycoprotcin with 3.3シ 6 hcxosci and,with “
YoNEMURA,I,MOToYAMA,T and HASEKURA,H 1989 ヽlode
a molccular mass of 76,600,it is calculatcd to havc of inhcritancc of lllaJor gcncs controling lifc span diffcrcnccs
13-15 hcxosc rcsiducs pcr molcculc. It combincs between two inbrcd strains of Drο s9′ み′ 滋 121`滋 ″οgas′ ′″ ―
Her`′ ′ αsII′ :207-214
arl121yヽ Vith ConA,and is assumcd to posscss polysac― YoNEMURA, I, ABE, M, ISHIDATE, R, IsHIYAMA, T, MO―
charidc chains 、vith mannosc corcs, 1lkc fctuin TOYAMA, T, HASEKURA, H and BoETTCHER, B 1990
1nflucncc of tcmpcraturc on thc inhcritancc of aoult
(NILSSON Ct al 1979) lifc span in D″ ο
s″ 力′ 力″Ogα s`′ ″―
/r7″ ′ ―〃θ
/′ r/7′ s fr2: H7-
α
Wc havc produccd an antibody against JP (un― 127
publishcd data)ThC ncxt stcp in our studics will bc YoNEMURA, I, MOToYAMA, T, HASEKURA, H and
BOETTCHER, B 1991a Relationship betwecn genotypcs of
thc dctcrnlination ofthc anlino acid scqucncc in thc
longcvity gcncs and dcvclopmcntal spccd in Drο sο ρ/1ア ル
activc part of JP which clongatcs lifc span,followcd ″ α″οgα s′ θr ―― Hcr`沈 ヶ 66: 143-149
″θ′
by dctcrnlination ofthc basc scqucncc of3綸 Aallclcs YoNEMURA, I, ｀4oToYAMA, T, HASEKURA, H and
BOETTCHER, B 1991b Cytoplasnlic in■ ucncc on thc cxprcs‐
and localization ofthc Jη 2И gcnclocus on a chromo― sion of nuclcar gcncs a∬ ccting lifc span in D″ οs)pЙ jル
somc Thc rcsults would hclp in dctcrnlining thc α72ο gα s′ ′″ ―― fferθ ″ r1 66: 259-261
″′′
mcchanism of gcnctic control of longcvity 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
Иcた 71011々 r/gο ″´
″お ― This study was supported by the Smoking or short lifc span ――Herθ ′ ‐
with gcnctically‐ dctcnnincd,10ng “
Rcscarch FOundation(Japan) ′αs ′′7:241-250