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Her`滋 ′
`“ `″ `″ `′
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
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 or longcvity potcntial,bcforc cmcrgcncc(YoNE‐
is undcr polygcnic control, thc lifc span of MURA Ct a1 1991a)ThC ncxt stcp was thc inding
D″ οsρ ρカメ
あ 777′ ル″Ogα s`ε ″(D771)haS bCCn shown to of a corrclation bct、 ′
een thc cxtcndcd prcscncc of a
bc grcatly innucnccd by a fcw maJor genes(YoNE 77 kDa protcin in cxtracts from young adult iD 727
MURA ct al 1986;LucKINBILL Ct al 1988)Re― and thc prcscncc of thc autosoma1 longevity allclc
ccntly,thc modc ofinhcritancc and the function of 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
dctail,and thc studics on lifcspan of D″ lifc span in D″ (YoNEMURA Ct al 1992)
ablc to bc undcrtakcn from an cntircly ncw aspcct Thc prcscnt rcpOrt dcscribcs dctails of thc purin‐
(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 chcnlical and bi01ogical charactcristics
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 NIlatcrials and mcthods
short― livcd nics,and adult lifespan bcing ablc to bc
prcdictcd at thc tilnc of cmergence Thcsc rcsults
D“ ′″″οgα srar
suggcst that thc function of thc J770 genes is to Two inbrcd strains of D 771 WCrC uscd,as in the
scgrcgatc thc gcnctically dctcrnlincd adult lifc span, 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)
″″′ A PROTEIN ASSOCIATED WITH LONGEVITY DIFFERENCE 253
笏呵 隆覇嬢琴…
D″ .werc kcpt at 27° C and werc givcn thcir 770
respcctivc food, accOrding to thc grOup, from day 662
5 of cmcrgcncc Thc dcad wcrc countcd evcry day, 1
whcn old food was changed、 vith ncw; thc culturc 450 .― ‐ ‐
tubc、vas rcnewcd cvcry、 veek Othcr details of thc
警鸞 摯 鸞
mcthod of culturing nics 、vcrc as in thc prcvious
rcport(YoNEMURA Ct al 1989)
257 攀 ・
‐
‐ _鰺
Fig l shows thc 10シ 6 SDS― PAGE pattcrns of Fig. 1. SDS― PAGE protein pattcrns of thc fractions at
protcins extractcd from D 777′ ′ α72ο gα s′ θr pupac and cach step in thc purincation of the 77 kDa protcin from
in thc fractiOns obtained at cach stcp in thc purin― pupac Of the long lived strain of D″ ,′ ノ
α770gα s′ ′
′
cation of thc 77 kDa protcin Lanc 2 shou/s thc Lanc l: Molccular wcight standards: ovOtransferrin
protcins in thc salinc cxtract frOm thc pupac (77 0 kDa),bo ne scrum albumin(66 2 kDa),ovalbu_
Lancs 3-5 show thc pattcrns of thc protcins in thc min(45 0 kDa),chymOtrypsinogcn A(25 7 kDa),myO―
fractiOns Obtaincd from I)EAE chromatOgraphy. globin(17 2 kDa),cytOChromc(12 3 kDa)
Lanc 2:PBS cxtract from pupac
Thc 77 kDa protcin was not dctectcd in FrA(lanc
Lanc 3-5:DEAE chromatography,Lanc 3:Fraction
3); a nlinOr prOportion of it was fOund in FrB
A (50 mM Tris― HCl eluatc)Lane 4: Fraction B
(lanc 4)among many othcr protcin、 it was a (450 mM Tris‐ HC l eluatc)Lane 5:Fraction C(eluate of
mttOr prOtein component of FrC(lanC 5),about 500 mM Tris― HCl contalning 0 5 M NaCl)
45シ 6 Lanc 6-7:ConA chromatography Lane 6:Fraction D
FrC was subnlittcd to COnA Scpharosc chro― (eluatC Of 20 mM Tris― HCl cOntaining 0 5 M NaCl)
matOr辱車井→曇lding FrIΣ ttanc 6),whiCh COn_ Lanc 7:Fraction E(cluate Of o 2 M α― methyl― D― man―
taincd a low cOnccntration Of thc 77 kDa protcin nosidc in 20 mM Tris HCl cOntaining 0 5 M NaCl)
among othcr protcins,and FrE(lanc 7),conSiSting Lane 8:thc 77 kDa protein purincd on gel■ ltratiOn
rr7ゎ /c2 ′ Protcin yicld at each step of the proccdurc uscd in thc purincation of the 77 kDa
protcin
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―
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
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
αわ′ Lifc spans of flics fcd On food supplcmcntcd、 vith di∬ crcnt amounts of thc 77 kDa protcin
「 `θ
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
(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 rrarθ ″′
α∫′′ア(1992)
¶一
Cont_― 一 tt lifc span J771χiS an X chromosomallongevity gene
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
protcin. Thc incrcasc in lifc span is silnilar in thc cmcrgcncc, as sccn in onc¨ diincnsional SDS‐
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 the 77 kDa protcin from day 5 onwards,providcs a
LINTS and SoLIMAN (1988). TherCfOre, thc in‐ protcin which is norlnally undetcctablc in the ny,
crease in life span observed here is highly unlikely but is present at carlicr stagcs
to be due to a supplement of methionine The 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 shown in rats that polypcptidcs 、vith molccular
from day 5 aftcr cmcrging, unlike Vitanlin E, 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
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′