1 of 7
`
`PENN EX. 2150
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`t"'.".
`
`-----· ---· --· -·····-·---:-······-------·--··-·· .
`
`Mechanism of MTP-Catalyzed Lipid Transport
`
`Biochemistry, Vol. 31, No. 39, 1993 10445
`
`1980; Swenson et al., 1988). The cytosolic nonspecific lipid
`transfer protein appears to operate by yet another mechanism
`(Nichols -& Pagano, -1983;.Gadella .&. Wirtz,.1991). This
`protein penetrates the surface of the membrane and enhances
`spontaneous polar lipid transfer by enabling lipid molecules
`to exinhe ·membrane mote readily. Although this· tatter
`mechanism may be reasonable for the transport of phospho(cid:173)
`lipids and polar sterols; iJ·is unlikely that such a mechanism
`would be feasible for the transport of insoluble triglycerides
`and cholesteryl esters. The goal of this study is to determine
`the·mechanisrn of MTP-catalyzed lipid transport.
`
`·-. ,_ .,
`
`r·:
`
`;..·
`
`Lipid Transfer Assay. TG and CE transfer was measured
`from donor to acceptor SUV in an assay· similar to that
`previously described by. Wetterau and Zilversmit. (1985).
`Donor membranes, acceptor membranes, 100 ng (0.7 pmol)
`of MTP, and S mg of BSA were adjusted to a total volume
`of 0.5 inL of as8ay buffer and incubated at 23 °C for 30 and
`60 min. Transfer reactions.were terminated.by addition of
`0.5 mLof DEAE (Whatman DE-52) suspension (1 :i ratio of
`DEAE: 15 mM Tris, pff 7.4 ), which .-binds the .negatively
`charged donor membranes. DEAE-bound donors· were then
`. pelleted by centrifugation at lS{)()()g for 2 miJJ: The ·[l'CJ·
`triolein or [l"Cjcbolesteryl oleate in an aliquot of the
`supcrnalant was measured by .seintillation counting. ,Aj>-
`MATERIAI.S AND METHODS
`~ranee of 14C: to. or 14C-Ciiin the supernatant represcntS
`Materl~Is: Ti:iolein, ~gg phosphatidylcboline, cholesteryl
`oleate, cardiolipin, and fatty acid free bovine svtuin albumin
`transfer of labeled neutral lipid from donor to aceeptor
`(BSA} were pu~~hased from Sigma (St. Louis, MO).· Egg
`membranes. Nons~ific lipid· transfer (lcss.than· 1%) and
`ph0sphaiidi~ acid, semisynthetic (Na+ .salt), :was purchased .
`nonprecipitated donor membranes .(less than 2%as determined
`from ~alrayl\;:lnc. ·{Pleasant- GJ!,p, ·PA). ~e (carboxyf-
`in c0n_trol experiments} were measured as lipid tran8fer in the
`1 4q~r!1*µi(.l.IAmCi/minol),[o_/eale·l~1~]chol~t~iyl~l~te
`absence of MTP and subtracted from tli:e totai 14C-labeled
`(52-mCi/mmol)., and [2-palmitoyl~9,I0-3H(N)]diJ!cllmi•
`neut~al lipid in thesupernat~nH~«;alculate_the ra~eofprotein·
`ioylphQ$phatidy~chc;>line (~O P/mmol) w~.re pui<:~~se(l frolll
`stiinulated · lij>id transfer. · · Beeause ·pH]DPPC. transfer is
`inslgniri<:8nt. r:elati~ to •:4¢l'f9 -,~r i4c,cE transfedn ·th.is
`New·sngi_anQ. NucJ~((HQffni11n ·~ta.t.c:S; IL). ,c_~ole!it~i'YI.
`J~pyrenedecanoate ~~s pil~~_sed: from Moleeula.r-ProbeS.
`,~y:[CE and TG:8retr8.11Sferre'd 3~SO ti~ f~ter~~an
`(J?ugen~. OR):· . All lipids -:were. sto~ u11der~·tJ2 .. sils: in ... · : PCi ·s~'Wette~ali. and :ZilverJmit· ( 1985)], [lHJDPPC :,was:
`chloroform·. at ,.,~Q :,0c .. : .
`. :.' . .. . .
`.: used: '!ls. jm . fu:d.icat.Or :of:. a~p~or vesicie: f~\/ery ·: in .th~
`' su~ritata_rit. Th~{~gh·rcicoVe..Y "of'['fiiDPPC.(rangedJrom
`. ' : isoiation"of AfTf .. )d.ti' was pu,ieyed frqID.'bov~#~;lire.~>&s.
`95%'to to~%) con.fitms that'PC transfer was insignifieant~:A
`originally. _descr._i!>ed .·(wetterau & Zilver5µiit.. i98~)- and
`sub5equently m9<1ified (\Vetterau et. al.,· .1991 b}.. :Pil-rified
`. S%vari8.tio1{in acpeptor vesicle recovery would have minimal
`· ·· MTP·was dialyzed.into 15 mM Tris. pH 7.4,"40 !llM NaCl;
`· effects on the ealculatcd' transfer rate:
`.To ~lculate the t~tal neutia.l ·lipi'd transfer, frr~t-ord~r
`l ·JQM ethyle.nediaminetetraacetatc; and 0.02% NaN3 buffer
`(hereafter teferted to as. a8say buffer}. assayed for prot~in
`.
`.
`·kineties were used (x, = .xoC-k' •. where:· Xo and x, equal the
`with the ~ierce.BCA-reagent (Rockford~.IL), and stored at
`4 oc. BSA ( 1 mg/m,L) was added to dilute MTP· solutions
`fraction oflabeled lipid in the donor membrane at times Q and
`to stabilize the protein.
`t, respectively;:ktXo =mass lipid transfered;whereXoeq.uals
`the.initial mass oflabeled lipid in the donor membrane): This
`PreptJration·of Donor and Acceptor ·¥embranes. Donor
`.
`corrc:Cts for the dilution pf radiolabeled lipid in the donor
`and.ac:cept<ir PC vesicles,coJJtaining either TO or·.CE were
`prep~red as follows .. 111 all kinetic studies, e.x:cept-othel'Wise
`SUV which results from the transfer o( unlabeled lipid from
`the. res:ul~s were expressed as
`indicated, donor meqibrancs contained egg PC, 0.25. moi %
`acceptor to donor. S_UV.
`velociticS, graphed in double-reeipro.Ca~ format (l/v. versus
`[l'C]triolein or 0.25 mol % [l'C]cholesteryl oleate, arid 5 inol
`% cardiollpiri to c;onfer a net i)egative charge, while accep~e>r
`l /[DON}), an~: cuiv~ .fltt~ h.Y -liilear. regres_sion. .
`membranes contained· egg .. PC, .. a- tr!lce ,_9f Pl-fJdipalmi~ .
`. :Experimental Design. Te> evaluate the kinetic mecha~iSm
`for '.fG and CE :transport, t~o-differe~t experimental· ap-
`toylphosphatidylcholine ([3HJDPl~C), and 0.2_5 mol % un•
`prbaches .were used: Both approaches account for acceptor
`labeled trioleinor cholesteryl oleate. Unilamellar.phospholipid
`·membrane-inhibition which was experimentally ~bserved (for
`· vesicles were prepared by bath sonication (4boratocy Supplies
`Co. Inc., Hicksville, NY) under a nitrogen atmosphere at
`example, see Figure 3, w}!ere·TG transfer d<:ereases \,Vith
`room temperature .. Following sonication, the heterogeneous
`increased coneentration of acceptor vesicles). DOnor mem· .
`unilamellar vesicles were fractionated by ultracentr:if ugation.
`branc inhibition which would result in a nonlinear. (concave
`using a modification orthe procedure desc;ribed by Baren~olz
`upward ~t low l / (DON]) double-teciprical -ploi was never
`observed. Ping-pong bi-bi kine~ics arc diagn9l1tic of a shuttle
`et a!. (I 977) to obtain a homogeneous population of small
`~cchanism, and random bi-bi kinetics are diagnostic of a
`unilamcllar vesicles (SUV). Sonicated vesicles in 6 mL of
`assay buffer were spun at 159000g for 2 h in a Beckman (Palo
`ternary complex mechanism for MTP-catalyzed lipid transport
`Alto, CA) TiS0.3 rotor. The SUV contained in the t<ip4 mL
`(Figure l).
`· ·
`of the centrifuge tube were removed by pipet. Typically, 45-
`65% of the original Jipid was recovered in this fraction. The
`bottom 2 mL and.pellet, which contained large unilamellar
`and multilamellar vesicles, respectively, were discarded.
`Purified vesicles were utilized in experiments within 5 h of
`preparation,
`Aliqu9ts of SUV were characterized by fractionation on a
`Bio-Gel A 15-M gel permeation column (fractionation range
`40000 to IS x 10') (Bio-Rad, Richmond, CA) to ensure
`purity of the vesicle preparation. SUV homogeneity was
`confirmed by incorporating both ( 14C]triolein and [3H]DPPC
`into vesicles and demonstrating that the 3H/ 14C ratio across
`t~e Bio-Gel A I 5- M peak of the purified vesicles was constant.
`
`(A) Approach 1. Initial velocity (v) measurements were
`inade at varying donor membrane concentrations ([DON])
`(25-400 pmol/mL) expressed as picomolcs ofTG or CE per
`milliliter, while acceptor membrane concentrations ({ACC])
`were held constant. Experiments were. then repeated at several
`{ACC] (4-1000 pmol/mL). The res_ults.were plotted as I/v
`versus 1 /(DON] to generate a seri~ of lines which can be
`comparedtothepatternspredictedfromtheequationsfor.the
`kineticmechanisms: eq l for ping-pong bi-bi kinetics.modified
`for acceptor-acceptor membrane inhibition (Segel, l 97Sa)
`and eq 2 for random bi-bi kinetics modified. for acceptor
`membrane inhibition [derived from rapid equilibrium as-
`
`_____________________ .................... ·-----
`
`2 of 7
`
`PENN EX. 2150
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`10446 Biochemistry, Vol. 32, No. 39, 1993
`
`Atzcl and Wetterau
`
`ping pong bi bi
`
`DON•
`
`DON .ACC
`
`ping-pong bi-bi kinetics
`
`I {Ko+ KA) 1 ( Ko)
`I
`f; = [OONj\ V mu
`.. + Viux 1 + K;
`random bi-bi kinetics
`(aKo + aK.A)·
`aKrJ<A
`I
`1
`-=---
`+
`[DON] 2 V mu
`o
`[DON]
`V max
`
`(3)
`
`+
`_1_· (I + aKo) (4)
`
`sU:mptio~s as outlined. in· Segel (l9.75b)}. ·
`·:
`
`P.ing-pong bi-bikinetjcs
`..
`l=-··i_. [~o(l +[ACCJ/Ki>J~.
`v . . [DON]
`..
`. V mu ·.
`. :V~.{l + [A?C]) (I)
`
`· .
`
`:·: ·.
`
`.
`
`. .
`
`K1
`. Vmax.
`random bl bi
`There are no variables in the. intercept .or slope term of eq 3
`E .+.DON• ~ .. E•DO!'I•
`so a double~reciproeal plot yields a straJght Jin~. Equation 4
`is ~iifferent from cq l in: that :the [DON,j2 ietm ·in the
`+
`+·
`· denominator causes tbe double~reciprocafpiot to:hepa,rabrilic
`ACC
`. ACC
`(o0neaveup).atlow[OON](~iido)ph.& F~mm;1979). When
`·~ ·
`·
`· >...v
`·
`·
`·
`SUbStrat.tconcenfr.atiO~.becom.. . e.v......,."';·,;,, ·1. ,h.·o.wever;the. term
`l·~A . ·
`.uio -.: . 1-_. ·;. /!E .
`K1 . .
`.
`E•,4.CC•AcC ~AfX +E·ACC +:ooN•""-r-.E•OON-ACC*;-;.ooN.
`-·J~
`" .· ·· ,
`. '. , ..... , ,. · .
`. . ~~~ c0ritairliitg"[DONi.:t·wUl:,;jlpp~ch ie!:o~ and. th~ .:dou.ble-
`· .. ·
`.. ·
`·
`· rccipi:ocal plot-will .~.~mil~r~-10 ~hiirc;if.!:q,:3 .. : A b_eneficlBt
`Fto~RB. l :. · Pil!J~Po~B bi-.bj· :(s:ti~ttJ~). aiid. faildol!l bi:~ (tcril<irY
`. :a~pect~f tni~· ~eoori~ ~PW~C~~;i~3.t.tf!.e slN.!pe of.t}ie·~~u~I~ ·.
`compl~)-meclianis!Ds~ In tbe.ping~p<ingbH>! mb:~anism.(top), Ko · '. .. reciproqil',plois ·is: lii>f::S.IC~t~i<f' by :!i¢eptor,·¢.o~peti~ive
`and KA·arc steail)"-statc.constantS ei(Ulil:to.k7<.1ri·'f~,)/k1(lcJ·'t"k1) ·
`·
`· ··
`· ..
`·
`... · · ·
`· ·
`.. ·,
`·
`· ·
`·
`a~d M~ + li7)/ k,(lc, t ki) #sj)Cctfyciy, and .Ki :is the di&$0!:iation
`. · iithibitiOn:as'.oecurs·<AiithAhe' ftl'Si:appr0ach. Klapia.'li ~mJY.
`1.n: tb°e' intetCeJWt(:rnf i# );ljth::e<Js: 3 "~n~ 4 .and· tli1;1s does not
`constant forthe'MJP::-'AC::C.C9mpla; In the oi!ldom bi-bi mccbaniSm
`influenee die shape ofthcf'curVe
`· ·
`·
`·
`·
`,.(bOtkim)l(i;,:K".ctK'D;ciKA.andKtare.Ciluano[EJlQONJ/~'.DPNJ.
`[El LA(;CJl,lE·AeCJ. lE·A~CllDQNl/tl}p<>~·h~·cCJ. lE,DQl'll~
`.. · . Equilibfiu'm Ge/Rf/'ira'tioli c;Ji;~,r,a~~~~~h~ .. T~:csti~ate
`. th~ affi. tnity'.or. MT.·P (or:subsira'te5', ·""11illbriu~ gCl filtra.tion
`[A~tl/[E·DOl1h\CC]; and fE·ACC}[A~CJ/(IMCC•ACC);re- .
`spcetively~·. A!>breViatiO!is:. E =' enzym:c; DON =:dono'r mcmbrane5; ·
`·
`-,.
`. AC<; .::acceptor ·membranes, and.• .. TO.or CE;
`chromatographywasj>etfofjnCd. Donor and acceptorvesiCles
`. ·: . . ·
`.
`were perpared and purified as d~cribed above.· A Scpbacryl
`S-300columri (Pharmacra, PiiicataW:ay,'NJ) (Ix 27-cm)\vas
`eqtiiiib111tedwithassa.ybuffer1ll0ileorassaybuffercontaining
`750 pmo~ of TG / inL,ofsubst~te '.~esicles .. M'.'f P (O.S p.g in
`JO mg of BSA) was loa<fed. onto the column, and its elution ·
`volu~e was m.easured in tlie abSence ~r·presence.ofvesiclC:S
`in the.elution buffer.· Ttie:elutfon priSitioiiS for vi:sfoles and
`MTP were· detennfoed ·by.measuring.radiaactiv.ity.and lipid
`transfer activity, resp_CctiveJy,. A sh;ft .in.tJie elution position
`ofMTP in th~ preseneeof vc:Sicles is indicative of MTP binding
`.
`.
`.
`.
`. .
`.
`.
`to vesicleS;·
`· ·The dissaciation.eonstants ( K~) for MTP-v:esicle coin pl exes
`were estimated from the shifdn the.'elution po$ition of. MTP
`using· the relationship Ko =.: {MT.P][VESJ/[MTP-,YES),
`where [MTP] eq'1alsthe fracti9n.ofthe.elution tim~ MTP is
`free; [MTP-VESJequaJs the fractionoftlieelutiori time MTP
`is.bo11-nd to .vesicle8, and. [YES] .equals the free vesicle
`concentration. Under experimental conditions, the total vesicle
`concentr~tion ·was 10 tim~ that of.MTP, assuming 3000 PC
`moleculc:S.per.vesicle (Huang & Mason, 1978) .. Therefore,·
`free [VES] was notsignificantly different·from·total [YES]
`(250 pmolofTG/mL), and the latter value was used in.the
`calculations.
`·
`·
`Fluorescent lipid Transfer. Assay. PC vesicles containing
`6 mot % cholesteryl 1-pyrenedecanoate were prepared by
`sonication.as described above except the vesicles were not
`isolated· by centrifugation following sonication. At· this
`concentration in the membrane, pyrene-CE is self-quenched.
`The fluorescence intensity of the VC$icles (0.36 nmol of CE
`in0.8 mLofassaybuffer) was measured at 380nm (excitation
`340 nm) in a Perkin-Elmer iuminescence spectrometer LS
`50B (Norwalk, CT). Excitation and e~ion· slit widths were
`set to 2.S nm, and the temperature was maintained at 23 °C.
`MTP (0.47 nmol) in 100 µL of assay buffer w-as added, and
`the time-dependent change in fluorescence intensity was
`measured. As a control, the change in fluorescence of the
`vesicles iri the absence of MTP was measured following the
`addition of assay· buffer.
`
`random bi-bi.kinetics
`
`v
`
`[DON] . ·.
`
`·
`
`..
`
`. .
`
`V ma• ·
`
`·
`
`:·
`
`! = _·_1_· -[aK0 (1 + [ACC]/K; + KA/[ACC])] +
`··v~.(1. + 1:~) (2)
`
`fn.!;>oth mec~anis~ at hi!li [ACC], aecepto~ membranes
`behave as competitive.'iilhibitors, and a series of nonparallel
`intersecting lines result. The slopes· of the intersecting lines·
`incr~se with increasing [ACC] in a oouilterelockwise mariner
`([ACCJ/K1 term dominates·stope)~ ·At low:[ACC]. (in the
`absence of acceptor membrane inhibition), [ACC]/ Ki becoriles
`insignificant., and this term 4oes not influence the slope. In
`this case, ping-pong bi-bi kinetics generates a series of parallel
`lines, while random bi-bi kineiics generates a series of
`intersecting lines with· the slopes ofthe lines decreasing with
`increasin~ [ACC].
`(B) Approach 2. In the second kinetic approach, [DON]
`was set equal to [ACCJ, and both substrates were varied
`simultaneously in a l: 1 ratio. Initial velocities were measured
`as a function ofsubstratc concentration. Actual concentrations
`of DON and ACC ranged from 2 to 1200 pmol of TG or
`CE/mL. Because [DON] is equal to {ACC], the donor term
`can ~ substituted for the acceptor term, and eqs I and 2
`reduce to eqs 3 and 4 for ping,pong bi-bi and random bi-bi
`kinetics, respectively.
`.
`·
`
`3 of 7
`
`PENN EX. 2150
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`Mechanism of MTP-Catalyzed Lipid Tran~porl
`
`Biochemistry, Yo/. 32, No. 39, 1993 10447
`
`- . ~
`
`~ 20
`··~· 10
`~ 0
`
`-10
`0
`
`30
`
`90
`60
`Time (min)
`FIGURE 2: Bidirectional and uiiidirectional lipid ~nsport. Lipid
`transport was investigated by· ~easuring the apparent 1."G. transfer
`overtime in the presence of lOOngofMTP. Donor membranes (300
`pniol ofTG/ril) oontained 0.25% 1.4C-TG, while il~ptor mcm·
`branes (lOO·pmolof l'G/mL) contafued either O.iS% !"C~TG (op~n ·
`circles) or' ooTG. ( 120 n.~ol/mLph~phatid~boline~. (5olid cli'Clcs).
`RESULTS .
`Assay Conditions,
`·Preliminary. experiments were . per(cid:173)
`form:c;d to establish conditiQns. for m,easuring initial velocities.
`At 2l°C; with 40Qng/mL ol'.le8sMTP.itr:igl~ride.irailsfer
`was· 1foear ~ith .time for. 60 'inin:at aifdoni>r concentrations
`~s~<(c~1{~oi:51i~r.): ·wit.ii in~~-tll~:4oo:ns/111i. MTi>,
`. or.fo(:11b8•ioii times' JQnge,t: !lia.n QQ ·min;:ti-ansfer v~ilus ilirie
`. deviat~ . fi:Om 'linearity.;. ,:Genc;f.lly; )hi$·, cor'rtsjio.nd~,f lo
`.g~tei'\tliin 3Q% trll~fer.; .· AU.:' kinetic·; ~x~~pts.,were
`perforfuC<I ai 2f 0.e..wi~·20o ng/mL (U .. ppioi/iO-)..>: Mn> ..
`Under ihese· C:oni;litil>ns: 3o9fi 1'8.nsfer. was ·n<)f exceeded: ·
`. · ti~id i~ansfe~ ·?r~leiDs. rria1 W-<;ffi<?i~ the :c~9h'il-iiie (bicii-
`... reCtional . movement) «)r ·:the ncf tran5fer (unidireetional
`· movem~nt) ·of iipid moleeules ·between membranes .. In o~r
`t~ansre; assays, theinitial TG conecntration.was o.2s mo!%
`in ~t~donor{radiolabeled TQ) a·nd a(x;ep~or (unlabeled 'J'.G)
`inembram;s. T().dete~ne wii~er MTP.promoted.excbange
`or net ·transier .of ro,. the uiila i>CJed id in acceptor vesicles
`·was repla~ with 14C-TG, and the ap~.rent timt>dependent
`transfer of ''C"TG was 1*~ured. If 14C~ TG exchange oc:Curs,
`the t•c~TG in·each\resfofo p0p\ll11t.ion would r~~ain consta,nt,
`regardless <>f:~TP-caialyW.l lipid tra11~p0ii Howe~er, if net
`lransf er occurs, one vesiclej~opiilatjo11 would be\x>me eiifj~l:icd
`in 14c-TG .. while the o~her wouid beci:ihte depleted. ·when
`radiolabeled T(J:~u inoo):pOtated in~ 1>.olhdoµQr and aceeptor ··
`me~branes; there. ~~s no tiin~d(t~ndent change in the ~~C- .
`TG <:Oote~t in ;acx~ptornieirlbniriciSas indic;ated by thea~nee
`o(~pparent TG tran8fer (F~gure 2). This dc;monsfrates lhllt
`MTP catalyzes 14C-TG excha·rige under thtse e~perimental
`conditions. Identical re5ulis (data not shoWTI) were obtained
`with CE transj>ort .. In subsequent analyses, first-order. kinetics
`were used (see Materials and. Methods) to calculate the total
`TG or CE transfer .. This corrects for the dilUtion or labeled
`lipid in the donor particles resuliiriB from lipid exchange~ When
`TG was omitted from acceptor vesicles, unidirectional trans(cid:173)
`port of TG from donor to acceptor. membranes was observed
`·
`(Figure 2).
`· Mechanism of MTP-Mediated Upid Tran.spoFI. Two
`different kinetic approaches were e~pfoyed to determine the
`mechanism· of. MTP-catalyzed lipid transport .. In the first
`approach. the donor membtaneooncentratfon was varied while
`the acceptor membrane concentration was held constant. This
`was.then repeated at varying acceptor membrane concen(cid:173)
`In the second· approac'1, donor and acceptor
`trations.
`membrane concentrations were set equal to each other and
`varied simultaneously in a t: 1 ratio. Each of.these approaches
`exhibits characteristic double-reciprocal plots that can be
`utilized to diagnose the kinetic mechanism as outlined in
`Materials and Methods.
`
`120
`
`. 0:03
`
`0.04
`
`.1i1QONJ (mllpmol TG)
`FJGURE 3: .Double-reciprocalplot-.apprQacb 1. .Plot of. l/v versus
`I /(DON) at varying a<Xeptor membranecqncentratioils. Data jloints.
`arc an average of tw.o initial v~ocity. I!le&surcments. Aa:cptor
`COt!CC11tations were 4 {solid triangleS), H>-(solid squarcs), 12 (solid
`circles), 90(open circles); 220(open9quarcs), and 436 (open triangles)
`pmol ofTG/mL Datli points·were fit to lines.by linear regre.ssion'.
`
`. ': ,•
`
`: iJ.2o
`0.30 . . . 0;40 .
`1llOONI. (irdfpmoi TIJI .
`FIGURE 4: DoubJC-rcciprocai pJot.:...:approacb 2 ... Plot of 1/v versus
`1/[DON))vhen [DONl=: (ACCJ . .TG transfer was measured as
`a function .of substra~c concentration. Data points are an average
`of two initial vclOcity measurements. Data· points were lit to a line
`b)'. linear regression (r '- ().997; y inteicept = O.OOSO). ..
`·
`Results of approach . .J are shown in· Figure 3. · A~ptor
`co~eentrations above.JOO,pmolof.TG/rnL r~l.llt in a series
`of. i~tersecting lines with 4ecreasing slopes· Witb decreasing
`(ACC]. This:is .diagilostii: of acecp~or·meinbrane inhi~it~on
`for both ·the ping~j>ong. bi:..bi .and .raD4om. bi~bi·mecbanisms.
`The change in slope ·is -_caused· by=·co~peli.tive ~hi~ti0n by
`acceptor. me111l>r!lnes ~s· [ACC) ~mes.s~if!Cl!nt retatiVe
`toK; ... At ~oW'[ACC], the double-reciprocalplot}'ields a ~es
`oflines tha~ appear parallel, with the V v .intercepts dccreasiitg
`with increasing [ACC). TJiese resultS.are consistent with a
`ping-pong methanisni; however, mechanism diagnosis using
`thisapproachisdifficultbecauseofthelimitedrangeof[ACC]
`wh.ich can be used. ·At high [ACC), there is substrate
`inhibition, and at low [ACC.), if is difficult to obtain acc;urate
`transfer rate8 .. Similar results were found for cholesteryl ester
`transfer (data not shown).
`·
`R.e8utts:of kinetic analysis by approach 2, in which [DON]
`= [ACC] and both are varied simultaneously, are shown in
`Figure 4. Thedouble-recii)rcical plot is a straight line which
`·is diagnostic of ping-pong bi-bi k:inejjcs (eq 3). Similarresults
`were obtained in two additional experiments performed with
`TG · and three experiments performed with CE (data not
`shown) .. In eq 3, Ki only influence:S the intercept, so a straight
`line results regardless of substrateinln"bition. Iflipid transport
`oceurred by a ternary complex mechanism,' the data would
`have fit a para?ola (eq 4) significantly better than a straight
`line(eq J)(Mannervik.1982). Theda ta were fit lo the generic
`equations 1/y =a +.b(l/x) and 1/y =a+ b(I/x) + c(I/x)2
`for a straight line and parabola, respectively. The parabolic
`solution gave an equation with a negative coefficient for (I/
`
`4 of 7
`
`PENN EX. 2150
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`10448 Biochemistry, Vol. 32, No. 39, 1993
`
`Atzel and Wetterau
`
`40
`
`30
`
`E
`
`~ 20
`l
`
`10
`
`100
`
`;:;. 80
`
`c ..
`
`Ill
`Q)
`
`~ 60
`~ 40
`Q)
`a:
`
`20
`
`0a
`
`9· 10 11 12 13 14 '15 16
`Elution volume (ml)
`FIGURE 5: MTP binding· to donor and acceptor membranes. A
`Sepbacry1S-300colu~ (l x 27 cm).wascquilibratcd in assay buffer,
`and the elution or MTP (O.S µg). was measure,Hn the. absence or
`presence or 25~ pmofof TG /mL of donor·µicmbrancs ( oiien Circles)
`or.ac;Ceptor mcmbrunes .. (solid c~) in ~e·elµtion.buffer. The
`eluti!)ti ~ks of di>nor pr aeceptor.v~icl~ alone; Ml'~,!l~nc, ·and
`a BSA·~.~ndard are indic;ated by ilrtows..
`·
`·
`.. x)2: . . i/j; =·.::o;c)()34,+ 2.33(1/x) ..;...·L1~(1/x)2, (:# 0.998,
`. C:ausll.ig ,i( to .~~i~il~i .. toJli.e; s~rai~t-~e~_ohitJon. of :l/J~·:=
`-0:<)949. +:·i;96(1 /~);; ~ ·o.~91~: :Foi ine:<:M~m'.di!lgnosis,
`the simj;iet'm·9d~I (eq' 3, ·i>in87P9n& bi~6ikinciiiCs) 'w!iS ch<>sen
`. over the p'iore eomj>lex inodelsince i)nefit'w~not'significaiitly
`bC:tter·t~·~ri-the. other (Ma'rinervik,.1982).
`· .• •.·.·
`··
`.
`..
`, Aj}Jnity;o/MTP for.,DoM,ra'nd1kc.eptor..Membranes.: The
`affinily<or MTP for. subSirate vesicles. was inve8tigated by
`"eqililibrium ·gel filtratfori cilk>matography: · tiie elution
`p<>Sitfon of MTP was measured on a Sephai::ryl S-Joo:eolumn
`equilibrated in assay buffer or assay buffer con'taining 250
`pmol of TG I mL of qoilQr Qr acceptor vesicles(Figure 5) .. ne.
`· elution o_f MTP was shifted to ~rlier. fractions i~ the preSc:nce
`of e,!uier dorior or acceptorv'eSi~l~; indfoatingth_at tvtTP. binds
`. to both'vesicfos. The affinity ofMTP for a~ptor inembnines,
`however, is' higherlhini its aJfinity (or donor membraneS as
`indi~ted by th'ela:rgei. shift Pi 't~e ·e1u~on piOSiiioil.of ~TP
`in :.the presence of: aceeptors; The estimated dissociation
`constants.{see Mater.ials.-and Metl:!ods) for·MTP-:<lonor and
`MTP-4ceeptor ~~icie·complexes are'·1~00.pmol ofTG/mL
`and 400 pinol of TG/rriL. respectively. The higher affinity
`for neutnil acceptot,vesicles.compared: to·~egatively .charged
`donor vesicles agrees witli the ob5erved substrate.inhibition
`at high concentrations ofacceptors, while no such effect was
`. observed with donors.
`. . . ..
`.
`. . . .
`. .
`The ability of cardiolipin (a negatively charged phospbO-:
`lipid) 'to decrease the.affinity of MTP for vesicles suggests
`that MTP·'"atalyzed. lipid transport may be :regulated by
`membrane charge. . To test this hypothesis, increasing C9n· ·
`centrations of cardiolipin were incorflorated into donor vesicles,
`. and the the effect liPon .TG transfer was determined .. Figure
`6 ·shows that as the content of cardiolipirifo donor membranes
`increases, the TG tra·nsferratedecteases. Similarresults were
`observed when phosphatidic acid replaced cardiolipin as the
`negatively chargCd phospholipid (data not shown).
`.
`.
`. Upid Binding Properties of MTP: Protein-mediated lipid
`transfer by a shuttle mechanism indicates that the lipi_~
`molecules are bound to the· protein as they are.transp0rted.
`To investiga~e the lipid molecule binding properties of MTP,
`a fluorescent assay was used to measure the time-dependent
`binding of neutral lipid to MTP. PC vesicles containing 6
`mol % pyrene-CE were prepared by sonication. The fluo(cid:173)
`rescence of pyrene-CE at 380 nm is dependent upon its
`concentration in the membrane (Wetterau et al., 1991 b). Al
`6 mol %, it is self-quenched. Pyrene-CE binding to MTP will
`be evident by an increase in fluorescence .upon· addition of
`
`.. -_
`··: ..
`
`;,:,
`
`30
`
`40
`
`0
`0
`
`10
`
`20
`Cardiolipin (mol%)
`FlauRE_6: Membrane charge rcglilates lipid transfer. Vesicles were
`prepared a_s described in Malerials and Methods except that they
`.were nOt Tractionat~ by centrifugaiiori. · ~rdiolipin content of. the
`.d~nor VC1Jic:Jcs.was var.i~ ~s show~:. '.JG ttaii.sfci; in the presence of
`200 ng/mL MTP was m~surecfat 23 °<;: in.~y buffer. ,Spontaneous
`lipi~ transfer (absence opYi:f P). wis less"tlian i% of the total.
`_ .......
`
`.·\
`
`·:.
`
`,''•
`
`.::
`
`.. ·
`
`<"
`
`.. :·
`
`12.0
`
`· . Time. (min)
`FiouRE 7: Time coursc·orpyr~ne-CE b.i.ilding to M'rP. MTP, 0.47
`nmol'(Solidcircles),or.a~ybufTer(!)pellcircleil)wasaddedt.ov~i'*5
`!X)nt.aining 0,36. nmol 0f pyrenc~CE, and the timc-dcpcndent ·change
`in· fluorescence wanncasurcd at.23·°C. Excitation and 'cinission
`wavelciigtbs were-.340 and 3~0 :nm; respcetlvdy .. The initial
`fl~tlT~CC int~itY: W8JI 237. .
`. ,. .
`.
`MTP to pyren~CE vesicles. wtiicb i~dicat~s that ~yrene-CE
`isreniovedfrom its self.:quenched envlronm~nt .. Figure 7 shows
`tl,lat when MTP is added to vc:Sicles, :a rapid incre&Se in
`.n~~.i:espe11!=e.is.~bserVCd,. Tilus,_ ~TJ>.biiJ.ds .pyreri.~tabeled
`chglesteryl ester. .
`·
`· ·
`·
`
`DISCUSSiON
`T~~- ind~pendent kinetic approaches were used. to dem·
`onstrate that MTP-catalyzed lipid transport is best described
`by ping~pong bi-bi kinetics, which indicates tha't MTP sbutties
`lipid molecules. between membranes. Sub,s~te inhibition
`complicated the analysis. In approach l, at high conccnfra·
`lions of aceeptor vesicles, both ping-pong bi-.bi and random
`bi-bi mechanisms exhibit incrwing slopes with i_nereasing
`[ACC) in a double-reciprocal plot. This occurs. because
`· [ACCJ:is significant relative to K;, thlis affecting the.slope
`terms of eqs 1 and 2. To.diagnose the kinetic.mechanism by
`t~is approach, (ACC] needs to be insignificant compared to
`K1. However, if [ACC] is too. low, accurate transfer rates
`cannot be determined. The first approach yielded a series of
`lines which appeared parallel (Figure 3) at [ACC] < 100
`pmol/mL, consistent with ping-po11g bi~bi kinetics. In this
`model, K1 i~ the dissociation: constant o( an acceptor-MTP
`complex. At [ACC] greater than 100 pinol/mL; [ACC) is
`significant relative to K; and substrate inhibition is observed,
`which is in agreement with the K; predicted from a direct
`measure of the MTP-acceptor .vesicle dissociation constant
`(400 pmol/mL) by equilibrium gel filtration.
`
`5 of 7
`
`PENN EX. 2150
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`. f" ........................ ,,,, _______ ,, .. : ... :._· ~----"'''''''""": ................... :,_~---
`. ~
`I.
`
`Moch~;•m of MTP-C"•lyzod L;p;d T""'"°''
`
`Biochemistry, Vol. 31, No. 39, 1993 10449
`
`~:
`' t
`
`L
`h
`~
`
`~
`,,
`i
`g
`..
`0
`i-
`
`>
`
`60
`
`40
`
`1000
`
`800
`
`600
`
`..
`
`.,
`l! ·a
`i ..
`!
`
`400 J
`
`200
`
`0.1
`
`02
`
`().3
`
`0
`
`0.A .
`
`1/(DONI
`
`FIGURE 8: Doublc·rci;ip~oail plot ~~ng.riindom bi~bi kinetics.
`The experimentally dctcl'mined KD·arid KA aild eq 4 .w~·usCd to
`calcula_tc the relationship bet.ween th~ initial_vc!QC_i~Y..and.{DON].
`The results are J>lottl)d as V _,/b versus l/(DQN) (solid'cirdes ). For .
`comparisOn, tlie 1 /(00N}1 term.in i:q 4 was ouiittcd~ ·~lid. JI maxfD
`versus L/(DON) -~.aneplott~ (open. circles).
`·
`.. ,
`·.
`.
`· .
`·-.
`·.
`· .·· · .. ·
`.
`. ·
`.. Approach" 2 is<more ·definitive• th~ appioich 1 for
`determining the kinetic mecbii.niSm bCcau5e the shapc:S of.the
`. double:reciprotalplot8 are.nm affected by.sµbsgate inhibition
`· 'and a large range of ·substrate ooricentratit>iiS c:Quld-:bC
`
`shuttle mechanism. MTP affinity for vesicles was sufficiently
`low that equilibrium conditions had to be used to detect MTP(cid:173)
`vesicle interactions (i.e.; stablC MTP~vesicle complexes could
`not be isolated). CETP, in contrast, has high affinity for its
`lipoprotcin substrates. Stable CETP-high-density lipoprotcin
`or CETP-"phospholipid vesicle complexes can be isolated·by
`gel permeation chromatography (Pattnaik & Zilvcrsmit,
`1979). The.high affinity that CETP has for membranes would
`enable the formation of ternary complexes in CEPT-mediated
`lipid transport .. ' .
`· .
`. ·
`·
`.
`Although MTP affinity for membranes' is low, transient
`membi:ane interaction dcies Occ:ur. MTP had lowei affinity
`for negatively charged vesicles than for: neutraily charged
`· vesi~les, suggesting that surface~charge plays ~ regulatory
`role-lD· ~TP-m.~mf?rane bincijng.and.lipid transfer. To test
`~is. TG ___ transfer: to. accepto_r vesi~ies_ was ~ea_ .. ·'.s.ur_ed usi~g-
`. danot inembranes.~iltairung_inc~sirig al!lounts o_( negatively
`charged phosphohp1d .. J~ci'eased negativ~ charge resulted in
`decrea.si:(I rates ofT:G ti-ansf ~.' T_be actjvities.of.C~TP (tall
`· et at., l986):and_ the cy~solic PC-specific transfcr·prot~in
`(van. den.~ela~r ct at,J97S;'Wirtz·ef ai., -1979;· DiCQrleto
`. et al,, I ?!71 are a,lso ~~gtiiated by membr~ile_surface charge.
`._,\-lter;at,lO!l,ofmen'lQ.r~nesurfa~ch~rge~Y ~e a~.a means
`
`. ~j;~;::~·~t ~;:~r:~7:~~~~~$.r:·4~~ti~s~:J,~::· • ~r~~u~~n:a:isj! ~~~:;~~~!~:t~~~~~~:~~~pd in~
`
`. gly(:eride,!as8ociattVto·:fonn' a·'lipoprot~ur Jiiirueie·"is··not ·
`not' affected ·by 'subst~ate inhibition: Th'e disurigwsti1ng ·
`. undi:rstoqd:· Stu4ies .. f>Crfor.m~ i~ Hep:Gi-<;en~ sµgg~t that
`parametedn this appi'oi;lch isJhe·aKi)'KA/{DO~P teitJfbfeq
`4~. When [J;>ONJ2.» ·aKi;KA; thii; term appr0ach~ '~ro and; . .
`· in-:thc initia1 stag~ of lipopr:~tein · a~em~ly,' aPQ. -~ .. Ctitran8-
`· .. eq 4 is simifar:to eq·3. ·Ther~fore, itiS neces!13ry:10 niairitafu .
`la~ioni)Uy as$Qciates wit1' lipid OJor6n et al., J 992); A .. smail
`substrate concentrations low enough so that this term ci<>es
`particle with 'the density ofiiigh--Oensity lipoproteins' appears
`not drop 9ut of eq 4. · To demonstrate that Uie substrate
`. to _be an intermediate in the ii!lSCmbly process (Bostrom et al.,
`concentrations used in the study-would generate a parabola
`1988; Bor6n et al., 1992). · Adqitional lipid is then.added to
`if MTP utilized a. random bi-bi mechanism; the estimate4
`. this particle tO fonn a mature lipopl'.Otein pa'rticle, possibly at
`a site distinct from apo B syntJ1esis:_(Bor~n et al., 1990). In
`:;a!uc;s of K,. (400 pmolof TG/mL) and Ko (1800 pmol of
`TG/inL) were used to calculate the relationship between 1 /v
`the abs,ence of sufficient oleic acid or triglyceride synthesis,
`apo B in a membrane-bound form or in a higb~density naseent
`and I/ (DON]- a_t lhe [DON] used· iii', the study. When
`calculated V ,,,..,:_/v values are graphed as a funCtion of
`· particle is degrad~ intrac.e!lularly (Boren et al.: 1990; Sato
`l/[DONJ, a parabola is generated which can be. easily
`et al., 19~; Dixon· et al., 1991). Studies·demonstratfog the
`absence of MTP in.abeta~ip()proteinei:nic subjects (Wetterau
`distinguished ·from the straight line which is generated when
`tlie l/(DON]2term is elimmated.frotri eq 4 (Figure 8). A
`el;llt,)992) _not 9nly su_p1>9rl a role for _MTP in VLDL and
`. sim.ilar parabolic cµrvc Was o~rved \vben.we varied a from
`Ch.YlOJnicron asse~bly but t_hey also suggest thatit iS ~~i.rCd.
`. However;•the preeise rolc·of-MTPis'rioi. known. MTP may
`I to 100 or wJicn KA and ·Ko. were mcreased or deCreasCd
`l 0-fold. This anlllysis demc>nsuatcs th.lit the experimental
`p1aya role fothe early stage8 oflipoprotein assembly, possibly
`conditions used in the. study were ·appropriate to determine
`mediatingtbetransportoflipid tona8';ent apo B peptide chains
`.yielding a stable, secretion-competent form of apo B. It may
`the !cinetic mechanism. The l'CSulting straight line obtained
`from this approach (Figure 4) is diagnostic-_or a· ping-pong·
`_also play.a role in·laUer stages of assembly. It is not clear
`whether apo B particles are bound· to the ER membrane or
`bi-bi mechanism; .
`if they are free in the ER lumen during the various stages of
`lipoprotein assembly. In either caile, MTP by virtue or' its
`shuttle mechanism of action is capable of transporting lipid
`to an acceptor particle which bas no d'rect contact with the
`ER membrane.
`
`. The lipid bi~ding properties of MTP were studied as an
`independent approach to investigate the mechanism of MTP·
`catalyzed lipid transport. A shuttle mechanism predicts that
`a stable MTP-lipid complex is an intermediate in the transfer
`reaction. A fluorescent assay demonstrated that MTPbinds
`neutral lipid molecules (pyrene-CE) rapidly (Figure 7). in
`agreement with thekineticalJydetermincd shuttle mechanism.
`Additional evidence for an MTP-lipid complex was obtained
`by chemical analysis of purified bovine MTP, which was found
`to contain a few molecules of bound lipid (Wetterau &:
`Zilversmit, 1985).
`MTP is a soluble protein found within the lumen of
`microsomcs. It is readily released from microsomes by basic
`pH