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MOU.ci L.in nmtM.M-oLoav, 10, 110-120
The Influence of Dichlorodiphenyltrichloroelhane, Polychlorinated Biphenyls and Anionic Amphiphilic Compounds on Stabilization of Sodium- and Potassium-Activated Adenosine Triphosphatases by Acidic Phospholipids
Chanles W. Sharp, Dohothy G. Hunt, Samuel T. Ceementm, anu William E. Wilnun
Pathologic Phyeiolvgy Branch, Xuiiongl Iuetitnle of Environmental Health Scttncee, Hfearvh Triangle Park, Xorth Carolina 17709 (Received May 4,1073)
SUMMARY
Sharp, Charles W., Hunt, Dorothy G., Clements, Samuel T., and Wilson, William IS.: Tlu* influence of dichlorodiphenyltrichlorocthane, polychlorinated bi phenyls and anionic amphiphilic compounds on stabilization of sodium* and potassium* activated adenosine triphosphatases by acidic phospholipids. Mol. Pharmacol. 10, 110-120 (1974).
Dichlorodiphonyltrichloroethane (DDT), extensively chlorinated biphenyls, deoxycholate, and piv'pholipnae A inhibited beef brain and rabbit kidney (Na+ 4* K+)-ATPases (EC 3.0.J.3). Phosphatidylserine or phosphatidylinositol but not phosphatidylcholine or phosphntulyl'.-thanulammc prevented or reverwed the inactivation of the ensymon by each of theso inhibitor*. Albumin protected against and reversed inactivation of (Na+ + K+)-ATPases by deo\ych>dttto, oleate, or dodecyl sulfate; however, this protein waa less effective against in* activation by the extensively chlorinated hydrocarbons. The extent of (Na+ + Iv^-ATPase inactivation by anionic nmphiphiles waa dependent upon the temperature at which an enzyme-inhibitor mixture waa incubated prior to assay, whereas inactivation by chlorinated hydroeurljona waa not affected by temperature. Our experiments lead to the hypothesis thot acidic phospholipids arc necessary for stabilization of the enzyme and that chlorinated hydrocurbons, deoxycholatc, and phospholipase A interfere with the stabilization process.
INTRODUCTION
The Midium- and potassium-stimulated adenosine triphosphatase |(Na+ + K*) ATPnw, EC 3.6.1.31, which is generally thought to comprise the membranal so* dium-pumping system (1), is inhibited by various chlorinated hydrocarbons such as DDT1 (2-4) and polychlorinated biphenyls
' The hblircvmtionB u*ed are'. DOT, 1,1,1* iriclilorx l,2 l)ia(^-clilor>>phaiiyl)lhnne; DDE, 1,1* dirlil'-ro 2,2 - bi(p ( hlor<i|>heny))clliylcno; PC'IIn, p..]yi'1iti)rimlcil biphenyl mixture* Ktirh as Arui lor 1221 or Arncior 12W.
Coto rxlii <1 UI1 )>y AeHtmie l'r. Ine. All r.*ln cl r*|iN>4uMl* In > l**n
I
(5, 0). The possible toxicological significance of inhibition of this enzyme by the chlo rinated hydrocarbons has received its most substantial support from studies of toxicity in eels. Kinter and co-workers (7-9) ob served that DDT and PCBs were capable of inhibiting eel intestinal mucosal and gill (Xa+ + K+)-ATPases to the extent that control of osmoregulation was luM, leading
to death. Since, under the conditions used for en
zyme inactivation, the chlorinated hyilrocarlKjn* exist as oil-in-water dispersions, ihc
y MONS 081950
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120 SHARP KT AL.
physical state of tho inhibitorM appem* to lx* of great importance. Bemuse the hy drocarbons are essentially insoluble in water, it is possible that (Xa+ + K+) ATPase inactivation results from interactions with either dispersed (micellar) or soluble (pre sumably monomeric) hydrocarbons. More over, enzyme inactivation by chlorinated hydrocarbons should be compared with that caused by oleate (10), dodccyl sulfate, or deosycholnte, as several of these anionic amphiphilca have been reported to effect
enzyme inhibition where they exist in micellar form (11).. Since phosphatidylserine stimulatesenzyme activity following (Na+ 4K+) ATPase exposure to phospholipase A (12, 13) or to deoxycholate (14-17), we have investigated the selectivity among phospho lipids for protection against and reversal of enzyme inactivation by chlorinated hydro carbons, several amphiphiles and phospho lipase A.
MATERIALS AND METHODS
Chemicals. Dodecy] sulfate, ATP, XADH, phosphoenolpyruvate, and deoxycholate were obtained from Sigma Chemical Company. Fraction V albumin (Armour) and defatted,
crystalline albumin (Sigma) were used inter changeably. Individual biphenyls were ob tained from Aldrich Chemical Company. Aroclor J 221 (biphenyl plus mono- and dichlorinMod biphenyls) and Aroclor 1234 (tri-, letra-, penta-, and hexachiorinated biphenyls) were obtained from Monsanto.
Phospliatidylserine was prepared from bovine brain (18). Brain phosphatidyiinositol and Lubrul WX were obtained from General Biochemicals, Inc. Other brain phospho lipids were obtained from Supelcu, Inc., Bellefonte, Pa. Each phospholipid was at least 90VA homogeneous by thin-layer chro matography in three systems (10-21). Phos phate contents were determined, according to Dittmer and Wells (22), to be 1.25 pmolcs/mg of phosphatidylserine and 1.20 pmoles/mg of phosphatidylcholine. Dry phospholipids were dispersed in 0.1 m TrisHC1 (pH 7.0), using a Teflon pestle and glass tube homogehizer, Pillowed by 30 sec of smic oscillation. Phospholipids were also dispersed by the technique of Stahl (23).
yhosjihttlipuxe .1. Phospholipase A (Viprra
usselli; Sigma Chemical Company) solu tion in 50^ glycerol was heated at 100* for 8 min prior to use (24). The extent of hy drolysis of the various phospholipids was determined by thin-layer ehromatugraphy (23) coupled with phosphate analysis (22). Phosphatidylserine was routinely used as a reference substrate for phospholipase A, as this pure phospholipid was hydrolyzed at least 10 times more rapidly than phos phatidylcholine under the conditions in dicated in the legend to Fig. 4.
(Na+ 4 K~)`A TPa*r /irejiuralion. Bovine brain (Xa- + K*)-ATPase was prepared according to a modification (26) of tho method of Skou (27). The enzyme was stored as an aqueous dispersion at --20* at a protein concentration of 30 mg/ml. Prior to assay, the enzyme was thawed and the protein concentration was reduced to 1 mg/ ml. Protein was determined by the technique of Lowry et al. (28).
Babbit kidney (Xu-* 4 K*)-ATPns* was prepared by a modification of the method of Jurgens*'!) and Skou (2tf). I11 the dooxycholute activation step 34 nni KC1 and 160 m.u Nad were present in addition to 0.0
mg/ml of deoxycholate, 2 m.u EDTA, and 25 imi imidazole at pH. 7.2. (Xa* 4- K+)* ATPase accounted for 85-00'.7 of the total ATPase activity in these preparations.
(Na* + K~)-ATP<tsf orfinty assay. Lac
tate dehydrogenase and pyruvate kinase were obtained from Sigma Chemical Com pany and were dialy zed against 0.02 u Tris-HC), pH 7.4, prior to use.
The assay medium wa* incubated at 37* for 4 min prior to initiation of the enzyme reaction. The reaction was usually started by adding 40 vtt (40 of beef brain (Xn+ 4 K+)-ATPase protein or 10 mR (10 al) of rabbit kidney enzyme protein to yield a final assay medium volume of 1 ml. Phos pholipid dispersion* nitd/or inhibitor solu tions were added in the sequences indicated in the tables and figures. Initial velocities were determined by measuring the rate of XADH oxidation at 340 nm (30). Final
concentrations nf component* of the incuba
tion medium were 3 mu ATP, 3 mM Mgt'h,
125 mu XnCl, 25 mu KC1, 1.5 m\i phew-
phoeitolpyrmate, 0.25 m.u XADH, 2 units
M0NS 06195x
OUT, mm, AXtt AAIPHJI'HILES as (Xn- + K+)-ATPA.SB INACTIVATORS
121
Tajilk 1
Putin (A* + A!*)-A TPute rcxponxtveneist to dixperxiona of oleale and chlorinated liyln,rnrbon*
Inhibitor! wti-o riiliodnced into the tuny medium ns lolutioii* in dimethyl sulfoxide. Ai rhe ai*ay cfiiirentriitinn of 1`r (v/v), dimethyl sulfoxide hud no effect on euxyme activity. (Na* + K_j-ATPaie ppaifir act ivity wan 40 inuln of Pi per milligram of protein per hour; enzyme protein coin filtration in the nscay whs 40*g,'ml. Ahmvv conditions are described under uatkiuala and uhthoda Hcsulis are menus ntnudard errors for three determinations.
Inhibitor
Inhibitor concentration
Order of addition of emyme and inhibitor to assay medium
Inhibitor added 15 sec (Na*- + K*)-ATP*se reaction iniuated bv
after initiation of adding enzyme at indicated intervals after
(Na* 4- K*)-ATPae
inhibitors
reaction
--------------------------------------------------- ------------
0.1mm
20 min
120 min
None Trii<>oh>ate
l)lT Arot'lor 1254 Ai"lor 1221
(i.tf PP>
100 20
10 15
100 40 4 44 4
42 4 48 ^ 4
% cenirct activity
43 3 44 i 47 4 40 * 4
42 4 40 4 53 * 3 48 . 3
43 4 11 = 4 55 4 >7-4
of lnctnte dohyclrogenn-e, 7 unit** of pyruvate kinase, mid 30 m.u Tris-HCl, pH 7.4.
In order lo ascertain the validity of the results obtained using the* coupled lactate ih-hydi'iigcnuw-pyruvate kinase system, we initially determined that the rate of NADU oxidation wn* unaltered when 0.15 m.M ADP was tilt'd a* substrate in the absence of (Xa- + K'f)-ATl>a.sc and in the presence of each amphiphilic compound, hydrocarbon, phospholipid, and combination thereof. Also, most of the data in Tables 3 and 4 were cor roborated by results from our phosphate unalvsi* lechnique (20b
UF.HlT.T*
(.W + K')-ATPome reeponeitxneu to unfc,-<tiofu,'/e vibstunces. Inactivation resulti-d from interaction of (Xa+ 4- Iv+)ATPase with oil-in-water disjwwiona, rather Ilian solutions, of fatty acids or chlorinated hydrocarbon*. The extent of (Xa* + K+)ATl'aw inactivation by chlorinated hydro carbons or by oleate was not appreciably influenced by the time at which the enzyme wu> exposed to inhibitors .(Table 1). Char ut|U"ms phases obtained by centrifugation of oleuie, J'CUs, or D1)T dispersion* (Table l) for 30 min at 100,000 X <j were not in hibitory.
Addil ion of ethanol solutions of DDT (31), 2*<liliinl)iphcii\ 1^ I'CB mixtures, or Tris-
oleatc to water resulted in the formation of dispersions whose particle sizes and. or num bers appeared to change continuou-Iy for several hours (31) (Fig. 1). The spectral alterations accompanying the change of phase of 2-chlorobiphcnyl (Fig. IA; re flected contributions from hydrocarbon in solution and from light scattering by micelles; however, the alterations accompanying the change of phase of oleatc (Fig. IB; reflected only the time-dependent increases in micelle numlicr and/or size (32).
In a concentration dependence tudv of (Xa~ + JvD-ATPastf inactivation, an in crease in the chlorinated hydrocarbon con centration resulted only in an increu.-e in amount of inhibitor in the dispersed phase (Fig. 2). However, the extent of enzyme inactivation increased as the extent of chlo rine substitution of biphenvl wa> increased
(Fig. 2). Concentration and temperature di prudence
of (Xa* + K')~ATPase inmtuotion bi/ anionic amphiphilic compounds. The curve
for concentration dependence of brain (Xa~ + K-)-ATPnsc inactivation by oh-ute had a different shape from the curve* t'-.r inactiva tion by dodecyl sulfnte(Fig. 3). Thi- may be due to the occurrence of enzvnv- innriivation at assay concentrations below- tin -critical micelle concentration for doderyl -ull'ate (ealeulated to be about 0,17 him from ref.
HONS 0H195i
Fro. 1. Absorption npnirn of 67 \r 2-chhrobiphenyl (d) ami 26 pH Trix-uhate {H) in clhm.ol , nil'/ in 1% cthunui in water (------)
KpecUnl idlerations in the ethanul-water solutions were determined nr the indirnted interval a .r . preparation. Hpeetra were recorded with n Heckman Acta V spectrophotometer.
Fro. 2. Concentration <lepf>.<lenre of bruin (A'o*- + K*)-ATi,at inhibition by DDT, Annlnr t.-.i,. Arorlor t22l, t-rhlorobtphfiiyl m.it biphtnyl
Ten mieiolltors u( a dimethyl sulfoxide solution of each vldorinnted hydrocarbon were added : nfler addition of 40 pg of eiuyme protein to initinle the ATPiise nanny.
122
Km. 5. Concentration dependence oj ire.- u\'a+ + K*)-ATPat inhibition by Tn. d sodium ihnlu yl mifnie (SDS)
Dispersions of eneyme plus inhibitor, b at 12.3-fold higher concentrations than di:r:: ; . ibeequeiit nssny, ware incobuled in a medium conra.r.jig 5 mat KCI, 24 m.u NaCI and 17 mu Trii-HCl. pH 7.5. Iimibnt'on we conducted fur 10 min at 0s r 37*; then 80 mI of thia mixture were added tv ''o) w) of the aseuy inixture (aea matkwals and xietho: in order to initiate the (Na* -f K*)-ATPi*e >.ctioti.
11), whereas micellar oleatc was r-, tired lu HTift inactivution.
Following prior incubation the ionic mnphiphilc* (but not the hydroearb-i in activated (Xa+ + K4)-ATPtt6ffl in a tem)H'r;itun-depcndcnt fashion (Fig. 3). 1:. eontmat with results obtained with c -iecyl sulfate, accentuated enzyme inaetiva;: n by tilcatc wna observed only at an n_i'jitor concentration greater than that requir-d to c/fert 40% inactivation.
The extent **C (Xw +- K.")-ATPa*r inac tivation hy deoxychnlftte depended up n the absolute quaniilioa of ensyme and inhibitor aubjcctod to prior incubation at 37 Table if). Temperature-dependent enzyme inac tivation wan only observed following prior incubation at deoxycholate concentr^'ions appreciably in exeeaa of the critical rv.yellc concentration (calculated to be ah'.>:- 1.2 rmi from data in ref. II); however. ;-avchnliilc enneeiitratioMK during the u.--uv -A-ere always Mow this level.
Pmtrrtion of (.Vo4 + K*')-.\Tl>u*< >. -n'tirt inuilivuUon hi/ anionic um/ihiphi!< m-
poum/s. I'lu^pImtidylNcnue awl ple-x.ati-
dylinositoP (not shown) protect'-i |>.,th (Xa+
f- K+)-ATPascs against inae-.iviuon by deoxycholato when prior inrubari ,-r. was con ducted at 37; however, phnpha*i Icholine and phosphatidyicthanolaniimr r. t shown) provided no protection (Table > . Bv con trast, each phospholipid protHi'-i the en zyme against inactivation hy ieatc or tkxlccyl sulfate. The indicated iebiomet ric relationships among eazymr. inhibitor, and protective phospholipid w*-- optimal for the observation of ph'*ph';.ipuJ selec tivity; however, identical re.-u!`- were ob tained over ft 4-fold range of eori'^Mrotioiia of either phosphatidylcholine <r ;>bo-phnti-
dylserine (i.c., from one-hall to rwicc the
* In each instance when protects..;. . - reversal
by phosphstidylinositol or phoMihir-v.teihanol-
amine is mentioned only in the
s>:ui.Ta,
at least two analyses were pcrtvrr:--4 mi cniio
did (he reeults using pho`p)in<). !'i r. .'ivtrttnine
differ significantly from those mil.: p*. -phatidyl-
choline. Tlie commercial lv>vir.e l-.,i phoa-
phnlidylinositnl consistently
iJ- ; --- ills sim-
ilnr t< those obtained with imr br:>:: ;'t. vlmt idyl-
serine; however, rommercial prep-
: - <4 plant
phosphatidyl inositol hnvc yielded vnr.air !t- results.
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MONS 081954
Taiim. 2 Influmn of prior incubation on extent of bruin (,V' -f K')-A TPu.se tnaclu n/io.. )., ./rost,,
Deoxychnlate cimcnilnitinit
(lining USSIIV
Standard assay'
Prior incnhalinti At multiples <d (do'.ychr>lM*i n
),->*
X3.1
X6.2
XUS
"* *"x>r
0* 37* 0* 37* 0* 37' (i 37'
0 04 m 235
510 1020
'.c control activity
100 92 SO 80 91 88 Si 57 91 85 84 74 74 04 73 72 79
85 73 58 W* 05 _7101__ Hi 21
07 04 50 __ 07 01 51 28 20 10
48
45______42_ 44 20" 17
&U
20
*21 10
12
4
* As described under m.\tkhials anu :u>;thoi**, 4()pR,'tnl of enzyme protein mwl th*- !`-ii<mifed .um-mt of deoxychnlule were added separately tu the cuvette in order tu obtain the results iii this column Con trol (Na* + K*)-ATFuse specific nativity was tffi/unvleg uf Pj per milligram of protem per hour Result h are averages of three determinations.
* For example, the multiple X 12.5, at 255 jm deoxycholate, indicates that 500 $ <-\ enzyme protein, 3200 mm deoxyeholaio, and 17 him Tris-CI, pll 7.5, were mixed and incubated fur 0 min .it O' or 37'; then an aliquot was diluted 12.5-fold into the Assay cuvette, containing a reaction mix' .re ?nrh time nssav conditions were identical with those of the atundard assay. Results below the short lint- represent !, lumperature depeudeuee effect following prior incubation.
Taui.k 3
Vhoepholipul protection of mammalian (.Va1- 4- K*)-A TPaeen agatimt ioa>` 1 oho,, t,,j
tcvtral anionic amphiphilic compoutult
Seethe legend to Pig. 3 for n general description of the incubation tcchuupie. Albumin r ph<upho.
lipitl whs added to the (Nn+ + K+)-ATPasc dispersion prior to addition of the inhibitor dilution. The
couecntrntious of chcIi component of the incubation mixture wne 12.5-fold greater
hrniu hikiuki
or 50-fold greater (rnbbit kidney enzyme) than during aasay. Respective specific u.iivitics r<-r ln-e/
brain and rabbit kidney (Niv4 + K^l-ATRases were 40 itud 140/mtoles of I'i per milligram <-f prutem pr hour, while corresponding protein concentrations were 40 and lOag/mi during assay Kiuyme rtivit\ unsay conditions are described under matkiuami and methods. The vtduea nre men;.' ->f *ix determina tion* (SUM S HP ,*).
Knsyme source unrl inhibitor
Tcm- No protective Phosphatidyl- I'hnsphalicyl- Albumin
pernture compound
choline
serine
(6.23 nig mb
(1.ft mg/ml) (1.6 mg r9'
Drain (beef) N inhibitor
Oloate, 1.25 mM Dodecyl sulfate, 0.84 m.u
Peoxycholale, 3.4 mM Kidney (rabbit)
No inhibitor
Dodecyl sulfate, 0.84 mM
Deoxycholate, 3.4 him
c % control activity
0 100
100 (92)
100 (129.
ion (9.1 j
37
100 (08}*
100 (96)
100 (140-
100 (91)
0 30
90 93
94
37 7
80 84
70
0 71
75 80
94
37 11
(57 00
88
0 54
71 83
73
37 19
15 62
60
0 100
100 UK)
100
37
100 (03)
100 (93)
100 (93/
100 <95/
0 90
100 100
102
37 23
100 100
92
0 58
92 99
97
37 22
18 93
90
" Values in parentheses are percentages of the 100% values at the top of t lie column honied "\u pro tective. compound" for cnc.lt enzyme preparation. All other values nre relative to the ippropnatn 100% value (i.o., the ones obtained with no inhibitor preaeut during prior incubation at 0; ;it 37*:
124
HONS 0S1955
\ VT-
IM>T, JH US, AMI AMPHIPHILES AS (Xll+ + KJ)-VnAE JNA(.TlV.\TOS
1 _>j
Taiiu: 4 I'hoy/tholi ftt'l uo-i nlOiiiui" jiruictiion of hrnin (An* +- K*)-/lTPaae nyut -/.' i-hiOilion by
. - vhloniinled bipheuylt and DDT
irchnUiii** are deforibed in the legend to Fig. 3. The (Nn* + K'l-ATPajie protein con cent rut inn \vm 40 .g ml during i ho Muy. Ynlno* are menus almirlurd error* of four determinations.
Inhibitor
Temperature during initial
incubation
No protective compound
Phospha tidylcholine (133 #ig/ml)
Thosphatidylserine (133 Mg/ml)
Ph-Hpha- Alliumin tid\ lir.osiiol (1000#<){/ml) (133 ns ml)
None HDT, 3.3 ppm Arwl'ir 122), 10 ppm Arucltir 1234, 5 ppm
c
0 37 0 37 0 37 0 37
100 100 (98) 40 8 45 * 3 44 sb 7 42 * 0 48 4 4(1 7
100 (92)* 100 (90) 70 8 70 =fc 2 08 7 no 8 07 0 70 -fc 8
\ iontrei activily
100 (135) 100 (145) 95 9 72 8
103 zt 7 08 * 4 95 9 88 8
100 M10) ICO -108/ 90 == 1
=5 (*' = (i
-5 10) - 9 95 = 5
100 (99) 100 (05) 4(1 ^ 5 49 * 0 06 * 5 05 4 07 5 GO t
Viilm-s in j>HmnheM*8 nro percentages of the control (Nnf + K*)-ATPaai* rr .vit\ shown at the top of Hip column heuded "No protective compound."
concentration* of phospholipid indicated in Table 3).
Albumin, at I times the ma.-s of the phos pholipids, effectively protected the brain enzyme again.-t **Mth of the anionic amphiphilcs (Table 3).
Protection of (A-a* -I- K*)-A TPa*t$ against inhibition by chlorinatcrl hyd.nrarbofit. The prior incubation technique was not optimal for observing sdectivu phospholipid protec
tion against (Xn* 4- K+)-ATPa*e inactiva tion by chlorinated hydrocarbons (Tabic 4). Both albumin and phosphatidylcholine pro tected against the chlorinated hydrocar bons. Furthermore, both photphatidylserine and pho*phntidylinositol activated the con trols (Tables 3 and 4). Since it was not possible to determine the extent of chlo rinated hydrocarbon influence on phospho lipid activation of the control.-, the validity of our Arbitrary correction of the values in the vertical columns of Tabic 4 could not be
determined. In contrast with the above results, acidic
phospholipids did not increase control (Xa* 4- K-J-ATPam* activity when l>oth enzyme and phospholipid were added directly to the assay medium (Table 5). Acidic phospho lipids M-Icctlvely protected the two (Xa+ 4K+)-ATPr*cr against inactivation !>y deoxyrholiite, DDT, mid Arodor 1 i?o4 (Tabic 5).
Albumin protected aguin-t 'mzynic inac
tivation by deoxycholati.- but not against
that caused by DDT r Arodor 1254
(Tabic 5).
Rtvursal of (Na*- -f-
TPaae inac
tivation by hydrocarbon* by amphiphiles.
Albumin and both acidic ami neutral phos
pholipids reversed brain (Xa+ 4- K+)-
ATPasc inactivation resuming from enzyme
exposure to dodccyl sulfate. oleate, Arodor
1221, or 2-chlorobiphctiyl (Tabic 0). Only
phosphatidylscrine or phv*phatidylinositoI
(not shown) effectively reversed inactiva
tion of the enzyme by DDT or Arodor
12.14. Albumin, at 2000 nft ml, reversed inac
tivation by deoxycholate. DDT, or Arodor 12.14.
The conditions described in Table 6 were
nearly optimal for demonstrating selectivity
among different compounds that could re
verse inactivation by tin dominated hydro carbons. For instance, a 5-fold increase in
phosphatidylcholine concentration gave only
15% greater reversal of inactivation by
DDT. Phosphatidylserine. at ;s0 jig/ml dur
ing assay, was essentially ; effective as it
was at 100 jig/ml,
Phmphnlipid netectivity in reversimj avd
protecting ayainst (Nn-- K~)-A TPase in
activation by phnspholipoa* A. Phosphutidyl-
scrinc and phnsphatidyUn*>~itol reversed the
inactivation resulting from (Xa+ 4- K*)-
ATPa.se exposure to pine-pic-lipase A (Fig.
**,' 1 / /. \ ' \'
j
* '*>: i
- tW-:
. 1`rotet'hoti "f iniiiiiintiliaii (A ' f A* >/! Tl'nsrt with inhibitor it ml. <r prnm-t we i-oiii/i'' ! /iiettn! mihj dnnntj assay
Hmin and kidney enzyme protein < fiilrulimi* wore 10 mid 10 ng/m). respectively, d:: ,u t lie nanny; respective specific net ivit ice were- tf) mid HO jin tiles of l*, per milligram i( protein perh r. Tlie reliction w;ih initiated by adding un/.yme the assay mixture, which contained protective <<; M 37*. Inhibitor solution (10 fA) iviw added immediate)) after t lie enzyme. Value* are mentis six determinations (SKM S &10rA. forem-h value).
Knuyme source and inhibitor
No o
Phosphatidyl- Phosphatkiylserine Alim
choline U00 Mg/ml)
(100/jg/ml)
(800,.
llmiu (lied) No inliibitor jJeoxyidiulntu, 000 >im
Dodecyl sulfate, UN) um Oleate, 100 hm Aroclor 1221, 20 ppm Aroclor 1254, U) ppm DDT, 3.5 ppm or 10 pm Kidney (rabbit) No inhibitor DeoxydioUte, 000 Doderyl sulfate, 100 ma Oleate, 100 ** Arorlor 1254, 10 pptn
DDT, 10 mm DDK, 10 M
c control activity
104 f.5 52 73 84 81
1U1 50 54 02 00 90
T\nu: 0
Hfvergal of enzyme inactivnlion />.. ohospholtpid.i amt nlbiitntn after crpc-mci </ Ur (A'fi* + A'') AT/*at to ( amphiphites amt chlortanted hydrocarbons
.**ix minutes after initiation of the etini:,* reaction in ilie presence of inliibitor (see mam in > - \mj Mi.rntms), 10 (<l of a phospholipid disperse' or a protein solution were added to the cuvette ){ lion velocitiea were measured for un sdditioiud >; min. Control (Nrn+ + K+) ATPase specific nctivi - wa.> 40 pinole# of Pi per milligram of protein \.-*-r hour; protein concentration during assay wus 4(1 ,i ml Values are meuns standard errum of six determinations
Inhibitor
Concentration No revening Phosphatidyl-
compound
choline
(100 pg/in!)
Phosphatidyl serine
(100 /ig/mt)
Allninnn -- . . _ .--
8U0 ng/ml 2000 .z ml
N' one Deoxyeholate Doderyl sulfate Olente DJ)T Aroclor 1264 Aroclor 1221 ?-Chlornhiphenyl
MU ppm
600 83 100 10 3.5 10 10 20
100 3S = 5 50 =. 1 34 = 4 44 a: 5 41 s <} 44 - j 45 s: >i
95 7 40 5 C9 5
f db c 50 * 3 45 3 m2 68 rfc 4
% control acln-ily
105 : 8 45 5 80 4 74 7 76 \ 71 5 95 4 83 5
97 8 48 5 SC * 4
71 .-t 8 47 5
51 0 62 2 til) * 2
65 -- 5
50 - 0 64 * 0
4). After fhcS-mm exposure to phosph<'lip;ts<t A, boIh phosphatidylcholine and ph'-'phalidylsorint' in thn brain (Nti+ + K+)-.\'n>:tsu pp'pimitinn were Jiydrolyzwl at Iriist DON, in
agreement with tin* < ibsrrvatinns nf T.miguehi and Tunoimirti (12).
Acidic phospholipids selectively pr*r. <nd the (Xa+ -+ K'4)-.\Tl'ttse8 against mm-nva-
MONS 081957
nor, pnis, and amphiimules as (Na+ -f K>)-ati*asb inactivaroics
127
Fio. 1- Prriftdi"" by a< ,-m'o phoapitolipult
<ifitinat mid r< i ,r*o/ /
(: of (,Ya* + /,f)-
A TPat>t prtjHtriiiioi rtpn*vd {PiofpAo/ipaifl A
flecf bruin .r rr.hiiit kidney (Na* |- K*)-
ATPstcs, lit protein concentrations of 0,5 mg/ml,
were incubated nr 37* in 0.5 mi of n medium con-
siMing of 50 m>t Trw-Cl tpH 7.0), 2 mM CC1
mid 4 mg of ulbumiit. Phospholipid concentration
win 0.(1 rng/ml of incubation mixture. After each
mixture (with or without phospholipid) *ii in-
cubnted for rt min, phoepholipueo A was added
to yield n final protein concentration of 2.5 pg/ml.
At \Uo indlrnted times 24 volumes of the phos
pholipase A-t rested nuxturo were withdrawn and mixed with I volume of n.l m L'DTA at 0* to atop
the hydrolysis by phospholipase A. Ueveretd of
(\a+ + Jtyi-ATIVc inactivation (indicated by
daHhed arrows), resulting from 1 min of ensyma
wpomire to phoaphttiipnse A. was effected by
mixing 5 volumes <-i : l>o EDTA-inaetivated reac-
linu mixture with 1 volume of tho appropriate
pluxiphnlipid dieperii't to give a final phoapholipid
concentration of 1.07 mg/ml. 'Although the re-
hiiII* am not shown in this figure, phosphatidyl
choline and phnspbatidyiethamdcimino were atao
milled nfter the S-min exposure to phoapholip-
iimi A. Neither neutral phospholipid effected
reversal of enzyme inactivation.) Aaaaye for
iXn* 4* K^l-ATlNi-e m-tiviiv wero porformod oa
described under mati.iii.w.s
mi:tioi>a, using
iili<tuols containing l"pg of hr:,in enzyme protein
or 10 k kidney eniymo pr-^fin per milliliter
of uaany mixture. Much point tlio average of
firm hy phospholipase A (Tig. 4). However, under identical assay conditions, about 40% of the pun* phoaplmtidyberine and loss than o% of the pure phosphatidylcholine were hydrolyzed in 8 min by phospholipase A. When it was mixed with >% Lubrol WX (w/tv), phosphatidylcholine hydrolysis by phospholipase A was inereused to 8-12%, but tilt; e.xt<;nt of protection against phos pholipase A inactivation was only slightly liiglier than indicated for pure phosphatidyl choline in Fig. 4.
The selectivity among phospholipids for protection of brain (NV + l\+)-ATI,aso against inactivation hy dcoxycholatc or for reversal of inactivation by phospholipase A was essentially identical whether the phos pholipids were dispersed in Tris-Cl (u.vtrhiai-s and methods) or in Tris-ethyleno glycol bis(0-nminocthyl ether)-W,.V'-tetraacetie acid (ROTA), followed by dialysis against Tris-CI, according to the technique of Stahl (23). The only obvious difference was that phospbutidylwrim* prepared according to Stahl's technique (23) was somewhat more efficient than our visual preparations in reversing inactivation hy phospholipase A.
niscvsjfioN
Inactivation of (.\'a~ -f K+J-ATPnsos subsequent to exposure to oiMn-watcrdispersions of chlorinated hydrocarbons (Figs. I and 2; Table 1) or of oleaie (Figs. 1 and 3; Tabic 1) presumably occurred as a con sequence of formation of mixed micelles comprising the enzyme and each of the inhibitors, No attempt was made to study the kinetics of the iplubiliorv phenomena, as such efforts would have required addi tional {unavailable) information relative to micelle dimensions, composition, and sta bilities.
The conditions of prior incubation with micellar dcoxycholatc accentuated the ex tent of (NV' + K*)-ATPasc inactivation even though a constant enzyme-inhibitor ratio was maintained /Table 2). However, neither dcoxycholatc n*>r dodecyl sulfate needed to exist in micellar form to effect (Na* -+- K'J-ATl'a.se inactivation -a rela-
flix detenuinntuma. I'S, photphiuidyberiur; PC, phrapliHlidylcholino; PI, |Au'Bphnlidyrm..niiel; l'l'', phoophuudylctliiinol nmiiuv
.T' V*
12s MIVKP ET ,vL.
tiun>hip implied by Jttrgenscn and Skon purified (Na* 4 K)-AT)W against ph-
(Hi.
'
pholipn>e A inactivation, but did not i
In view of its binding capacity for doderyl port the effect* of other phospholipid
sulfate (33-35), albumin was anticipated 10 Since many factor*, including detergen
protect against (Tables 3 and 5) and reverse (37) and orgnnii- solvents (3b), can influrn.
(Table 0) inactivation of the (Nn+ + I\')- the rates of hvdrol} *i* of pure phnspliolipi*
ATl'iisea by dodocyl sulfate and the other by pho*phulipaw A or phospholipase C (23
anionic amphiphilca.
the selectivity among phospholipids for pr
The selective protection by acidic phos tection against (X* f K*)-ATPav i:
pholipids agninnt enzyme inactivation by .activation was considered to be of limit'
d<'M\ychoIate (Tables 3 and 5) (suggested unfulness for this inve*tii'tion.
that inactivation involved cither extraction The responsiveness of (Na+ 4- K*
or dissociation of phospholipids retjuired for ATTase to DDT and the more extensive
normal stabilisation. The phospholipid selec substituted biphenyls differed from that i
tivity waq obviously related to some property anionic amphiphilic compounds in sever
of the steroid ring of deoxycholate rather important respects. Albumin provided vei
than to its carboxyl group. Although neither little protection against or reversal of i t
phosphatidylserine nor albumin reversed zvme inactivation by DDT or Aruclor 12'
(Table) (Xa+ + K+)-ATPaw>inactivation (Table* 5 and 6), presumably because of
by deoxycholate under conditions in which relatively low affinity for extensively chi
these compounds could provide protection rinated hydrocarbon*. Anionic ainphipluL
(Table 5), the reversal by a higher concentra compounds could accentuate (Xa+ + K*
tion of albumin indicated that the enzyme ATPase inactivation in a fempentture-d-
lmd not been irreversibly inactivated.
pendent fashion (Fig. 3; Tallies 2 and 3
The nonselective phospholipid protection but the chlorinated hydrocarbons were ui
against, and reversal of, inactivation by able to do so (Table 4). Temperature d-
olente or dodecyl sulfate (Tables 3, 5, and G) pendente was assumed to reflect the influem
.suggested that the inactivation was un of variation* in numbranat phospholipi
related to interference with (Na+ + K'j- hydrocarbon pha.se fluidity on the caw*
ATPase stabilization by acidic phnsphi ilipid*. enzyme inactivation.
Tin* lack of selectivity among phospholipids The structure of chlorinated hydroerr
for reversal of (Xa+ 4- K+J-ATPase in;n- bon dispersions probably differs from tl.
tivation by these two amphiphiies resemb^d micellar structure of the anionic amphiphil
ob'rvationa on enzyme inactivation by in thAt no ionizable groups can exist At tl.
phospholipase C (23). Stahl observed that eurface of hydrocarbon dispersion* How
maximum inactivation of brain (Na* + ever, it has not been possible to defit.
K"yATPase resulted when phospholipase nn.v unusual properties of oil-in-wati
C hydrolysed 95% of the enzyme-associated dispersion# of DDT (31). The X-ray di
pho'phatidylcholine and 65% of the pho*- fraction pattern of DDT sedimented ft\o
])hatidylserineplusphosphatidylimwitol (23). aii oil-in-wntcr di*p*r$ion, such a* the on
Although pure phospholipids were hydro in Table 1, was identical with that
lyzed at markedly different rates by pho.s- crystalline DDT.* The parking pattern i
phnlipnae C (30), they were equally effective the crystal lattice >.f DDT did not reve;
in reversing (Na+ 4- K4)-ATJ'ase inactiva any unanticipated potential for into
tion by phospholipase C (23).
molecular dipole-dipole or dipole-induce
The selectivity among phospholipids in dipole interactions (39).
protecting against (Xn+ 4- K+J-ATPase in regard to th* mohviilnr a**ociulio
inactivation by phospholipase A (Tig. 4) capabilities of the hydrocarbons, DDT ea
could le explained, in part, on the busi* of a**ocinte to siguifi'-ant exti-nt* with ind"!
apparent substrate preference of phospln*lipax* A fur (lie pure phospholipid*. Hokin and Hoxum (13) hud observed that pure
(40) , phen.'l (40), and phosphate grouj (41) . However, Indrogen bonding of ehl* rinated hydrocarbon* to phospholipid pho-
pho-phatidylcholinc protected their highly
* X. Mnrosoff, prronml c>>nmonirniion.
MQNS 081959
I
( f ill klSiliiilaia AV i
UDT, pens, AND A.MPHIPUILES AH (Na+ + K"* ) -ATP.\.-E IN.VCTl VATOIW
129
phntr groups must be unrelated to (Xn+ + K^-ATPa*** inactivation, since DDE (Tabic .*>) iind tin* PCHh arc incapable of such inter actions (i.e., t lii'M* inhibitors do not possess l>c*hydryl hydrogens).
The acidic phospholipid selectivity for protection against ami reversal of (Xa* + K+)*.\TlY*t* inactivation by DDT, Aroelor
1254, and deuxyebolute obviously mimics thut fur reversal of enzyme inactivation by phospholipase A. Sine** this selectivity cannot be explained on the basis of structural or other properties of the inhibitors, it must reflect properties of the (Xa+ + I\+)ATPaao. These studie* support the hypothe
sis that the extensively chlorinated aromatic hydrocarbons DDT and Aroclor 12">4 inac-. tivnte membrane (Xn^ + K+J-ATTases by interfering' with ilie stabilizing function of ucidic phospholipid*.
ACKNOWLEDGMENTS
Wu iliank Dm. It. W. Alliers and X. K. Wilaun (or several helpful dbcuaaiona relevant to thie Invealigniicn. Dr, P. W, Alhro performed gat viir*imai"t(r:iphic analyse* of the Aroclor mixture*.
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4
. -y '. rV*4
---mnpu'i mi
wp--jwjWj^isaiW'Wenw1'11 -''MJ + z-V-ir .'.* ' VX-ti '-``S'
HONS Od1960
.