Document jNkga8gQMrm152mGyq22Q039Q

." * .' ^ a*'.S-,::' , v/-v- J, * '^", '. r,?,'.-V '~'i'r-J2^': '-'-`'v-ov '`^v-'^'j.* tf-i-V *'';* V->.`> . .',., * . - *- <v". j * ' ;5^- ^ . V. / 43. r'y. `-.Si.iV- aj ',, t\ ,v.; * ' . '. . .*.--,*... *. * , ^ '. * '. -4- :^t--. llOLtCtun I'H.OIM acology, 10, 110-129 The Influence of Dichlorodiphenyltrichloroefhane, Polychlorinated Biphenyls and Anionic Amphiphilic Compounds on Stabilization of Sodium- and Potassium-Activated Adenosine Triphosphatases by Acidic Phospholipids Changes W. Shake, Dorothy G. Hunt, Samuel T. Clements, and William E. Wilson Pathologic Physiology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 17709 (Received May 4, 1973) . SUMMARY Shake, Charles W., Hunt, Dorothy G., Clements, Samuel T., and Wilson, William E.: The influence of dichlorodiphenyltrichloroethane, polychlorinated bi phenyls and anionic amphiphilic compounds on stabilization of sodium- and potassiumactivated adenosine Iriphosphatases by acidic phospholipids. Mol. Pharmacol. 10, 119-120 (1974). Diclilorodiphenyltrichloroethane (DDT), extensively chlorinated biphenyls, deoxycholate, and phospholipase A inhibited beef brain and rabbit kidney (Nn+ -j- K+)-ATPases (EC 3.0.1.3). Phosphatidylserine or phosphatidylinositol but not phosphatidylcholine or phospluitidvlethanolaminc prevented or reversed the inactivation of the enzymes by each of these inhibitors. Albumin protected against and reversed inactivation of (Na+ + K+)-ATPases by deoxycholate, oleate, or dodccyl sulfate; however, this protein was less effective against in activation by the extensively chlorinated hydrocarbons. The extent of (Na+ + K+)-ATPase inactivation by anionic amplnphiles was dependent upon the temperature at which an enzyme-inhibitor mixture was incubated prior to assay, whereas inactivation by chlorinated hydrocarbons was not affected by temperature. Our experiments lead to the hypothesis that acidic phospholipids arc necessary for stabilization of the enzyme and that chlorinated hydrocarbons, deoxycholate, and phospholipase A interfere with the stabilization process. . INTRODUCTION The sodium- and potassium-stimulated adenosine triphosphatase [(Na+ + K+)ATPase, EC 3.6.1.3], 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 1 The fibbrevirttiong aged are: 01JT, 1,1,1Irichloro-2,2-biB(p-clilor`>ph<?iiyl)<!lhnno; DDE, 1,1 - diclil'iro - 2,2 - Imb(/> - chlorophenylH'thylcnc; PCIJs, p.'tyi'lilorinutcd biphenyl mixtures such ns Aroclor 1221 or Aroclor 1254. (;>, 6). 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. Ivinter and co-workers (7-9) ob served that DDT and PCBs were capable of inhibiting eel intestinal mucosal and gill (Xn+ + K+)-ATPascs to the extent that control of osmoregulation was lost, leading to death. Since, under the conditions used for en zyme inactivation, the chlorinated hydro carbons exist as oil-in-water dispersions, the * -WS.jh;r'. 4. -.NW-vy. - -t&i/.' V. . 4 "J'r.'f1' - * V .. . xv i- * i. M k r,i., f, 'G':v1 j-V:T '* ` 85-vv,.A.%riv J ?; s.'oj i *\ **', r*'T '3 C,-4Wr. V .Ti* "*-1? y 'r `PifcJ'i t- .S\ > t'- ;i ' t. J- -//> ' ^ -rV.5 V- STLCOPCB4009783 -hii-it i ti' W r-'iV*1-- Tri*1 rl^v~,^'"i^ti ' r- V*^-' t -r 120 SHARP RT AL. physical state of the inhibitors appears to be of groat importance. Because the hy drocarbons arc essentially insoluble in water, it is possible that (Xa+ + K+)-ATPnsc 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), dodecyl sulfate, or deoxycholate, as several of these anionic amphiphiles have been reported to effect enzyme inhibition where they exist in micellar form (11).. Since phosphatidylserine stimulates enzyme activity following (Na+ + K+) ATPase exposure to phospholipase A (12, 13) or to deo.xvcholatc (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. MATEHIALS ANI) METHODS Chemicals. Dodecyl sulfate, ATP, NADH, phospboenolpyruvate, 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 1221 (biphenyl plus mono- and dichlorinated biphenyls) and Aroclor 12o4 (tri-, tetra-, penta-, and hexachlorinated biphenyls) were obtained from Monsanto. Phosphatidylserine was prepared from bovine brain (13). Brain phosphatidylinositol and Lubrol WX were obtained from General Biochemicals, Inc. Other brain phospho lipids were obtained from Supclco, Inc., Bellefonte, Pa. Each phospholipid was at least 90 7r homogeneous by thin-layer chro matography in three systems (19-21). Phos phate contents wore determined, according 'to Dittmer and Wells (22), to be 1.25 pmoles/mg of phosphatidylserine nnd 1.20 ^moles/mg of phosphatidylcholine. Dry phospholipids were dispersed in 0.1 m TiisHC1 (pH 7.0), using a Teflon pestle and glass tube homogenizer, followed by 30 sec of sonic oscillation. Phospholipids were- also dispersed by the technique of Stahl (23). Phospholipase .1. Phospholipase A (Vipern russelli; Sigma Chemical Company) solu tion in 50 f7 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 chromatography (25) 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+ -f- K~)-A TPase preparation. Bovine brain (Xa- + K+)-ATPase was prepared' according to a modification (26) of the method of Skou (27). The enzyme was stored os on 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 I mg/ ml. Protein was determined by the technique of Lowry el al. (28). Babbit kidney (Xu4 -f K*)-ATPnse was prepared by a modification of the method of Jorgensen and Skou (29). Jn the deoxycholut.e activation step 34 him KC1 nnd 100 imi NaCI were present in addition to 0.0 mg/ml of deoxycholate, 2 m.u JEDTA, and 25 dim imidazole at pH.7.2. (Xa* -P K+)ATPa.se accounted for 85-90(7 of the total ATPase activity in these preparations, (Na+ -f K~yATPase acliriiij assay. Lac tate dehydrogenase and pyruvate kinase were obtained from Sigma Chemical Com pany and were dialyzed against 0.02 ,m Tris-HCl, pH 7.4, prior to use. The assay medium was incubated at. 37 for 4 min prior to initiation of the enzyme reaction. The reaction was usually started by adding 40 ug (40 al) of beef brain (Xn+ 4- K+)-ATPase protein or 10 ng (10 al) of rabbit kidney enzyme protein to yield a final assay medium volume of 1 ml. Phos pholipid dispersions -and/or inhibitor solu tions were added in the sequences indicated in the tables and figures. Initial velocities were determined by measuring the rate of NADH oxidation at 340 nm (30). Final concentrations of components of the incuba tion medium were 3 m.u ATP, 3 m.u MgC); , 125 mu XnCl, 25 mu KCI, 1.5 mu plios- pboenolpyruvate, 0-25 m.u XADH, 2 units ' 1 . DSW 025829 If'l'J'K ft r*'- T. STLCOPCB4009784 UDT, Pl'HS, AND A.MPHJl'HILES AS (Xn^ + K+)-ATPASE INACTIVATORS 121 Taui.k 1 llniin -f- K*)-A TPuse responsiveness to dispersion* of oleatc and chlorinated hydrocarbon* Inhibitors were introduced into the assay medium as solutions in dimethyl sulfoxide. At the assay concentration of 1`r (v/v), dimethyl sulfoxide had no effect on enzyme activity. (Xa'1 + K"i-ATPase upecific act ivity was 40 pmoles of Pi per milligram of protein per hour; enzyme protein concentration in the assay was -iO/ig/ml. Assay conditions are described under materials and methods. Results are means standard errors for three determinations. Inhibitor Inhibitor concentration Order of addition of enzyme and inhibitor to assay medium Inhibitor added 15 sec after initiation of (Xa+ + K+)-ATPase reaction (Na+ + K*)-ATPase reaction initiated by adding enzyme at indicated intervals after inhibitors 0.1 min 20 min 120 min e.v ppm Xoe Tris-ol^nie DDT An.".-lor 1254 Apwlor 1221 100 20 10 15 100 40 db 4 44 4 42 4 48 db 4 % control activity 43 db 3 44 4 47 4 49 4 42 rfc 4 4G 4 53 3 48 A 3 43 4 43 = 4 53 = 4 57 = 4 of lactate dehydrogenase, 7 units of pyruvate uleatc to water resulted in the formation of kinase, ami .'10 ni.u Tris-HCl, pH 7.4. dispersions whose particle sizes and, or num In order to ascertain the validity of the bers appeared to change continuously for result? obtained using the coupled lactate several hours (31) (Fig. 1). The spectral dehydrogenase-pyruvate kinase system, we alterations accompanying the change of initially determined that the rate of XADH phase of 2-chlorobiphcnyl (Fig. 1A) re oxidation was unaltered when 0.15 m.u A DP flected contributions from hydrocarbon in was used as substrate in the absence of solution and from light scattering by micelles; (Xa^ + K+)-ATPase and in the presence however, the alterations accompanying the of each amphiphilic compound, hydrocarbon, . change of phase of oleate (Fig.- IB) reflected phospholipid, and combination thereof. Also, only the time-dependent increases in micelle most of the data in Tables 3 and 4 were cor number and/or size (32), roborated by results from our phosphate In a concentration dependence study of analysis technique (20). (Xa~ + K*)-ATl>ase inactivation, an in-, UF.SIT.TS crease in the chlorinated hydrocarbon con centration resulted only iu an increase in (AV' + K^)-ATPase responsiveness to amount of inhibitor in the dispersed phase water-insoluble substances. Inactivation re (Fig. 2). However, the extent of enzyme sulted from interaction of (Xa+ -f Iv+)- inactivation increased as the extent of chlo ATPaso with oil-in-waler dispersions, rather rine substitution of bipbenvl was increased than solutions, of fatty acids or chlorinated (Fig. 2). * hydrocarbons. The extent of (Xa+ + K+)- Concentration and temperature dependence ATPase inactivation by chlorinated hydro of (Xa+ + K+)-ATPase inuclhvtion bp carbons or by oleatc was not appreciably anionic amphiphilic compounds. The curve influenced by the time at which the enzyme for concentration dependence of brain (Xa* was exposed to inhibitors (Table 1). Clear + K")-ATFase inactivation by oleatc had aqueous phase? obtained by centrifugation of a different shape from the curve tbr inactiva oleatc, PCIls, or DDT dispersions (Table 1) tion by dodecyl sulfate(Fig. 3). This may for 30 min at 100,000 X y wore not in be due to the occurrence of enzyme inactiva hibitory. tion at assay concentrations below the critical Addition of ethanol solutions of DDT (31), micelle concentration for doih-cyl sulfate 2-ehlorobiphenyl, FOB mixtures, or Tris- (calculated to be about 0.17 imi from ref. f*. , ` 4 :'v * t: ' h, - T'/cv'-t <v.' L'V-> s* _ : ' VvT,v ] vy ' V** ; u .. .. \\ c-'V5- J -v * -vr:- r.lvf *:> re ' fVVii 2%i:-.*f.r-*'-.-v,->X0; 4:vVv?- >/l'.S',' -- v *<^ 11 ' ** * * .x- ' V . i * PA . 1- W A " >ij.7;^ > >. - JT- OSW 025830 STLCOPCB4009785 rv-* *v,.-' _ : * * wv. <71 .-' -V ' ,', ' Wove^^^P*) Fig. 1. Absorption spectra of 67 pit 2-chlorobiphenyl (/l) and 26 *i.v Tns-olcate (B) in ethanol and in 1% ethanol in water (------) Spectral alterations in the ethanol-water solutions were determined at the indicated intervals a::.': preparation. Spectra were recorded with a Beckman Acta V spectrophotometer. Fig. 2. Concentration dependence of brain (A'a+ -t- K+)-ATBase inhibition by DDT, Aroelor t26i. Arorlor 1221,2-chlorobiphenyl and biphenyl Ten microliters of ft dimethyl sulfoxide solution of each chlorinated hydrocarbon were added sec after addition of 40 /ig of enzyme protein to initiate the ATPase assay. 122 STLCOPCB4009786 Khi. 3. Concentration dependence of tre,- t.Ya+ + K+)-ATPa$e inhibition by Triij todium ilnilicyl Kidfatc (SDS) Dispersions of enzyme phis inhibitor, b v. at 12.5-fold higher concentrations thaD ditri:.? subsequent assay, "ere incubated in a medium conts.ir.ing 5 tom KCI, 24 nut NiCl and 17 imi Tris-HCl, pH 7.5. Incubation was conducted for 10 min at 0' r 37; then 80 pi of this mixture "ere added vOO p\ of the assay mixture (see matkjijals and metho: s in order to initiate the (N'a+ +- K+l-ATPase reaction. 11), whereas micellar oleate was r-'.uired dylinositol2 (not .shown) protected both (Na+ lo effect inactivation. + K+)-ATPases against inactivation by Following prior incubation the anionic deoxycholate when prior iucubari was con amphiphiles (but not the hydrocarb' r-> t in ducted at 37; however, phosphatidylcholine activated (X'a+ + K+)-ATPases in r-. tem and phosplmtidylcthanolamine n-:t shown) perature-dependent fashion (Fig. 3). In con provided no protection (Table 3 . By con trast with results obtained with c -iccyl trast, each phospholipid protected the en sulfate, accentuated enzyme inactive;: n by zyme against inactivation by !<-ate, or oleatc was observed onlj- at an iiAibitor dodecyl sulfate. The indicated sodchiomet- concentration greater than that requl'-d to ric relationships among enzyme, inhibitor, effect 40% inactivation. and protective, phospholipid wc-r- optimal The extent of (Xif- -F K~)-ATPa.v.- inac- for the observation of phospholipid selec tivalioti by deoxycholate depended u;:- . n the tivity; however; identical results were ob absolute quantities of enzyme and inhibitor tained over a 4-fold range of concentrations .subjected to prior incubation at 37 'Table of either phosphatidylcholine or phusphati- . 2). Temperature-dependent enzyme inne- - dyiserine (i.e., from one-half to twice the tivation was only observed following prior incubution at deoxycholate concentrations appreciably in exeess of the critical micelle ' In each instance when prniecti"v r reversal by phospliatidylinositnl or phosphatidylethanolamine is mentioned only in the -ext mksui.ts, concentration (calculated to be Hbou" 1.2 at. leHst two analyses were performed Jr. no case m.w from data in ref. 11); however, deoxycholute concentrations during the u^av wore always below this level. Protection of (Nay + /t+)-. 1 77Vo n -uiiH inactivation by anionic amphiphilic mnpuunds. Phosplmtidylseriue and pho-phati- did the results using phosphatidyle-r. melamine differ significantly from those omi.e j,r. -phatidylcholine. The cominercinl hovi:.e rr:.i:i phosphntidylinositol consistently provided r--nhs sim ilar to those obtained with <>nr bra::, th -pliatidylacrinc; however, eommercial preps ra*: :of plant phosphatidylinositol have yielded variable results. j '.if; 'v ' > **. | ' I*' ; '4 * .yi r* V >*i \ * r'. X is* if. vlr;. -'V %* A KJ * x< '*> - b*. .V.: .vi! '**'*, . -J ii*U**. Previn i-" ' `S OSW 025832 *V'-*'-:-T /- .**, -.'j- iy &-'***- I *- --- - STLCOPCB4009787 ' , fecv.>- ; v Taiii.k 2 Influence of prior incubation on cxlcnl of brain. {/Yd* + K*)-ATPuse inactivation by <leoxt/t'hofaft Deoxycholate concentration during ussay Standard assay' Prior incubation at multiples of (deoxycholate] in .-tnndard a? sa\"* 1 11 !i ^tr, 1 's ; X oo - : i1 X3.1 X6.2 X2s 0 0 37 O' 37 t I Oj Q . p.V 0 01 128 255 510 1020 f/c control activity 100 92 80 89 91 88 8-3 87 91 88 81 74 74 04 73 72 79 02 85 73 58 00 05 71 01 40 21 07 04 56 07 01 51 28 20 10 48 45 42 44 29 17 S 14 4 20 21 10 12 4 " As described under mathrials and mktiiods, 40pg/ml of enzyme protein mid the designated aiiinunt of deoxycholute were added separately to the cuvette in order to obtain the results in this column. Con trol (Nid + K+)-ATPuse specific activity was lift pinoles of Pi per milligram of protein-per hour. Results are averages of three determinations. 1 For example, the multiple X12.5, at 255 /*.m deoxycholute, indicates that. 500 pg of enzyme protein, 3200pM deoxycholate, and 17 him Tris-CI, pH 7.5, were mixed and incubated for 0 min at 0 or 37"; tben an aliquot was diluted 12.5-fold into the assay cuvette, containing a reaction mixture such tlmt assav conditions were identical with those of the standard assay. Results below the short lints represent the temperature dependence effect- following prior incubation. Tadnr; 3 Phospholipid protection of mammalian (Xa+ + A'+)-yl TPoses against ina<?:> at it,,, l,,j several anionic amphiphilic compounds See- the legend to Fig. 3 for n general description of the. incubation technique. Albumin or phospho lipid was added to the (Na+ 4- K+)-ATPase dispersion prior to addition of the inhibitor solution. The concent rations of each component of the incubation mixture was 12.5-fold greater ibeef brain enzyme > or 50-fold greater (rabbit kidney enzyme) than during assay. Respective specific activities for beef brain and rabbit, kidney (!W + K+)-ATPnses were 40 and 140 amoles of 1R per milligram of protein pr hour, while corresponding protein concentrations were 40 and 10 pg/ml during assay. Fnr.yme activity ussay conditions are described under matnkials and mktiiods. The values are meat ? of six determina tions (SKM S 10%). Knzyme source und inhibitor Tcm- Xo protective Phosphatidyl Phosphatidyl- Albumin perature compound choline serine (6.25 nig ml/ (1.6 mg/ml) (1.6 mg r~l > Drain (beef) No inhibitor Oleatc, 1.25 ntxi Dndecyl sulfate, 0,84 niM Deoxycholate, 3.4 iwm Kidney (rabbit) No inhibitor Dodecyl sulfate, 0.84 him Deoxycholate, 3.1 tmi C % control activity 0 100 100 (92) 100 (129/ 100 793 J 37 100 (98) 100 (96) 100 (140/ 100 (01) 0 39 90 93 94 37 7 89 84 70 0 71 75 80 94 37 11 07 GO 88 0 54 71 83 75 37 19 . 15 62 00. . 0 100 . 100 100 100 37 100 (93) 100 (93) 100 (93/ 100 795) 0 96 100 100 102 37 23 100 100 92 0 58 92 09 97 37 22 18 93 90 " Values in parentheses are percentages of the 100% values at- the top of t he column headed "Xu protcctive compound" for each enzyme preparation. All other vnlues ore relative to the ; `propria te 100% value (i.0., the ones obtained with no inhibitor present dol ing prior inc ubation at 0" i ir at 37!. 124 DSM 025833 STLCOPCB4009788 Taiilk 4 Phnxphntipid un'l albumin protection of brain (.Vd` +- K'r)-ATPasc again.-: inhibition by chlorinated biphenyls and DDT lueubnthm ifChnitiues are described in the legend to Fig. 3. The (Na+ + K')-ATPase protein con centration was 40 ng ml during the assay. Values "re means standard errors of four determinations. Inhibitor Temperature during initial incubation No protective compound Phospha tidylcholine (133 Mf/ml) Phosphatidylserine (133 ng/ml) Phospha- Albumin lidelinosiiol (1000 /ig/ml) (133 ml) None DDT, 3.5 ppm Aroelor 1221, 10 ppm Aroelor 1254, 5 ppm X .0 37 0 37 0 37 0 37 100 100 <0S) 46 i 8 45 3 44 =fc 7 42 0 48 4 ' 40 7 % control activity 100 (02)* 100 (Of5) 70 8 70 =b 2 08 7 (vfl tis 8 100 (135) 100 (145) 95 9 72 db 0 103 7 98 4 100 M10) .100 i IOS; 90 - 3 7*1 -=z 5 --0 93-5 07 0 70 db 8 95 9 88 db 8 10) - 9 95 = 5 100 (99) 100 (95) 40 5 49 0 06 5 65 4 67 5 66 =t 4 Values ill parentheses are percentages of the control (Nn+ -f K+)-ATPase activity shown at the top of the eolunin headed "No protective compound." concent rat ion? of phospholipid indicated in Table Id). Albumin, at 4 times the mass of the phos pholipids, effectively protected the brain enzyme against each of the anionic amphiphile.s (Table 3). Protection of (A'a4- -f- K+)-A TPases atjainst inhibition by chlorinated hydrocarbons. The prior incubation technique was not optimal for observing selective phospholipid protec tion against (Xa4- + K+)-ATPase inactiva tion by chlorinated hydrocarbons (Table 4). Both albumin and phosphatidylcholine pro tected against the chlorinated hydrocar bons. Furthermore, both phosphatidylserinc and phosphatidylinositol 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 tire controls, the validity of our arbitrary correction of the values in the vertical columns of Table 4 could not be determined. -. In contrast with the above results, acidic phospholipids did not increase control (Xa+ 4- lv'd-ATPase activity when both enzyme and phospholipid were added directly to the assay medium (Table 5). Acidic phospho lipids selectively protected the two (Xa+ + K+)-ATPascs against inactivation by deoxycholatu, DDT, and Aroelor J254 (Table 5). Albumin protected against enzyme inac tivation by deoxycholate but not against that caused by DDT r Aroelor 1254 (Table 5). Reversal of (Na+ + K~)-A TPase inac- Ovation by hydrocarbons or by amphiphiles. Albumin and both acidic and neutral phos pholipids reversed brain (Xa+ + Iv+)- ATPase inactivation resulting from enzyme exposure to dodooyl sulfate, oleate, Aroelor 1221, or 2-chlorobiphenyl (Table 0). Only phosphatidylserinc or phosphatidylinositol (not shown) effectively reversed inactiva tion of the enzyme by DDT or Aroelor 1254. Albumin, at 2000 pg ml, reversed inac tivation by deoxycholate. DDT, or Aroelor 1254. The conditions described in Table 6 were nearly optimal for demonstrating selectivity among different compounds that could re verse inactivation by the chlorinated hydro carbons. For instance, a 3-fold increase in phosphatidylcholine concentration gave only 15% greater reversal - of inactivation by DDT. Phosphatidylserinc, at 80 Mg/ml dur ing assay, was essentially as effective us it. was at 100 fig/ml, Phospholipid selectivity in reversing and protecting against (Na^ -- K~)-A TPase in activation by phospholipase .4. Phosphatidyl- serine and phosphatidylinositol reversed the inactivation resulting from (Xn+ + K4)- ATPast: exposure to phospholipase A (Fig. >T . , .i . i \ f.U*r \ V r V. :.k '> *. * y ; A ' ' *' STLCOPCB4009789 r_>c SKA UP JET AL. T\jii/i: 5 Protection of mnnimnlian. (V;' ~ K^y-ATPnsrs u-ith inhibitor und/ur protective ' corny< 'fi present only during unsay limin and kidney enzyme protein cm.mitriitions were 40 rind 10 respectively, during the assay; respective eperilic activities were 40 and 140 pn dies of P; per milligram of protein per 1; ;r. The reaction was initiated by adding enzyme to (lie assay mixture, which contained protective compound, at 37. Inhibitor solution (10 pi) wns added immediately after the enzyme. Values are means of r,,ur to six determinations (SliM <; 10% for each value). Enzyme source and inhibitor Brain (heef) No inhibitor Deoxycholate, 000 aim Dodecyl sulfate, 100 mm Oleate, 100 aim Aroclor 1221, 20 ))pm Aroclor 1254, 10 ppm DDT, 3.5 ppm or 10 aim Kidney (rabbit) No inhibitor yMDeoxyeholnte, 000 Dodecyl sulfate, 100 eM Oleate, 100 mm Aroclor 1254, 10 ppm DDT, 10 a>m DDE, 10 aM No protective compound 100 42 z~ 41 34 :>-S -It; 100 34 *24 17i1t', Phosphatidyl- Phosphatidylserine choline (100/jg/ml) (100/ig/ml) % control activity Alburzin (800 y ml) ` 07 104 49 05 r.N 48 52 79 05 73 80 54 84 34 45 81 41 47 72 47 98 101 97 30 50 GO 51 04 90 04 02 99 09 99 07 03 90 05 72 05 07 Tadlk 0 Reversal of enzyme inactivation l,.- phospholipids and albumin after exposure nf bruin 1(A' K') ATPrise to (wi*'-V atnpkiphiles and chlorinated hydrocarbons .Six minutes after initiation of the enzyme reaction in the presence of inhibitor (see mati.iiia:wo methods), 10 *d of a phospholipid disperse.:; or a protein solut ion were added to the cuvette, lb -vtion +velocities were measured for an additional n min. Control (N n+ K+)-Al'Pase specific nctivi v was 40 pinoles of Pi per milligram of protein per hou r; protein concentration during assay was 40 Values are means standard errors of six determinations. ml. Inhibitor N one Jjeo.xycholate Dodecyl sulfate Oleate DDT Aroclor 1251 Aroclor 1221 2-Chlorobiphenyl Concentration M-V ppm GOO S3 100 10 3.5 10 10 20 No reversing compound 100 3S = 5 50 =. 4 34 = 4 44-5 41 = 0 44 = 5 45 - 0 Phosphatidylcholine (100 fig/ml) ------Phosphatidylscr\ne Albumin ---- 1(100 jug/ml) 800 Aig/ml 2000 yi ml % control activity 95 7 105 8 10 5 45 5 09 5 80 4 08 G 74 - 7 50 db 3 45 3 70 .-fc 4 71 5 00 2 08 i 4 95 1 83 5 97 S 48 5 85 =fc 4 71 db 8 47 5 51 0 02 2 09 2 05 rr 5 59 ~ 6 04 - e 4). After the S-miu exposure to phospholipase A, both phosphatidylcholine and phosphatiilyJscrino in the brain (Nu+ + K+)-ATPase preparation were hydrolyzed at least DO %, in agreement with the observations of T.-miguclii and Tonomura (12). Acidic phospholipids selectively protected the (Xn+ -f- Iv+)-ATPases against imtetiva- nor, pens, and amphiphiles as (Na+ -f- K+)-atpase ixactivatous 127 Fia. 4. Prolcdi'io by acidic phospholipids apuiiist 0>id ru,:r,,al <; iimclicaiion of (A'a* + /v+)- ATPase preparative* exposed phospholipase A Hoof brain "V rabbit kidney (Na+ K+)- ATPnees, at protein concentrations of 0.5 mg/ml, wore iueubatpd nr 37 in 0.5 nil of n medium con sisting of 50 him Tris-Cl (pH 7.0), 2 him CaClj and 4 mg of albumin. Phospholipid concentration was 0.(1 mg/ml of incubation mixture. After each mixture (with or without phospholipid) was in cubated for 11 min, phospholipase A was added to yield a final protein concentration of 2.5 jtg/ml. At the indicated times 24 volumes of the phos pholipase A-treatcd mixture were withdrawn and mixed with 1 volume of (1.1 m EDTA at 0 to stop the. hydrolysis by phospholipase A. llevcrsal of (N*a+ + K+)-ATPaec inactivation (indicated by dashed arrows), resulting from $ min of enzyme exposure to phospholipase A, was effected by mixing 0 volumes of ;he EDTA-inaetivated reac tion mixture with 1 volume of.the appropriate phospholipid dispersion to give a final phospholipid concentration of 1.07 mg/ml. (Altlwygh the re sults are not shown in this figure, phosphatidyl choline and phospharidylethari'tiuminc were also added after the S-min exposure to phospholip ase A. Neither neutral phospholipid effected reversal of enzyme inactivation.) Assays for (Xa1 + K+)-ATPase activity were performed an described under mati;i:i.w.s and methods, using aliquots containing 4(1 ag of brain enzyme protein or 10 jug of kidney enzyme protein per milliliter of assay mixture. Each point Is tlie average of tian by phospholipase A (Fig. 4). However, under identical assay conditions, about 40% of the pun' phosphatidylserine and less than 5% of the pure phosphatidylcholine were hydrolyzed in 8 min by phospholipase A. When it was mixed with >% Lubrol WX (w/w), phosphatidyleholine hydrolysis by phospholipase A was increased to 8-42%, but the extent of protection against phos pholipase A inactivation was only slightly liighcr than indicated for pure phosphatidyl choline in Fig. 4. The selectivity among phospholipids for protection of brain (Xa- -f- K+)-ATPase against inactivation by deoxycholato or for reversal of inactivation by phospholipase A was essentially identical whether the phos pholipids were dispersed in Tris-Cl (maTEHIAES AND METHODS) Of ill THs-CthyloilO glycol bis(|8-aminocthyl ether)-#,A:'-tetraacetic acid (EGTA), followed by dialysis against Tris-Cl, according to the technique of Stahl (28). The onh- obvious difference was that phosphatidylserine prepared according to Stahl's technique (23) was somewhat more efficient than our usual preparations in reversing inactivation by phospholipase A. DISCUSSION Inactivation of (A"a~ + K+)-ATPases subsequent to exposure to oil-in-water disper sions of chlorinated hydrocarbons (Figs. I and 2; Tabic I) or of oleate (Figs. I and 3; Table 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 inliibition 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 deoxycholate accentuated the ex tent of (Nn1' +' K+)-ATPasc inactivation even though a constant enzyme-inhibitor ratio was maintained (Table 2). However, neither deoxycholnte nor dodecyl sulfate needed to exist in micellar form to effect (Na+ + K'J-ATPa.sc inactivation---a rcla- six determinnrimo. PS, phospbaridylserine; PC, phosphatidylcholine; PI, phnsphatidylimisilol; PM, phospluitidy let hanol amine. vi.y.* U. `77:V:e' v< CV;-r77. jv.'KV.-v 2 7v - 7" r!.:" ' t^w7V?v-.vV,'"'-wy'.`.'V; v' * >-!T .-:; >, atv,'-fV'.'~7 " %%'-.; . .'.. ...'. *. 1 -v 7/ ?,,rVi s v ! * tv. .. .>l*y ~ .7.. .r M 7V'- - r' : y * '.;viV; -- * -* V yJ y ; )', r*-.*. . v\ 4 ' *4 ' ` " '''''a .Y-vw'-.e- A-?-,-:-7 w-. Vv -'' ;-'-V- -'4 >* 'V:'.'vv %'':-7?, 7/77. 1 -' !':'>V*'f;v.VpV; -vit. "o vv.-` .>>.;'} s/VC-''i.--*j-.'/Jj < i *. : 'r1. > . *.4',fyy\j4 . ; 12S sii.VliP KI AL. ti'>n^})i)) imjjlieil by Jorgenson and .Skou purified (Xirr -f K-yATl'iise against ph> (Jli. _ ' pholipase A inactivation, but did not i in vi(nv of its binding capacity for dodf-cyl port the effects of other phospholipid sulfate (33-35), albumin was .anticipated to Since many factors, including detergen protect against (Tables 3 and 5) and reverse (3T) and organic solvents (3b), can iufluen. (Table 0) inactivation of the (Nn+ -f K ')- the rates of hydrolysis of pure phnsplioliph ATi'ases by dodccyl sulfate, and the other by phospholipase A or phospholipase C (23 anionic ampliiphiles, the selectivity among phospholipids for pr TJ>c selective protection by acidic phos fection against (Xa~ T KX)-ATPase i: pholipids against enzyme inactivation by .activation .was considered to be of limit< dcnxyeholate (Tables 3 and 5) suggested usefulness for this investigation. that inactivation involved cither extraction The responsiveness - of (Xa+ 4- K- or dissociation of phospholipids required for ATPase to DDT and the more extensive normal stabilization. The phospholipid selec substituted biphenyls differed from that t tivity was obviously related, to some property anionic amphiphilic compounds in sever of ilie steroid ring of deoxycholate rather important respects. Albumin provided vet than to its carboxyl group. Although neither little protection against or reversal of ei phosphatidylserine nor albumin reversed zynie inactivation by DDT or Aroclor 12-' (Table 6) (Xa+ + K+)-ATPase inactivation (Tables 5 and 6),' presumably because of by deoxycholate under conditions in which relatively low affinity for extensively chi these compounds could provide protection rinated hydrocarbons. Anionic amphiphiL (Table 5), the reversal by a higher concentra compounds could accentuate (Xa+ + K~ tion of albumin indicated that the enzyme ATPase inactivation in a tcmperature-d> had not been irreversibly inactivated. pendent fashion (Fig. 3; Tables 2 and 3 The nonselcctive phospholipid protection but the chlorinated hydrocarbons were ui against, and reversal of, inactivation by able to do so (Table 4). Temperature di olente or dodccyl sulfate (Tables 3, 5, and (ij pendence was assumed to reflect tin- influem suggested that the inactivation was un of variations in mcmbranal phospholipi related to interference with (Na+ + K~)- hydrocarbon phase fluidity on the ease < ATPase stabilization by acidic phospholipids. enzyme inactivation. The lack of selectivity among phospholipids The structure of chlorinated hydroc.v for .reversal of (Xa+ + K-1)-ATPa.se inac bun dispersions probably differs from tl. tivation by these two amphiphile.s resembed micellar structure of the anionic amphiphil observations on enzyme inactivation by in that no ionizable groups can exist at th phospholipase C (23). Stahl observed that surface of hvdiucarbon dispersions. Hon maximum inactivation of brain (A'a~ + ever, it has not been possible to dffii: K~.j-ATPase resulted when phospholipase any unusual properties of oil-in-wat< C hydrolyzed 95% of the enzyme-associated dispersions of DDT (31). The X-ray di. phosphatidylcholine and 65% of the phos- fraction pattern of DDT sedimented fr< phatidylserjneplusphosphntidylinositol (23). an oil-in-water dispersion, such as the on Although pure phospholipids were hydro in Table 1, was identical with that < lyzed at markedly different rates by phos crystalline DDT.1 The packing pattern i pholipase C (36), they were equally effective the crystal lattice ,.,f DDT did not rove, in reversing (Xa+ + K+)-ATPase inactiva any unanticipated potential for into? tion by phospholipase C (23). molecular dipole-dipole or dipole-inducc The .selectivity among phospholipids in dipole interactions (39). protecting against (Xu4 -f K+)-ATPase In regard to the molecular associatio inactivation by phospholipase A (Fig. 4) could be explained, in part, on the basis of apparent substrate preference of phospho lipase A for the pure phospholipids. Hokin and Hexuiri (13) had observed that pure capabilities of the hydrocarbons, DDT ea associate to significant extents with ind<<! (40), phenyl (40), and phosphate grott; (41). However, hydrogen bonding of chf rinated hydrocarbons to phospholipid phos phosphatidylcholine protected their highly *X. Morosoff, personal communication. a:, - ' "-v .!... . v. . fc* . St4 r '.JI? ^ 7--C-> '4.1 id- vSJte s|. ..x -rr.i ".r'fi **- 1 M DSW 02583? rev -.--r;p.. .; . - .***% -..***!1 ......... ~'~t- .v. i ^ STLCOPCB4009792 r> , .kv i < " . *.'' b- ',, --T- 'f > X ' } ' *>: '*-, ' _ .. . , 4_ ^ ^ *'.-.- f * . . ~r j-: . . *'- . .. ';* : *;.\*t;<t.<'.->**. . v^v'-i 'VV i. vVc-r-o.; ~h;.*>-. . : ?; . -Vv;v';:^ >cr!; ,;l. iv*; X.'". *:' . > iiit )1 illlihn011*iiil'iW**-'**-"I3t-v7i(f ,'j,.'.Si^c-^A.ry, . UDT, PHIS, AND AMPH1PHILES AS (Na+ + K4)-ATP.\~E INACTIVATORS 129 phntc groups must be unrelated to (Xn+ + ID )-ATP;ise inactivation, since DDE (Tabic A) and the PCEs are incapable of such inter actions (i.e., these inhibitors do not possess heitzhydryl hydrogens). The acidic phospholipid selectivity for protection against and reversal of (Xn+ + K+)-ATlYse inactivation by DDT, Aroelor 1254, and deoxycholate obviously mimics 14. Fcnstcr, L. J. <k Copenhuvcr, J. H. Jr. (19(i7) Biochim. Biophys. Acta, 137, 400-408. 15. Tanaka, It., Sakamoto, T. <fc Sakamoto, Y. (1971) J. ilcn.hr. Biol., 4, 42-51. 16. Wheeler, K. P. A Whittam, It. (1970) J. Physiol. (!.,!.;, 207, 303-328. 17. Kimelberg, H IC. & Papahadjopoulous, D. (11)72) Biochim. Biophys. Ado, 282, 277-292. 18. Sunders, H. (1907; Biochim. Biophys. Ada, 144, 485-487. that for reversal of enzyme inactivation by 19. Skidmore, W. II. & Entenman, C. (1962) J. phospholipase A. Since this selectivity cannot Lipid Ret., 3. 471-475. he explained on the basis of structural or 20. Skipaki, V. P., Peterson, It. F. & Barclay, M. ' other properties of the inhibitors, it nmst. (1904) Biockon. ./., 90, 374-378. reflect properties of the (Xn+ + K+)ATPases. These studies support the hypothe sis that the extensively chlorinated aromatic 21. Skipski, V. P., Sruolnwe, A. F. & Barclay, M. (1907) J. Lipid Res., 6, 295-299. 22. Dittmer, J. C. A Wells, M. A. (1909) Methods Emymol., 14, 482-530. hydrocarbons DDT and Aroclor T2'>4 inac- . 23. Stahl, W. L. (11*731 Arch. Biorhem. Biophys., liviite membrane (X.rt"+ K+)-ATl'asc.s by 154, 56-67. interfering' with the stabilizing function of 24. Imai, Y. & Sato. It; (1900) Biochim. Biophys. acidic phospholipids. ' Acta, 42, 104-105. 25. Itntiser, G., Flei-chcr, S. & Yamamoto, A, ACKNOWLEDGMENTS (1970) Lipids, 5, 494-490. We thunk Drs. It. W. Alliers and X. K. Wilson 26. Wilson, W. 17., Sivitz, W. I. & Ilanna, I.. T. for several helpful discussions relevant to this (1970) Mol. Pharmacol., 6, 449-459. Investigation. Dr. P. \V. Albro performed gas 27. Skoit, J. C. (K"'.2p Biochim. Biophys. Ada, chromatographic analyses of ihe Aroclor mixtures. 58, 314-325. HEFEIiENCES ' 2,8. Lowry. O. H... Ro-rebrough, X. J., Farr, A. L. & Itandull, It. J. il951) ./. Biol. Chan., 193, ]. Albers. 11. W. (1907) Annu. Rev. Biockcm., 205-275. 36, 727-756. 29. J0rgensen, P. L. ck Skou, J. C. (1969) Biochem. 2. Koch, li. B. (1909) J. A'ewrochem., 16, 209-271. Biophys. Res. Cnmmun., 37, 39-46. 3. Mntsurnuin, F. & Patil, K. C. (1909) Science, 30. Barnett, 11. E. 1970) Biochemistry, 9, 4044 166, 121-122. 4648. J. Mntsitmura, F., Bratkowski, T. A. Jt Patil, 31. Wilson, W. E.. Fishbcin, L. & Clements, K. C. (1969) Bull. Environ. Contain. Toxicol., S. T. (1971) Science, 171, .180-182. 4, 202-270. 32. Bull, II. B. (1904) An Introduction to Physical 5. Yap, H. H., Desaiah, D., Cutkomp, L. K. & Biochemistry, p. 170, Davis, Philadelphia. . Kuril. It. B. (1972) Persped. Environ. Health, 33. Stcinhardt, J. <v- Reynolds, J. A. (1969) 1, 105-168. ' ' Multiple Equilibria in Proteins, pp. 84-124, 6. Yap. II. II., Desaiah, D., Cutkomp, L. IC. Academic Pre?s. New York. ' & 'Kuril, R. B. (1971) Kat. Aetr Biol., 233, 34. Reynolds, J. A. <fc Tnnford, C. (1970) Pror.. 61-62. - Natl, Acad. Sri. C. S. A., 66, 1002-1007. 7. Janicki. It. II. & Kintcr, IV. B. (1971) Science, 35. Nelson, C. A. '1971) J. Biol. Cltaji., 246, 173, 1146-1147. .' 3895-3001. .. S. Janicki, It. H. & Kinter, W. B. (1971) .Yat. 36. Stahl, W. L. (1973) .4r<7i. Biochem. Biophys., ' Xcw Biol. 233. 14S-149. 154,47-55.' ' ' 9. lunter, IV. B., Merkens, L. S., Janicki, It. H. 37. Magee, W. L., Gallai-Ilatchurd, J.. Sanders, & Guarino, A. M. (1972) Pcrspect. Environ. H. & Thompson. It. H. S. (1902) Biochem. J., Health, 1, 109-173. H3, 17-25. 10. Ahmed, K. & Tliomas, B. S. (1971) J. Biol. 38. Magee, W. L. <k Thompson, R. H. S. (1900) Chun., 246, 103-109. Biorhem. J., 77 , 520 534. 11. Jorgensen, P. I,. & Skott, J. C. (1971) Biodiim. 39. Delac.y, T. P. <k Kennnrd, C. H. J.. (1972) B tophus. A chi, 233, 30G-38O. J. Chan. S"C. Perkin Trans. I, 2, 214-8-2153. 12. Taniguchi, K. & Tononutra, Y. (1971) J. 40. Wilson, X. K. A- Wilson, W. E. (1972; Sci. Biochnn. (Tokyo), 69, 543-557. Total Environ., 1, 245-251. 13. Hokin, L. K. A Ilexutn, T. D. (1972) Arch.. 41. Tinsley, I. J.. IJ;tf|iie, Jt. it Sehmedding, 1), Biochem. Biophys., 131, 453-463. (1971) Srienr-. 171, 145-147. ry . >. C:'-. .<'0*'x-' w:*- *- . . ' ' ; *, r ^ .. '* OSM 025838 -- _ ,, T. i "V C * . . A- y >: r, S " r if >. r*. t I *> 't-t-1' , r* . c.t rl. A i. STLCOPCB4009793