Pergamon Prass
Life Scieacae Vol . 21, pp . 1559-1568 Printed is the II .S . A.
AN INHIBITOR OF OPIATE RECEPTOR BINDING FROM HIIMAN
ERYTHROCYTES IDENTIFIED AS A GLIITATHIONE-COPPER COMPLE% Giovanni Marzullo and Arnold J . Friedhoßß Millhauaer Laboratories oß the Department oß Psychiatry New York IIniveraity School oß Meäicine New York s N .Y . 10016 II .S .A . (Received in final form September 28, 1977) A "peptide-like" inhibitor oß opiate receptor binding and oß N-methyltranaßerase previously purißied by ua ßrom rabbit brain was also found in human red blood cells . Boiled extracts oß erythrocytes were ßractionated on Sephadex ßollowed by chromatography oß the active ßractiona on silica gel layers . Both, a migrating ninhydrin-positive spot and a naturally blue substance which did not migrate ßrom the origin coincided with the active ßractions . The blue substance was identißied as copper and the ninhydrin-positive material was identißied as oxidized glutathione . While glutathione per se has no eßßect, copper and other transition metals are potent inhibitors oß opiate receptor binding . A mixture oß glutathione and copper plus serum albumin in proportions simulating erythrocyte extracts gave results identical to the latter . Several other laboratories have extracted, from various tissues and body ßluida~ "opiate-like peptides" which are distinct ßrom the ß-LPH derived endorphins . In view oß our Bindings it is possible that metal bound to glutathione or to other peptide ligand may be a complicating Bettor in some oß these studies . During an investigé,tion on the ßormation oß N,N-dimethyltryptamine in rabbit xe observed that the enzyme N-methyltransßerase is strongly inhibited by a dialysable factor Bound in brainy as well as in other tissues from this animal . When purißied by gel ßiltration on Sephadex the major active principle ßrom brain had an apparent molecular weight oß 1500 and was digeste$ .by trypsin and carboxypeptidase A (1~2) . Later we observed the.t this "peptide-like" Bettor also inhibited the stereoapecißic binding oß opiate drugs to brain receptors (3,4) . The Bettor ßrom our laboratory dißßered ßrom both the enkephalin pentapeptidea identißied by Hughes et .al . (5) and from the analgesic C-ßragment o~ ß-LPH (~-endorphin)~6r The enkephalins and ~-endorphin had no eßßect on N-methyltransßerase and were sepa.ra le from our peptide by chromatography . Supported by funds Brom grant # MH 08618 Brom NIMH . x'S9A
1560
Copper, Glutathione and Opiate Receptors
Vol . 21, No . 11, 1977
Several other laboratories have observed "opiate-like peptides" distinguiaheable ßrom the enkephalins and ßrom p-endorphin and having apparent molecular weights intermediate between the above . These have been described in the rat pituitary (7 8), in human spinal ßluid (9), in various tissues ßrom rat (10~, in guinea pig ileum (11) and in human urine (12) . Moreover, a small ßactor unlike enkephalin was described in human blood serum by Pert et .al . (13) and termed "anodynin" . A similar factor from serum was recently reported by Schulz et .al . (14) . In the present report we ßirst demonstrate that the inhibitory ßactor earlier found by us in brain and other tissues from rabbit is also present in human erythrocytes . Subsequently we present the chemical analyses which have led to the identification oß the Bettor as a complex oß glutathione with copper, the metal constituting the inhibitory component . Methods Extraction and Purißication . Freshly drawn, un-heparinized human bloo was can rißuge at 1700 x g ßôr 10 min . followed by removal of serum and bußfy coat using aspiration . One volume of boiling hot distilled water was added to the packed cells and the tube was immediately heated in a boiling water bath ßor 4 min . with continuous stirring using a glass rod . After cooling on ice the pasty extract was~centrißuged at 105,000 x g ßor 60 min . Samples of the supernatant were ßractionated by gel filtration . Typically a 5 ml sample was applied on a 5 .5 x 37 cm column oß Sephadex G-25 (Pharmacia Chemical, ßine grade) equilibrated and eluted with 0 .25 mM NaP04 pH 7 .9 . Collection was in 8 .8 ml fractions at 16 ml/hr . Blue dextran and phenol red were used in some cases to determine void and wash volumes . The fractions were assayed directly or aßter concentration by lyophilization . Receptor Binding Assays . Bindins to opiate receptors was assayed as described by Simon et .el . (15) with modifications . Whole brains ßrom male Sprague-Dawly rata were homogenized in 6 volumes oß 0 .05 M Tris-HC1, pH 7 .4, and centrifuged at 105,000 x g ßor 20 min . The supernatant was removed by aspiration and its volume was replaced with fresh Tria bußfer, .followed by resuspension in a ground ~glasa üomogeüfzer . The binding assay mixture contained 100 ~1 oß membrane ßrâvtion, Tria-HC1 buf~er at 0 .05 M, eit#er dextrorphan or levorphanol at 2 x 10-7 M, H -etorphine at 10- M, plus test ßractions to a total volume oß 1 ml . The mixtures were pre-incubated for 5 min ., before addition oß labelled etorphine and incubation for 15 min . The samplés were filtered through Whatman GF/B discs under auction ßollowed by ßour rapid washes with 2 ml cold Tris bufßer . Stereospecific binding was determined as the difference between binding in the presence oß dextrorphan and in the presence oß levorphanol . Other sass s . N-methyltranaßerase was assayed as described previously 2 with slight modifications . Twenty ~1 enzyme ßrom rabbit . lung (1 :6 saline homogenate centrifuged at 105,000 x g ßor 60 min .) were added to a reaction mixture containing 5 ~ul S-adenosylmethionine-C 14 (S .A . 50 mCi/mmole), 0 .66 mM N-methyltryptamine, 50 mM NaP04 pH 7 .9 plus test ßraotions to a total volume oß 135 ~ul . After 30 min. incubation the reaction was terminated by addition oß 100 pl 0 .5 M borate bufßer pH 10, followed by extraction oß reaction products into ethylacetate and counting . Abbreviations : DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid) ; GSH, reduced glutathione ; GSSG, oxidized glutathione .
Vol . 2i, No . 11, 1977
Copper, Glutathioae sad Opiate Receptors
156 1
Teats ßor copper included reactions with o-tolidine and with alizarin blue as described by Feigl (16) and a quantitative assay with dithizone according to Brumby and Massey (17) . Total glutathione (reduced plus oxidized form) was assayed with glutathione reductase in the presence oß TPNH and DTNB, the absorbency change due to oxidation oß the latter compound being ßollowed at 412 m~ (18). . Chemicals . All chemicals were ßrom Sigma Chemical Co . except ßor HH -e~orphine obtained ßrom Amersham/Searle, dextrorphan and levorphanol, a gißt ßrom Hoffmann LaRoche, S-adenosylmethionine-C14, from New England Nuclear, 0-tolidine and alizarin blue ßrom Fisher . Results Figure 1 shows the inhibitory activity oß boiled extracts from human red blood cells aßter ßractionation on Sephadex G-25 . The enzyme N-methyltranaßerase and the binding of etorphine to brain membranes are inhibited by the same Sephadex ßractiona in a parallel ßashion . Molecular weight estimations oß the active peak on columns calibrated with somatostatin, bacitracin and oxytocin gave a value oß 1500 . This is identical to the apparent molecular wei ht oß the major inhibitory peak from brain as previously reported (2~ . For both, N-methyltranaßerase and receptor binding, the degree oß inhibition showed a linear relationship to the concentration oß active ßraction up to about 85 96 inhibition : Under the conditions described ßor Fig . 1, the relative potency of the most active ßraction, calculated as the volume oß unconcentrated sample necessary ßor 50 q6 inhibition, was 0 .21 pl per ~1 of total mixture ßor the enzyme reaction and 0 .54 pl/~1 for the binding reaction . Following ßractionation on Sephadex, the samples were concentrated by lyophilization and chromatographid on silica gel layers . Fig . 2 shows a tracing oß the chromatographic. result for individual fractions and also indicates the inhibitory activity oß each sample . A ninhydrin-positive spot, marked % (Rß- a15), appears to peak out in parallel with the most active fractions . Additional pink spots, marked Y and Z1-4, ßollow the % material and appear to correlate with the two small activity peaks which ßollow the major ßactor . In addition to the pink ninhydrin spot a blue colored material, which did not migrate ßrom the origin, also appeared and ßaided in parallel with the major active ßractions . This spot is marked B in Fig . 2 . This coloration appeared beßore application of ninhydrin and was independent oß chromatography since a faint blueviolet color could be seen in sufßiciently concentrated samples beßore chromatography . It seemed possible that the blue spot might represent copper ions . Spot tests were perßormed using the o-tolidine reaction and the alizarin blue test as described by Feigl (16) . Both teats were positive with the color intensities appearing to parallel the activities oß the ßractiona . Copper was subsequently assayed quantitatively using the dithizone reaction . As is shown in Fig . 3, a direct correapondencè is Bound between the copper levels and the inhibitory activities oß the ßractions . This correlation is also indicated ßor thé smaller activities which ßollow the major peak . The eßfects oß salts oß copper and oß other divalent cations was subsequently tested in receptor binding assays as shown in Fig . 4 .
156 2
Copper, Glutathione and Opiate Receptors
rt~cnoa ra.
901 . 21, No . 11, 1977
rucr~a+
No.
FIG. 1 . Gel ßiltration on Sephadex G-25 oß boiled extracts ßrom human erythrocytes . Five ml oß extract was applied as described under Methods . Unconcentrated samples oß the ßractiona were assayed ßor inhibition oß N-methyltranaßerase and oß opiate receptor binding as described above . FIG . 2 . Thin layer chromatography oß the ßractions ßrom Fig . 1 aß~rconcentration and lyophilization . The relative activity oß each ßraction is shown above the chromatogram . Layers of Silica Gel F-60 (Merle) were used with chloroßorm : methanol : NH40H (40 : 44 : 17) as the solvent system .
Mercury, silver, copper, zinc and lead, in this order of potency, are inhibitory at concentrations in the micromolar range . Calci~m, magnesium and manganese, however, have no eßßect even at 5 x 10- M . The EC50 ßor the binding inhibition by CuC12 was 4 .8 x 10-5 M. The EC50 ßor peak ßractiona ßractiona ßrom erythrocytes calculated on the basis of their copper content was Bound to be 3 .5 x 10-5 M, in ßair agreement with the above value .
The possibility that the activity peak might represent simple inorganic copper was tested by loading CuClZ on the Sephadex column . No inhibitory activity was Bound at the elution volume oß the unknown peak . Moreover, the metal did not elute ßrom the column but remained bound to the Sephadex and visible as a faint blue ring at the top of the column . A wash with penicillamine, a strong copper chelator, was required to regenerate the column ßor subsequent use .
Vol . 21, No . 11, 1977
Copper, Glutathione and Opiate Receptors
1563
~ti c:
nL ~K . M~ . My . Ca
FIG. 3 . Opiate receptor inhibition and copper concentration in ~ëSephadex fractions described in Fig . 1 . FIG . 4 . Effects of various divalent cations on opiate receptor i~nding .
This evidence suggested that the ninbydrin-positive spot which appeared concomitant with copper formed a complex with this metal and this accounted for its elution location . A literature search for natural peptide chelatora of copper suggested glutathione as a candidate . This tripeptide,,which is present in all cell types forms extremely stable complexes vit}i copper and with other transition cations (19) . Reduced and oxidized glutathione were chromatographed next to concentrated peak fractions on silica gel layers using the solvent system already described in Fig . 2 plus a second solvent (n-propanol : NH40H ; 60 :40) . The % spot showed the same migration as oxidized glutathione in both solvent systems (Rf values : 0 .15 in the first and 0 .27 in the second) . Reduced glutathione is oxidized in the presence of cations and/or upon boiling, therefore the tentative finding of high levels of the oaidiaed form was not surprising . Total glutathione (oxidized plus reduced forms) was subsequently essayed enzyme,tically using glutathione reductase with TPNH in the presence of DTNB .
As is shown in Fig. 5, glutathione levels in the Sephadex fractions parallel the inhibitory activity and, therefore, the copper levels . This finding indicated that a glutathione-copper complex could fully account for the elution behavior and for the inhibitory activity of the unknown factor .
Copper, Glutathioae and Opiate Receptors
1564
Vol . 21, No . 11, 1977
eq
q 0
`5
su a
a w
a fRACTION
NO.
FRACTION
NO.
FIG . 5 . Opiate receptor inhibition and levels oß glutathione in the Sephadex ßractions described in Fig . 1 .
FIG . 6 . Opiate receptor inhibition, copper levels and glutathione levels in Sephadex fractions obtained from a mixture oß glutathione, copper and serum albumin simulating erythrocyte extracts . More deßinitive evidence was sought by ßractionating and testing a mixture oß glutathione and copper, in the presence oß serum albumin, in proportions simulating the composition oß erythrocytes according to published values . A solution was prepared containing 4 .5 ~ bovine serum albumin, 0 .54 mg/ml reduced glutathione aßter Srivastava and Beutler, 20) plus 0 .081 mg/ml cupric sulßate ~aßter Markowits et .al ., 21) . A sample equivalent to 5 mT erythrocyte extract was boiled, centrißuged and ßractionated by the same procedures as described ßor the natural samples . The results, shown in Fig . 6, are analogous to those obtained ßrom blood samples . Copper, glutathione and the inhibitory activity coincide in a single peak occurring at the same elution volume as the unknown . In a ßurther test the light absorption spectra oß peak ßractions from both, the natural extract and the artißicial mixture were examined . Both samples exhibited an absorption maximum around 600'm}T . This is in agreement with the results oß Proneck (22) on the formation oß a blue-violet complex absorbing at 590 myx upon mixing copper and oxidized glutathione at a pH slightly basic . A final test was run to determine whether glutathione serves only as an inert carrier oß copper or whether it modifies the inhibition by the metal . Table I shows that, at the concentration range
Vol . 21, No . 11, 1977
Copper, Glutathione and Opiate Receptors
1565
found in the active fractions, neither form of glutathione has an effect iII the absence of copper . Moreover, at these concentrations neither form of the tripeptide has an effect on the extent of inhibition by copper . TABLE I Lack of Effects by Oxidized and Reduced Glutathione on Opiate Receptor Binding or on the Degree of Inhibition due to Copper . - _ Bound 3H-etorphine ~ of Control Additions None
6 .50 (± 0 .21) .
GSSG (5x 10-5 M) " 10 x " )) " 50 x " -5 GSH 5 x10 M " 10 x " ~ 50 x " Cu~ (5 x 10- 5 M) + GSSG 5 x 10 -5 M " + 10 x " " " + " ~50 x " ~ " n + GSH 5x10 -5M " " + " 10 x " ~ .. .. + n 50 x "
6 .53 6 .23 6 .46
Cu~ (5x10-5 M)
3 .38 (±0 .08)
100
52 .0 (±1 .3) 100 .1 103 .9 96 .8
6 .54 6 .75 6 .29
100 .5 95 .8 99 .4
3 .10 3 . 21~ 3 .43
47 .6 49 .4 52 .7
3 .42 3 .82 3 .62
(± 3 .2)
52 .6 58 .7 55 .9
(* (moles/mg tissue ± S .E .) Discussion A "peptide-like" factor originally described by us in rabbit brain as an endogenous inhibitor of N-methyltranaferase .(1,2) and later also found to inhibit opiate receptor binding (3,4) has been shown here to be present as well in human red blood cells . Purification and chemical analyses on the material from erythrocytes have shown that the inhibitory activity is due to a complex of copper with oxidized glutathione, the metal accounting for the inhibitory action . Identification of the peptide as glutathione is based on co-chromatography with the authentic compound, as well as on activity with glutathione reductase, an enzyme of high substrate specificity. Identification of the bound metal as copper is based on reactions with three specific reagents, o-tolidine, alizarin blue and dithizone . The presence of copper in the complex is further supported by the characteristic blue-violet coloration and the light absor tion maximum around 600 m~ (22) . Li et .al . (23,24) and Proneck p (22) have studied the comp~lexation of copper by both forms of glutathione in pure solutions . Reduced glutathione is oxidized by cupric ions and the oxidized form complexes the metal at molar ratios either 1 :1 or 1 :2 and with an extremely . high formation constant (Log K1=14.2) . Although glutathione can also form strong complexes with other transition cationa, at physiological pH 's complexation with copper is favored over the other metals (25, 19, 22) . It must be noted, however,
1566
Copper, Glutathione and Opiate Receptors
Vol . 21, No . 11, 1977
that glutathione is contained in excess oß copper in erythrocytes and in most tissues . Therefore complexes with other metals may be contained, in addition to copper, in the activity peak . Moreover, since copper ions tend to bind to Sephadex, it is possible that a ßraction oß the metal eluting with glutathione is not derived from the erythrocytes but is picked up ßrom the Sephadex where it may accumulate as an impurity contained in the phosphate bufßer salts . An asaesment of the proportion oß non-copper metal complexed or oß the ßraction of extraneous copper included xill require more detailed analyses . In our experiments the oaidized-glutathione-copper complex, which would have a molecular weight oß 676 assuming a 1 :1 molar ratio, elutes ßrom Sephadex at a volume corresponding to about 1500 daltons . This discrepancy could be explained iß two molecules of the complex combine to ßorm a dimer . This has been ßouad to occur with copper complexes oß certain amino acids and peptides ßrom human serum (26) .
The present Bindings explain the ubiquitous distribution of the inhibitory Bettor since all cell types contain high concentrations oß glutathione and considerable amounts oß copper . These results also explain the higher levels oß inhibitory activity in newborn rabbit tissues than in adult (2) since copper levels in newborns are several Bold higher than in mature animals . Furthermore, the lack oß specißicity oß the inhibition is explained since any enzyme or receptor activity involving an essential -SH group may be inhibited . Simon et .al . (15,27) have already demonstrated the presence of an essential -SH group at the opiate réceptor site . Our results point out an almost exact correspondence between the order of inhibitory eßßectivenesa oß the metals (Hg >Age Cu~Zn ~Pb) and their aßßinity ßor protein SH groups as reported by Slotz (28) . Several investigators have reported on the inhibition oß opiate receptor binding by Bettors which are distinguisheable ßrom the enkephalins and from the larCer p-LPH ßragments . Some of these reports have not included evidence oß naloaone-reversible analge sia ae tested in vivo or in whole tissue preparations . Since glutathione present in all tissues, it may be expected that copper or other transition metal complexed to this peptide may either account ßor, or be included and interßere in the inhibitory activities extracted by other investigators ßrom various tissues (7,8,10,11) . This would not apply to the human serum factor observed by Pert et .al . (13) since glutathione is not present in serum . However, certain amino acids Bound in human serum ßorm strong multiple complexes with copper (26) and these might interßere in studies oß'~,nodynin" . Similarly, the inhibitory preparations ßrom cerebral spinal ßluid (9) and ßrom human urine (12) might involve also peptide bound metals . Our glutathione-copper complex was tested ßor analgesic activity using the guinea pig ileum assay. Preliminary results have shown a morphine-like inhibition prevented, although sot reversed by naloaone (Puig, Marzullo, Musacchio and Friedhoßf, unpublished) . This eßfect appears similar to that of an alkylating anesthetic, as might be expected ßrom the common action oß these agents on essential sulßhydryl groups at the receptor sites (15,29) .
is
Vol. 21, No . 11, 1977
Copper, Glutathione aad Opiate Receptors
1567
References 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 .
G .MARZULLO, H .ROSENGARTEN and A .J .FRIEDHOFF, Trana . Amer . Soc . Neuroch . 7 75 (1976) . S GMARZi~H .ROSENGARTEN and A .J .FRIEDHOFF, Liße 5çi . 20 775-784 (1977) . H .ROSENGARTEN, G.MARZULLO and A .J .FRIEDHOFF, in Pharmach . Biochem. and Behavior Yol . ~, Suppl . 147-150 (1977 . --.ROB ASE GARTEN,G . tZULLÖ and A .J .FRIEDHOFF, Trane . Amer . Soc . Neuroch. 8 79 (1977) . .HIIGHES, T .SMÎTH, $ .1~ .gOSTERLITZ, L .A .FOTHERGILL, B .MORAh J and H .R .MORRIS, Nature 2~ 577-579 (1975) . C .H .LI and D .CHUNG, Proc . Nat . Aced . Sçi . U .S .A . 7 3 11451148 (1976) . H .TESCHEMACHER, R.E .OPFfEIM, B .M .CO% and A .GOLDSTEIN Liße Sçi . 16 1771-1776 (1975) . B .M .COS, R .E .OPHEIM, H .TESCHMACHER and A . GOLDSTEIN Liße Sçi . 16 1776-1777 (1975) . L .TERENIUS and A .1fAHSTROM, Acts Phvsiol . Scand . 94 74-81 (1975) : I .J .WADJA, A .NEIDLE, S .EHRENPREIS and I .MANIGAULT, is Ouiatea and Endogenous Opioid Peptides , H .il.gdaterlitz, Ed . ElsevierNorth Holland, Amsterdam, pp . 129-136 (1976) . M .i~USTER, Naunyn-Schmiedeberg s Archiv . Pharmach . Suppl . 297 R53 (1977) . A .GOLDSTEIS and B .M .CO%, Fed . Proc . Abatr . 1010 (1977) . C .B .PERT, A.PERT and J .F . ALId~tA~I,Proc . Nat . Aced . Sçi . U .S .A . 7 3 2226-2230 (1976) . 1~ SCHULZ, M .WUSTER and A .HERZ, Liße Sci . 2~ 105-116 (1977) . E .J .SIMON, J .M .HILLER and I . EDELMAN, Proc . Nat . Aced . Sçi . U .S .A . 7 0 1947-1949 (1973) . ~.FEIGL and Y .ANGER in S of Testa in Inor snit Anal sis pp . 209-217, Elsevier, Amsterdam 1972 . P .E .BRUMBY and Y.MASSEY in Methods in Enzvmology 10 471-474 (1967) . F . TIETZE, Anal . Biochem . ~ 502-522 (1969) . and Medicine J . SCHUBERT in Co er and Peroxides in Radiobiolo C .C .Thomas, Springßield, I11 . pp . 5-1 19 4 . S .g .SRIYA3TAYA and E .BEQTLER, Bioch . Bio s . Res . Comm . 28 659-664 (1967) . H .MARSO~TITZ, G .E .CARTWRIGHT and M .M .WINTROBE, J . Biol . Chem . 234 40-45 (1959) . .PRONECg, J . Amer . Chem . Soc . 97 3839-3841 (1975) . P N .C .LI, O .GAWRON and G .BASCU~IS, J . Aiaer . Chem . Soc . ~6_ 225229 (1954) . J .M .üTHITE, R .A .MANNING and N .C .LI, J . Amer . Chem . Soc . 2367-237 0 (1956) . R .J .P . WILLIAMS, Biol . Rev. 28 381 (1953) . B .SARSAR and T .P .~RUCg,inThe Biochemiatr oß Co er, J .Peieach, P .Eisen and X .E .Blumberg, s ., Acad . Press pp .1 3-194 (1966) . E .J .SIMON and J .GROTH, Proc . Nat . Aced . Sçi . U .S .A . 72 24042407 (1975) . I .M .ghOTZ in Mechanism oß Enz e Action, 1d .D .McElroy and B .Glass Eds ., John Hopkina eas, Baltimore, pp .257-285 (1954) . G .L .CRAYISO and J~ .M .M[TSACCHIO, Life Sçi . 18 821-828 (1977) .
Life Scieacae Vol . 21, pp . 1559-1568 Printed is the II .S . A.
AN INHIBITOR OF OPIATE RECEPTOR BINDING FROM HIIMAN
ERYTHROCYTES IDENTIFIED AS A GLIITATHIONE-COPPER COMPLE% Giovanni Marzullo and Arnold J . Friedhoßß Millhauaer Laboratories oß the Department oß Psychiatry New York IIniveraity School oß Meäicine New York s N .Y . 10016 II .S .A . (Received in final form September 28, 1977) A "peptide-like" inhibitor oß opiate receptor binding and oß N-methyltranaßerase previously purißied by ua ßrom rabbit brain was also found in human red blood cells . Boiled extracts oß erythrocytes were ßractionated on Sephadex ßollowed by chromatography oß the active ßractiona on silica gel layers . Both, a migrating ninhydrin-positive spot and a naturally blue substance which did not migrate ßrom the origin coincided with the active ßractions . The blue substance was identißied as copper and the ninhydrin-positive material was identißied as oxidized glutathione . While glutathione per se has no eßßect, copper and other transition metals are potent inhibitors oß opiate receptor binding . A mixture oß glutathione and copper plus serum albumin in proportions simulating erythrocyte extracts gave results identical to the latter . Several other laboratories have extracted, from various tissues and body ßluida~ "opiate-like peptides" which are distinct ßrom the ß-LPH derived endorphins . In view oß our Bindings it is possible that metal bound to glutathione or to other peptide ligand may be a complicating Bettor in some oß these studies . During an investigé,tion on the ßormation oß N,N-dimethyltryptamine in rabbit xe observed that the enzyme N-methyltransßerase is strongly inhibited by a dialysable factor Bound in brainy as well as in other tissues from this animal . When purißied by gel ßiltration on Sephadex the major active principle ßrom brain had an apparent molecular weight oß 1500 and was digeste$ .by trypsin and carboxypeptidase A (1~2) . Later we observed the.t this "peptide-like" Bettor also inhibited the stereoapecißic binding oß opiate drugs to brain receptors (3,4) . The Bettor ßrom our laboratory dißßered ßrom both the enkephalin pentapeptidea identißied by Hughes et .al . (5) and from the analgesic C-ßragment o~ ß-LPH (~-endorphin)~6r The enkephalins and ~-endorphin had no eßßect on N-methyltransßerase and were sepa.ra le from our peptide by chromatography . Supported by funds Brom grant # MH 08618 Brom NIMH . x'S9A
1560
Copper, Glutathione and Opiate Receptors
Vol . 21, No . 11, 1977
Several other laboratories have observed "opiate-like peptides" distinguiaheable ßrom the enkephalins and ßrom p-endorphin and having apparent molecular weights intermediate between the above . These have been described in the rat pituitary (7 8), in human spinal ßluid (9), in various tissues ßrom rat (10~, in guinea pig ileum (11) and in human urine (12) . Moreover, a small ßactor unlike enkephalin was described in human blood serum by Pert et .al . (13) and termed "anodynin" . A similar factor from serum was recently reported by Schulz et .al . (14) . In the present report we ßirst demonstrate that the inhibitory ßactor earlier found by us in brain and other tissues from rabbit is also present in human erythrocytes . Subsequently we present the chemical analyses which have led to the identification oß the Bettor as a complex oß glutathione with copper, the metal constituting the inhibitory component . Methods Extraction and Purißication . Freshly drawn, un-heparinized human bloo was can rißuge at 1700 x g ßôr 10 min . followed by removal of serum and bußfy coat using aspiration . One volume of boiling hot distilled water was added to the packed cells and the tube was immediately heated in a boiling water bath ßor 4 min . with continuous stirring using a glass rod . After cooling on ice the pasty extract was~centrißuged at 105,000 x g ßor 60 min . Samples of the supernatant were ßractionated by gel filtration . Typically a 5 ml sample was applied on a 5 .5 x 37 cm column oß Sephadex G-25 (Pharmacia Chemical, ßine grade) equilibrated and eluted with 0 .25 mM NaP04 pH 7 .9 . Collection was in 8 .8 ml fractions at 16 ml/hr . Blue dextran and phenol red were used in some cases to determine void and wash volumes . The fractions were assayed directly or aßter concentration by lyophilization . Receptor Binding Assays . Bindins to opiate receptors was assayed as described by Simon et .el . (15) with modifications . Whole brains ßrom male Sprague-Dawly rata were homogenized in 6 volumes oß 0 .05 M Tris-HC1, pH 7 .4, and centrifuged at 105,000 x g ßor 20 min . The supernatant was removed by aspiration and its volume was replaced with fresh Tria bußfer, .followed by resuspension in a ground ~glasa üomogeüfzer . The binding assay mixture contained 100 ~1 oß membrane ßrâvtion, Tria-HC1 buf~er at 0 .05 M, eit#er dextrorphan or levorphanol at 2 x 10-7 M, H -etorphine at 10- M, plus test ßractions to a total volume oß 1 ml . The mixtures were pre-incubated for 5 min ., before addition oß labelled etorphine and incubation for 15 min . The samplés were filtered through Whatman GF/B discs under auction ßollowed by ßour rapid washes with 2 ml cold Tris bufßer . Stereospecific binding was determined as the difference between binding in the presence oß dextrorphan and in the presence oß levorphanol . Other sass s . N-methyltranaßerase was assayed as described previously 2 with slight modifications . Twenty ~1 enzyme ßrom rabbit . lung (1 :6 saline homogenate centrifuged at 105,000 x g ßor 60 min .) were added to a reaction mixture containing 5 ~ul S-adenosylmethionine-C 14 (S .A . 50 mCi/mmole), 0 .66 mM N-methyltryptamine, 50 mM NaP04 pH 7 .9 plus test ßraotions to a total volume oß 135 ~ul . After 30 min. incubation the reaction was terminated by addition oß 100 pl 0 .5 M borate bufßer pH 10, followed by extraction oß reaction products into ethylacetate and counting . Abbreviations : DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid) ; GSH, reduced glutathione ; GSSG, oxidized glutathione .
Vol . 2i, No . 11, 1977
Copper, Glutathioae sad Opiate Receptors
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Teats ßor copper included reactions with o-tolidine and with alizarin blue as described by Feigl (16) and a quantitative assay with dithizone according to Brumby and Massey (17) . Total glutathione (reduced plus oxidized form) was assayed with glutathione reductase in the presence oß TPNH and DTNB, the absorbency change due to oxidation oß the latter compound being ßollowed at 412 m~ (18). . Chemicals . All chemicals were ßrom Sigma Chemical Co . except ßor HH -e~orphine obtained ßrom Amersham/Searle, dextrorphan and levorphanol, a gißt ßrom Hoffmann LaRoche, S-adenosylmethionine-C14, from New England Nuclear, 0-tolidine and alizarin blue ßrom Fisher . Results Figure 1 shows the inhibitory activity oß boiled extracts from human red blood cells aßter ßractionation on Sephadex G-25 . The enzyme N-methyltranaßerase and the binding of etorphine to brain membranes are inhibited by the same Sephadex ßractiona in a parallel ßashion . Molecular weight estimations oß the active peak on columns calibrated with somatostatin, bacitracin and oxytocin gave a value oß 1500 . This is identical to the apparent molecular wei ht oß the major inhibitory peak from brain as previously reported (2~ . For both, N-methyltranaßerase and receptor binding, the degree oß inhibition showed a linear relationship to the concentration oß active ßraction up to about 85 96 inhibition : Under the conditions described ßor Fig . 1, the relative potency of the most active ßraction, calculated as the volume oß unconcentrated sample necessary ßor 50 q6 inhibition, was 0 .21 pl per ~1 of total mixture ßor the enzyme reaction and 0 .54 pl/~1 for the binding reaction . Following ßractionation on Sephadex, the samples were concentrated by lyophilization and chromatographid on silica gel layers . Fig . 2 shows a tracing oß the chromatographic. result for individual fractions and also indicates the inhibitory activity oß each sample . A ninhydrin-positive spot, marked % (Rß- a15), appears to peak out in parallel with the most active fractions . Additional pink spots, marked Y and Z1-4, ßollow the % material and appear to correlate with the two small activity peaks which ßollow the major ßactor . In addition to the pink ninhydrin spot a blue colored material, which did not migrate ßrom the origin, also appeared and ßaided in parallel with the major active ßractions . This spot is marked B in Fig . 2 . This coloration appeared beßore application of ninhydrin and was independent oß chromatography since a faint blueviolet color could be seen in sufßiciently concentrated samples beßore chromatography . It seemed possible that the blue spot might represent copper ions . Spot tests were perßormed using the o-tolidine reaction and the alizarin blue test as described by Feigl (16) . Both teats were positive with the color intensities appearing to parallel the activities oß the ßractiona . Copper was subsequently assayed quantitatively using the dithizone reaction . As is shown in Fig . 3, a direct correapondencè is Bound between the copper levels and the inhibitory activities oß the ßractions . This correlation is also indicated ßor thé smaller activities which ßollow the major peak . The eßfects oß salts oß copper and oß other divalent cations was subsequently tested in receptor binding assays as shown in Fig . 4 .
156 2
Copper, Glutathione and Opiate Receptors
rt~cnoa ra.
901 . 21, No . 11, 1977
rucr~a+
No.
FIG. 1 . Gel ßiltration on Sephadex G-25 oß boiled extracts ßrom human erythrocytes . Five ml oß extract was applied as described under Methods . Unconcentrated samples oß the ßractiona were assayed ßor inhibition oß N-methyltranaßerase and oß opiate receptor binding as described above . FIG . 2 . Thin layer chromatography oß the ßractions ßrom Fig . 1 aß~rconcentration and lyophilization . The relative activity oß each ßraction is shown above the chromatogram . Layers of Silica Gel F-60 (Merle) were used with chloroßorm : methanol : NH40H (40 : 44 : 17) as the solvent system .
Mercury, silver, copper, zinc and lead, in this order of potency, are inhibitory at concentrations in the micromolar range . Calci~m, magnesium and manganese, however, have no eßßect even at 5 x 10- M . The EC50 ßor the binding inhibition by CuC12 was 4 .8 x 10-5 M. The EC50 ßor peak ßractiona ßractiona ßrom erythrocytes calculated on the basis of their copper content was Bound to be 3 .5 x 10-5 M, in ßair agreement with the above value .
The possibility that the activity peak might represent simple inorganic copper was tested by loading CuClZ on the Sephadex column . No inhibitory activity was Bound at the elution volume oß the unknown peak . Moreover, the metal did not elute ßrom the column but remained bound to the Sephadex and visible as a faint blue ring at the top of the column . A wash with penicillamine, a strong copper chelator, was required to regenerate the column ßor subsequent use .
Vol . 21, No . 11, 1977
Copper, Glutathione and Opiate Receptors
1563
~ti c:
nL ~K . M~ . My . Ca
FIG. 3 . Opiate receptor inhibition and copper concentration in ~ëSephadex fractions described in Fig . 1 . FIG . 4 . Effects of various divalent cations on opiate receptor i~nding .
This evidence suggested that the ninbydrin-positive spot which appeared concomitant with copper formed a complex with this metal and this accounted for its elution location . A literature search for natural peptide chelatora of copper suggested glutathione as a candidate . This tripeptide,,which is present in all cell types forms extremely stable complexes vit}i copper and with other transition cations (19) . Reduced and oxidized glutathione were chromatographed next to concentrated peak fractions on silica gel layers using the solvent system already described in Fig . 2 plus a second solvent (n-propanol : NH40H ; 60 :40) . The % spot showed the same migration as oxidized glutathione in both solvent systems (Rf values : 0 .15 in the first and 0 .27 in the second) . Reduced glutathione is oxidized in the presence of cations and/or upon boiling, therefore the tentative finding of high levels of the oaidiaed form was not surprising . Total glutathione (oxidized plus reduced forms) was subsequently essayed enzyme,tically using glutathione reductase with TPNH in the presence of DTNB .
As is shown in Fig. 5, glutathione levels in the Sephadex fractions parallel the inhibitory activity and, therefore, the copper levels . This finding indicated that a glutathione-copper complex could fully account for the elution behavior and for the inhibitory activity of the unknown factor .
Copper, Glutathioae and Opiate Receptors
1564
Vol . 21, No . 11, 1977
eq
q 0
`5
su a
a w
a fRACTION
NO.
FRACTION
NO.
FIG . 5 . Opiate receptor inhibition and levels oß glutathione in the Sephadex ßractions described in Fig . 1 .
FIG . 6 . Opiate receptor inhibition, copper levels and glutathione levels in Sephadex fractions obtained from a mixture oß glutathione, copper and serum albumin simulating erythrocyte extracts . More deßinitive evidence was sought by ßractionating and testing a mixture oß glutathione and copper, in the presence oß serum albumin, in proportions simulating the composition oß erythrocytes according to published values . A solution was prepared containing 4 .5 ~ bovine serum albumin, 0 .54 mg/ml reduced glutathione aßter Srivastava and Beutler, 20) plus 0 .081 mg/ml cupric sulßate ~aßter Markowits et .al ., 21) . A sample equivalent to 5 mT erythrocyte extract was boiled, centrißuged and ßractionated by the same procedures as described ßor the natural samples . The results, shown in Fig . 6, are analogous to those obtained ßrom blood samples . Copper, glutathione and the inhibitory activity coincide in a single peak occurring at the same elution volume as the unknown . In a ßurther test the light absorption spectra oß peak ßractions from both, the natural extract and the artißicial mixture were examined . Both samples exhibited an absorption maximum around 600'm}T . This is in agreement with the results oß Proneck (22) on the formation oß a blue-violet complex absorbing at 590 myx upon mixing copper and oxidized glutathione at a pH slightly basic . A final test was run to determine whether glutathione serves only as an inert carrier oß copper or whether it modifies the inhibition by the metal . Table I shows that, at the concentration range
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Copper, Glutathione and Opiate Receptors
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found in the active fractions, neither form of glutathione has an effect iII the absence of copper . Moreover, at these concentrations neither form of the tripeptide has an effect on the extent of inhibition by copper . TABLE I Lack of Effects by Oxidized and Reduced Glutathione on Opiate Receptor Binding or on the Degree of Inhibition due to Copper . - _ Bound 3H-etorphine ~ of Control Additions None
6 .50 (± 0 .21) .
GSSG (5x 10-5 M) " 10 x " )) " 50 x " -5 GSH 5 x10 M " 10 x " ~ 50 x " Cu~ (5 x 10- 5 M) + GSSG 5 x 10 -5 M " + 10 x " " " + " ~50 x " ~ " n + GSH 5x10 -5M " " + " 10 x " ~ .. .. + n 50 x "
6 .53 6 .23 6 .46
Cu~ (5x10-5 M)
3 .38 (±0 .08)
100
52 .0 (±1 .3) 100 .1 103 .9 96 .8
6 .54 6 .75 6 .29
100 .5 95 .8 99 .4
3 .10 3 . 21~ 3 .43
47 .6 49 .4 52 .7
3 .42 3 .82 3 .62
(± 3 .2)
52 .6 58 .7 55 .9
(* (moles/mg tissue ± S .E .) Discussion A "peptide-like" factor originally described by us in rabbit brain as an endogenous inhibitor of N-methyltranaferase .(1,2) and later also found to inhibit opiate receptor binding (3,4) has been shown here to be present as well in human red blood cells . Purification and chemical analyses on the material from erythrocytes have shown that the inhibitory activity is due to a complex of copper with oxidized glutathione, the metal accounting for the inhibitory action . Identification of the peptide as glutathione is based on co-chromatography with the authentic compound, as well as on activity with glutathione reductase, an enzyme of high substrate specificity. Identification of the bound metal as copper is based on reactions with three specific reagents, o-tolidine, alizarin blue and dithizone . The presence of copper in the complex is further supported by the characteristic blue-violet coloration and the light absor tion maximum around 600 m~ (22) . Li et .al . (23,24) and Proneck p (22) have studied the comp~lexation of copper by both forms of glutathione in pure solutions . Reduced glutathione is oxidized by cupric ions and the oxidized form complexes the metal at molar ratios either 1 :1 or 1 :2 and with an extremely . high formation constant (Log K1=14.2) . Although glutathione can also form strong complexes with other transition cationa, at physiological pH 's complexation with copper is favored over the other metals (25, 19, 22) . It must be noted, however,
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Vol . 21, No . 11, 1977
that glutathione is contained in excess oß copper in erythrocytes and in most tissues . Therefore complexes with other metals may be contained, in addition to copper, in the activity peak . Moreover, since copper ions tend to bind to Sephadex, it is possible that a ßraction oß the metal eluting with glutathione is not derived from the erythrocytes but is picked up ßrom the Sephadex where it may accumulate as an impurity contained in the phosphate bufßer salts . An asaesment of the proportion oß non-copper metal complexed or oß the ßraction of extraneous copper included xill require more detailed analyses . In our experiments the oaidized-glutathione-copper complex, which would have a molecular weight oß 676 assuming a 1 :1 molar ratio, elutes ßrom Sephadex at a volume corresponding to about 1500 daltons . This discrepancy could be explained iß two molecules of the complex combine to ßorm a dimer . This has been ßouad to occur with copper complexes oß certain amino acids and peptides ßrom human serum (26) .
The present Bindings explain the ubiquitous distribution of the inhibitory Bettor since all cell types contain high concentrations oß glutathione and considerable amounts oß copper . These results also explain the higher levels oß inhibitory activity in newborn rabbit tissues than in adult (2) since copper levels in newborns are several Bold higher than in mature animals . Furthermore, the lack oß specißicity oß the inhibition is explained since any enzyme or receptor activity involving an essential -SH group may be inhibited . Simon et .al . (15,27) have already demonstrated the presence of an essential -SH group at the opiate réceptor site . Our results point out an almost exact correspondence between the order of inhibitory eßßectivenesa oß the metals (Hg >Age Cu~Zn ~Pb) and their aßßinity ßor protein SH groups as reported by Slotz (28) . Several investigators have reported on the inhibition oß opiate receptor binding by Bettors which are distinguisheable ßrom the enkephalins and from the larCer p-LPH ßragments . Some of these reports have not included evidence oß naloaone-reversible analge sia ae tested in vivo or in whole tissue preparations . Since glutathione present in all tissues, it may be expected that copper or other transition metal complexed to this peptide may either account ßor, or be included and interßere in the inhibitory activities extracted by other investigators ßrom various tissues (7,8,10,11) . This would not apply to the human serum factor observed by Pert et .al . (13) since glutathione is not present in serum . However, certain amino acids Bound in human serum ßorm strong multiple complexes with copper (26) and these might interßere in studies oß'~,nodynin" . Similarly, the inhibitory preparations ßrom cerebral spinal ßluid (9) and ßrom human urine (12) might involve also peptide bound metals . Our glutathione-copper complex was tested ßor analgesic activity using the guinea pig ileum assay. Preliminary results have shown a morphine-like inhibition prevented, although sot reversed by naloaone (Puig, Marzullo, Musacchio and Friedhoßf, unpublished) . This eßfect appears similar to that of an alkylating anesthetic, as might be expected ßrom the common action oß these agents on essential sulßhydryl groups at the receptor sites (15,29) .
is
Vol. 21, No . 11, 1977
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References 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 .
G .MARZULLO, H .ROSENGARTEN and A .J .FRIEDHOFF, Trana . Amer . Soc . Neuroch . 7 75 (1976) . S GMARZi~H .ROSENGARTEN and A .J .FRIEDHOFF, Liße 5çi . 20 775-784 (1977) . H .ROSENGARTEN, G.MARZULLO and A .J .FRIEDHOFF, in Pharmach . Biochem. and Behavior Yol . ~, Suppl . 147-150 (1977 . --.ROB ASE GARTEN,G . tZULLÖ and A .J .FRIEDHOFF, Trane . Amer . Soc . Neuroch. 8 79 (1977) . .HIIGHES, T .SMÎTH, $ .1~ .gOSTERLITZ, L .A .FOTHERGILL, B .MORAh J and H .R .MORRIS, Nature 2~ 577-579 (1975) . C .H .LI and D .CHUNG, Proc . Nat . Aced . Sçi . U .S .A . 7 3 11451148 (1976) . H .TESCHEMACHER, R.E .OPFfEIM, B .M .CO% and A .GOLDSTEIN Liße Sçi . 16 1771-1776 (1975) . B .M .COS, R .E .OPHEIM, H .TESCHMACHER and A . GOLDSTEIN Liße Sçi . 16 1776-1777 (1975) . L .TERENIUS and A .1fAHSTROM, Acts Phvsiol . Scand . 94 74-81 (1975) : I .J .WADJA, A .NEIDLE, S .EHRENPREIS and I .MANIGAULT, is Ouiatea and Endogenous Opioid Peptides , H .il.gdaterlitz, Ed . ElsevierNorth Holland, Amsterdam, pp . 129-136 (1976) . M .i~USTER, Naunyn-Schmiedeberg s Archiv . Pharmach . Suppl . 297 R53 (1977) . A .GOLDSTEIS and B .M .CO%, Fed . Proc . Abatr . 1010 (1977) . C .B .PERT, A.PERT and J .F . ALId~tA~I,Proc . Nat . Aced . Sçi . U .S .A . 7 3 2226-2230 (1976) . 1~ SCHULZ, M .WUSTER and A .HERZ, Liße Sci . 2~ 105-116 (1977) . E .J .SIMON, J .M .HILLER and I . EDELMAN, Proc . Nat . Aced . Sçi . U .S .A . 7 0 1947-1949 (1973) . ~.FEIGL and Y .ANGER in S of Testa in Inor snit Anal sis pp . 209-217, Elsevier, Amsterdam 1972 . P .E .BRUMBY and Y.MASSEY in Methods in Enzvmology 10 471-474 (1967) . F . TIETZE, Anal . Biochem . ~ 502-522 (1969) . and Medicine J . SCHUBERT in Co er and Peroxides in Radiobiolo C .C .Thomas, Springßield, I11 . pp . 5-1 19 4 . S .g .SRIYA3TAYA and E .BEQTLER, Bioch . Bio s . Res . Comm . 28 659-664 (1967) . H .MARSO~TITZ, G .E .CARTWRIGHT and M .M .WINTROBE, J . Biol . Chem . 234 40-45 (1959) . .PRONECg, J . Amer . Chem . Soc . 97 3839-3841 (1975) . P N .C .LI, O .GAWRON and G .BASCU~IS, J . Aiaer . Chem . Soc . ~6_ 225229 (1954) . J .M .üTHITE, R .A .MANNING and N .C .LI, J . Amer . Chem . Soc . 2367-237 0 (1956) . R .J .P . WILLIAMS, Biol . Rev. 28 381 (1953) . B .SARSAR and T .P .~RUCg,inThe Biochemiatr oß Co er, J .Peieach, P .Eisen and X .E .Blumberg, s ., Acad . Press pp .1 3-194 (1966) . E .J .SIMON and J .GROTH, Proc . Nat . Aced . Sçi . U .S .A . 72 24042407 (1975) . I .M .ghOTZ in Mechanism oß Enz e Action, 1d .D .McElroy and B .Glass Eds ., John Hopkina eas, Baltimore, pp .257-285 (1954) . G .L .CRAYISO and J~ .M .M[TSACCHIO, Life Sçi . 18 821-828 (1977) .
