(From the Gastroenterology Division, Cornell University Medical College, New York 10021)

Transcription

1 ROLE OF HEPATIC ANION-BINDING PROTEIN IN BROMSULPHTHALEIN CONJUGATION* BY N. KAPLOWITZ, I. W. PERC -ROBB,~ ANn N. B. JAVITT (From the Gastroenterology Dvson, Cornell Unversty Medcal College, New York 10021) (Receved for publcaton 29 May 1973) The transfer of Bromsulphthalen (BSP) (Hynson, Westcott, and Dunnng, Inc., Baltmore, Md.) from plasma to hepatocyte may be medated n part by a proten that can be solated from the cell supernatant fracton of a lver homogenate. By vrtue of ts bndng capacty/or a varety of organc anons, ncludng BSP, ths proten, desgnated "Y" proten (1) or lgandn (2), s beleved to be a determnant of the hepatc uptake process. Ths vew s supported by the assocaton of "physologcal" jaundce wth a low concentraton of Y proten n fetal and neonatal lver (3, 4), as well as phylogenetc evdence demonstratng a correlaton between the rate of clearance of BSP from plasma and the presence of Y proten n lver (5). BSP s excreted n ble manly as the glutathone conjugate that s syntheszed n the lver (6). The conjugaton step s consdered to be rate-lmtng n the overall hepatc transport of BSP from plasma to ble (7, 8). Lver cytosol has been shown to contan a soluble enzyme that catalyses the conjugaton of BSP wth glutathone--the glutathone S-aryltransferase enzyme (9, 10). Durng studes of the bndng of BSP and other anons to Y proten, duplcatng precsely the condtons descrbed n detal by Lev et al. (1), BSP conjugaton wth glutathone was found to occur. Further evaluaton of ths phenomenon, whch forms the bass of ths report, has led us to the concluson that proten may act by both bndng and conjugatng BSP. Materals and Methods The lver donors were albno Wstar stran rats weghng approxmately 250 g. The lvers were perfused n stu wth 0.01 M phosphate buffer, ph 7.4, 0.25 M sucrose. 25% lver homogenates were prepared n the same buffer and the supernatant fracton was harvested after centrfugng at 100,000 g for 60 ran. Gel fltraton was performed on Sephadex G-75 (Pharmaca, Uppsala, Sweden) columns 30 X 2.9 cm usng 0.01 NI phosphate buffer, ph 7.4, contanng 0.I M NaC1 as moble phase. Flow rate was 28 ml/h, 10 fractons/h. BSP was obtaned from Vtarne Co., Inc., New York, and [2-3H]glycne glutathone was obtaned from New England Nuclear, Boston, Mass. (specfc radoactvty, 285 mc/#mol). * Ths work was supported by the Natonal Insttutes of Health grant AM :~ Unversty Clncal Chemstry Department, Royal Infrmary, Ednburgh, Scotland. I.W. P-R. was supported by a Natonal Insttutes of Health Internatonal Travelng Fellowshp Research Award. THE IOURNAL OF EXPERIMENTAL MEDICINE VOLUME 138,

2 484 KAPLOWITZ ET AL. BRIEF DEFINITIVE REPORT Glutathone S-aryltransferase actvty was measured usng ether [ass]bsp and glutathone as eosubstrates (9) or 3,4-dchlorontrobenzene and glutathone as cosubstrates (10). : : - RESULTS Usng BSP bndng as a proten marker, three proten fractons may be dentfed wth eluton vol of 75, 95, and 135 ml, respectvely (Fg. 1). The second proten corresponds to Y proten. [ah]glycne glutathone was eluted as two peaks: a mnor peak (eluto n vol 75 ml) and a large symmetrcal peak (eluton vol 95 ml). The latter peak s shown to correspond closely wth the presence of BSP bound,to proten (Fg. ). Snce a smlar pattern of glutathone bndng was also found n the absence of BSP, the glutathone bndng does not represent bound BSP-glutathone conjugate. 04- o 113, "S 02- z <~ co Pc - 0 g on I ot ELUTION VOL (ml), 500 E o ~ 300 >- H > I-- FIG. 1. Eluton pattern from Sephadex G-75 column of lver cell supernatant (4.0 ml) wth added BSP (4.0 rag) and [ah]glycne glutathone (2.5 #C). I00 O te Fractons correspondng to eluton vol 65-80, , , and ml were combned and glutathone S-aryltransferase actvty was assayed usng BSP as substrate (Table I). Actvb" greater than control was present only n the combned fractons correspondng to Y proten. When glutathone S-aryltransferase actvty was assayed wth 3,4-dchlorontrobenzene n each ndvdual fracton from the Sephadex column, a close correspondence wth the eluton pattern of [ah]glycne glutathone was found (Fg. 2). Enzvme actvty was found only n the fractons correspondng to Y proten. DISCUSSION Sephadex gel fltraton permts separaton of protens accordng to ther molecular weghts (11). The addton of the organc anon, BSP, to the hepatc supernatant fracton undergong molecular sevng has led to the dentfcaton

3 KAPLOWITZ ET AL. BRIEF DEFINITIVE REPORT 485 TABLE I Conjugaton of BSP and Glutathone by Lver Supernatant Proten Fractons Eluton vol Radoactvty pss] BSP BSP conjugate Percentage conjugaton ml cpm % , , , pss]bsp (1 ram) and reduced glutathone (8 ram) were ncubated n 0.01 M phosphate buffer, ph 7.4, at 37 C for 60 ran together wth an alquot of the proten-contanng fractons. Conjugated and unconjugated BSP were separated by thn-layer chromatography and radoactvty was estmated by lqud scntllaton countng. Nonenzymc conjugaton of BSP and glutathone produces approxmately 5% conjugaton under these condtons T s ~_ I.C- o u "a L 2.,o00 r v E u >- ~o.~- ~ 0.2 m I ooo b O m c~ ~" o ELUTION VOL (ml) FIG. 2. Eluton pattern of lver cell supernatant (2.0 ml) wth added [3H]glycne glutathone (2.5/zC). The maxmum enzyme actvty has been arbtrarly gven a value of 1.0 and actvty present n each fracton s plotted as a proporton of ths maxmum. of a seres of anon-bndng protens, one of whch has been called Y proten (1) or lgandn (2). We have demonstrated that the eluton volume of the BSP markng Y proten and the eluton volume of glutathone are the same. Ths suggests that glutathone bnds to the same proten as BSP and that glutathone may therefore be used as an alternatve marker for Y proten durng gel fltraton. Snce BSP and glutathone are cosubstrates for the hepatc supernatant enzyme, glutathone S-aryltransferase, the fndng of nearly supermposable peaks for these two substances correspondng to hepatc supernatant Y proten suggested the possblty that enzymc actvty mght resde n these fractons. Usng two dfferent assay systems, ths enzyme actvty was found to be I I

4 486 KAPLOWITZ ET AL. BRIEF DEFINITIVE REPORT present n those fractons contanng Y proten, and the pattern of actvty corresponded wth the pattern of glutathone; no actvty was detected n any other fractons (Fg. 2). We are thus led to conclude that a close relatonshp, f not dentty, exsts between Y proten and glutathone S-aryltransferase. Further purfcaton of the enzyme wll permt us to make an exact comparson of ts physcal and chemcal propertes wth those publshed for Y proten (2). Ths newly proposed functon for Y proten as a glutathone-conjugatng enzyme should lead to a reapprasal of ts role n the hepatocellular uptake of organc anons from plasma. The sgnfcance of bndng of anons other than BSP must be assessed wth partcular reference to possble nhbton of enzymc actvty as well as to other anons actng as substrates for the enzymes. For example, blrubn and cholecystographc agents have been shown to be bound by Y proten and thus mght nhbt the rate-lmtng glutathone conjugaton step n BSP transport. Ths would provde an explanaton for the clncal mparment of plasma BSP clearance seen n lver dsease or durng blary tract radography. Thus, lgandn may partcpate n the hepatc transport process n two ways: as an ntracellular bndng proten n the hepatc uptake of organc anons and as an enzyme that further metabolzes certan of these compounds. SUMMARY Usng gel fltraton, the bndng of both glutathone and Bromsulphthalen (BSP) to a lver-soluble proten was found to be dentcal. BSP-conjugatng actvty (glutathone S-aryltransferase) was present only n the fractons correspondng to the two proten-bound markers. Usng a hghly senstve assay, wth 3,4-dchlorontrobenzene, the pattern of glutathone S-aryltransferase actvty was found to concde wth Y proten. Ths evdence suggests that Y proten, or lgandn, has a dual role n hepatc transport: a specfc enzymc functon n the conjugaton of certan anons wth glutathone n addton to a transport functon n the ntracellular bndng of organc anons. REFERENCES 1. Lev, A. J., Z. Gatmatan, and I. M. Aras Two hepatc cytoplasmc proten fractons, Y and Z, and ther possble role n the hepatc uptake of blrubn, sulfobromophthalen, and other anons. J. Cln. Invest. 48: Ltwack, G., B. Ketterer, and I. M. Aras Lgandn: a hepatc proten whch bnds sterods, carcnogens and a number of exogenous organc anons. Nature (Lond.). 2114: Lev, A. J., Z. Gatmatan, and I. M. Aras Defcency of hepatc organc anon-bndng proten as a possble cause of non-haemolytc unconjugated hyperblrubnaema n the newborn. Lancet. 2: Lev, A. J., Z. Gatmatan, and I. M. Aras Defcency of hepatc organc anon-bndng proten, mpared organc anon uptake by lver and "physologc" jaundce n newborn monkeys. N. Engl. J. Med. 283:1136.

5 KAPLOWITZ ET AL. BRIEF DEFINITIVE REPORT Levne, R. I., H. Reyes, A. J. Lev, Z. Gatmatan, and I. M. Aras Phylogenetc study of organc anon transfer from plasma nto the lver. Nat. New Bol. 231: Javtt, N. B., H. O. Wheeler, K. J. Baker, O. L. Ramos, and S. E. Bradley The ntrahepatc conjugaton of sulfobromophthalen and glutathone n the dog. J. Cln. Invest. 39: Combes, B The mportance of conjugaton wth glutathone for sulfobromophthalen sodum (B SP) transfer from blood to ble..1. Cln. Invest. 44: Whelan, G., J. Hoch, and B. Combes A drect assessment of the mportance of conjugaton for blary transport of sulfobromophthalen sodum. J. Lab. Cln. Med. 75: Combes, B., and G. S. Stakelum A lver enzyme that conjugates sulfobromophthalen sodum wth glutathone. J. Cln. Invest. 40: Booth, J., E. Boyland, and P. Sms An enzyme from rat lver catalysng conjugatons wth glutathone. Bochem. J. 79: Tselus, A., J. Porath, and P. Albertsson Separaton and fractonaton of macromolecules and partcles. Scence (Wash. D.C.). 141:13.