~~~~~~~~~~~~~~~~2- ~~~~~~~~~~~~~~~~~10. go 3 NAFM

Transcription

1 2528 orrectons Proc. Natl. Acad. Sc. USA 73 (1976) orrecton. In the artcle "A 15-hydroxyprostaglandn dehydrogenase specfc for prostaglandn A n rabbt kdney" by H. G. Oen, E. A. Ham, M. E. Zanett, E. H. Ulm, and F. A. Kuehl, Jr., whch appeared n the Aprl ssue of Proc. Nat. Acad. Sc. USA 73, , an error was made by the PROEEDINGS Offce. Fg. 1B was shown as Fg. 2B and Fg. 2B was presented as Fg. 1B. The correct sequence s gven below. go 3 A ~~~~~~~~~~~~~~~~~ B 2 ~~~~~~~~~~~~~~~~ mnudts jg proten/ml ncubaton FIG 1. Tme and proten dependence of rabbt kdney paplla PGDH. (A) Dependence on ncubaton tme usng 3MM PGA1 and 13 gg of proten per.2 ml of ncubaton volume. (B) Dependence on enzyme proten usng 3MM PGA1 and 2 mn ncubaton tme. - I X9 I, a _ XL e NMD (FM) FIG 2. omparson of NAD dependence of PGDH of rabbt kdney paplla and bovne lung. (A) NAD dependence of rabbt kdney paplla PGDH usng 3 MM PGA1 and 13,g of proten per.2 ml of ncubaton volume. (B) NAD dependence of bovne lung PGDH usng ether 2.8 AM PGE1 or 3.MgM PGA1 and 2.3,g of proten per.2 ml of ncubaton volume. NAFM

2 Proc. Nat. Acad. Sc. USA Vol. 73, No. 4, pp , Aprl 1976 Bochemstry A 15-hydroxyprostaglandn dehydrogenase specfc for prostaglandn A n rabbt kdney (prostaglandn A dehydrogenase/rabbt kdney paplla/duretc agents/hypertenson/prostaglandn metabolsm) H. G. O1EN, E. A. HAM, M. E. ZANETTI, E. H. ULM, AND F. A. KUEHL, JR. Merck Insttute for Therapeutc Research, Rahway, New Jersey 765 ommuncated by Josef Fred, January 26,1976 ABSTRAT Examnaton of a soluble fracton derved from homogenates of rabbt kdney paplla revealed the exstence of a 15-hydroxyprostaglandn dehydrogenase specfc for A-type prostaglandns. Prostaglandns of the E- and F-seres were not substrates for ths enzyme. In agreement wth publshed data, the 15-hydroxyprostaglandn dehydrogenase(s) derved from the kdney cortex were found to degrade all prostaglandns examned (PGE, PGF, PGA) n the presence of added cofactor NAD. Thus t s evdent that n ths speces the kdney 15-hydroxyprostaglandn dehydrogenases are anatomcally compartmentalzed so that the paplla s able to metabolze only A-prostaglandns n contrast to the cortex whch s capable of degradng E-, F-, and A-type prostaglandns by ths metabolc pathway. Although there are many catabolc pathways for the prostaglandns (PGs), converson of the 15(S)-hydroxy group to a keto-functon by 15-hydroxyprostaglandn dehydrogenase (PGDH) s consdered to be both the ntal and major route for ther transformaton to nactve metaboltes (1). Ths enzyme(s) has been shown to be present n most tssues examned, although t s partcularly abundant n the lung, the organ most thoroughly studed n ths regard (2, 3). Studes revealed that prostaglandns of both the E-type (PGEs) and the F-type (PGFs) are almost completely nactvated by ths enzyme durng a sngle passage through the lung. On the other hand, prostaglandns of the A-type (PGAs) survve crculaton through the lung, a fact leadng to the suggeston that PGAs, as opposed to other prostaglandns, may be crculatng hormones (4, 5). Nevertheless, upon n vtro exposure to the acton of PGDH solated from the lung, PGAs are almost as readly converted to ther 15-keto metaboltes as are other prostaglandns (6). Although the reason for ths s not clear, the recent reports that trtated PGAI has an affnty for blood protens, exceedng that of PGEs and PGFs, may relate to ths phenomenon (7, 8). The affnty of PGAI for plasma protens may not permt ts exposure to, and degradaton by, the lung PGDH. Despte the falure of trtated PGAs to be sgnfcantly metabolzed when perfused through the lung, PGA2 has been reported to be rapdly converted to more polar metaboltes when perfused through the rabbt kdney (9). Thus, f the ablty of PGA2 to survve crculaton through the lung s attrbutable to the affnty of PGA2 for plasma protens, then ths suggests the exstence of a new enzyme that undergoes an unque metabolc nteracton wth the PGAs n the kdney. The partal purfcaton and characterzaton of a kdney PGDH, specfc for PGAs, s the substance of ths report. MATERIALS AND METHODS Trtated prostaglandns, specfc actvty of >6 /mmol, Abbrevatons: PGs, prostaglandns of type A, B, E, or F; PGDH, 15-hydroxyprostaglandn dehydrogenase. 117 were obtaned from New England Nuclear orp., Boston, Mass. 15-Keto-PGA1 was prepared by acd treatment of 15- keto-pgei or enzymatcally usng rabbt paplla PGDH. The use of ether method produced a product that was dentcal by mass spectrometry. 15-Keto-PGEI and 13,14-dhydro-PGE, (PGEo) were prepared by Dr. D. Taub and assocates of Merck & o., Rahway, N.J., accordng to the procedure of AnggArd and Samuelsson (1). PGB1 was prepared by the method of Andersen (1). Other prostaglandns were purchased from Ono Pharmaceutcals, Osaka, Japan. Ethacrync acd, dhydroethacrync acd, dhomoethacrync acd, 2,3-dchloro-4-(2,2-dacetylvnyl)phenoxyacetc acd, mersalyl acd, and furosemde were obtaned from Dr. E. J. ragoe of Merck Sharp and Dohme, West Pont, Pa. NAD, NADP, NADH, and NADPH were purchased from P-L Bochemcals, Inc., Mlwaukee, Wsc. Frozen rabbt cortex and paplla tssue was obtaned from Pell-Freeze Bologcals, Inc., Rogers, Ark. Preparaton of PGDH from Rabbt Kdney Paplla or ortex. All procedures were carred out at 4 unless otherwse stated. Rabbt kdney cortex and paplla tssue, dssected from fresh kdneys for mmedate use or obtaned commercally n frozen form, were used n these experments wth ndstngushable results. Tssue was mxed wth an equal volume of.5 M sodum phosphate, 1 mm EDTA, ph 7.4 buffer, and homogenzed wth a Polytron PT-1OST. entrfugaton of the homogenate at 1, X g for 3 mn was followed by centrfugaton of the resultant supernatant fracton at 1, X g for 6 mn. Sold ammonum sulfate was added to the 1, X g supernatant fracton to 3% saturaton and strred for 1 hr. entrfugaton at 16, X g for 3 mn gave a supernatant fracton whch was brought to 7% saturaton by the addton of more ammonum sulfate, care beng taken to mantan the ph at 7.4. The suspenson was strred overnght and the precptate obtaned by centrfugaton at 16, X g for 3 mn. The precptate was dssolved n phosphate-edta buffer, dalyzed aganst the buffer, subdvded, and stored at -9. It was found that 1 g of ether cortex or paplla tssue provded about 1 mg of enzyme-actve proten n the fnal preparaton. The paplla preparaton was found to be free of NADH oxdase. Bovne Lung Prostaglandn Dehydrogenase. Ths enzyme was prepared accordng to the method of Saeed and Roy (11) wthout the acetone precptaton step and stored at -9 untl used. One gram of lung tssue was found to produce 1 mg of fnal proten. Dehydrogenase Assay (Isotope Method). The dehydrogenatons were carred out n a fnal volume of.2 ml of.5 M sodum phosphate, 1 mm EDTA, ph 7.4 buffer, and 4 Al of methanol. Trtated prostaglandn (about 3,

3 118 Bochemstry: Oen et al. Proc; Nat. Acad. Sc. USA 73 (1976) Table 1. A comparson of the propertes of the PGDHs derved from rabbt kdney and bovne lung Rabbt kdney paplla Rabbt kdney cortex Bovne lung Km K Inhbtor Km K Inhbtor Km K Inhbtor ompound (MM) (AM)* type (Um) (jm)* type (AM) (,MM)* type PGAI PGA n.d. 6t 1 PGE1 n.s PGE2 n.s. n.d. n.d. n.d. 3t n.d. PGEO n.d. 11 n.d. n.d. n.d. 4 PGFa n.s. 49 n.d. n.d. 14t 11 PGF2a n.s. 34 n.d. n.d. 6t 3 PGB1 n.s. 3 n.d. n.d. n.s keto-PGA1 n.s. 11 n.d. n.d. n.d. n.d. 15-keto-PGEI n.s. 81 n.d. n.d. n.d. n.d. Assays were performed as descrbed n Materals and Methods (sotope method) wth 2 mn ncubaton tmes. Incubatons wth paplla PGDH contaned 4.2 mm NAD and 13,qg of proten per.2 ml; Vmax = 2. yg of 15-keto-PGA1/mg of proten per mn. Incubatons wth cortex PGDH contaned.42 mm NAD and 25 Ag of proten per.2 ml; Vmax =.6 Ag of 15-keto-PGA1/mg of proten per mn. Incubatons wth lung PGDH contaned 4.2 mm NAD and 5-1,ug of proten per.2 ml; Vmax =.15,g of 15-keto-PGA1/mg of proten per mn. n.s. = not a substrate. n.d. = not determned. = compettve nhbtor. * Usng PGA1 as a substrate. t Values as determned by Matschnsk et al. (ref. 18). cpm), NAD, and enzyme were added separately n buffer solutons whle unlabeled prostaglandns (for amounts see legends to fgures) and nhbtors were added separately n methanol solutons. The reactons were ntated by the addton of the enzyme and contnued at 37, usually for 2 mn. The ncubatons were termnated by the addton of.2 ml of.35 M ctrc acd, to adjust the ph to 4, wth 1ljg of carrer substrate and 2 jg of carrer product dssolved n 5 gl of methanol. The ncubatons were extracted once wth 4 ml of ethyl acetate and the extracts were washed twce wth.5 ml of water and taken to dryness wth a stream of ntrogen. The resdues were analyzed by thn-layer chromatography on Analtech slca-gel GF plates (25 j) usng ethyl acetate-sooctane-acetc acd (5:5:.5) when [3H}PGAs and [3H]PGB1 were substrates, and system F-VI of Andersen (12) when [3H]PGEs and [3H]PGFs were substrates. The carrer substrates and products were vsualzed wth ultravolet lght and/or odne vapor and these zones were counted n ethyl alcohol-toluene (3:7) phosphor usng a Packard Lqud Scntllaton counter. Recoveres were of the order of 85%, correctng for slca-gel blanks, and a reproducblty of 42% was obtaned. Prostaglandn Dehydrogenase Assay (5 nm A Method). Ths assay was performed by a modfcaton of the method of Vonkeman et al. (13). Incubatons were carred out n.5 ml of.25 M Trs-HI at ph 9. wth 118 jm PGAI and 1 mm of NAD, when requred. Reactons were ntated by the addton of paplla enzyme. After 2 or 6 mn at 37,.5 ml of 2 M NaOH was added, and the maxmum absorbance at 5 nm was recorded usng a Beckman K25 spectrometer. Maxmum absorbance was reached wthn 15 sec of mxng, and then rapdly declned. RESULTS Substrate specfcty and knetc propertes As shown n Table 1, for the prostaglandns examned, only those of the A-seres were metabolzed by the rabbt paplla enzyme preparaton; PGEs and PGFs were completely resstant to the acton of the paplla enzyme. Ths metabolsm of 4. I. -. k a ea mnutes NAD %M FIG. 1. Tme and proten dependence of rabbt kdney paplla PGDH. (A) Dependence on ncubaton tme usng 3 MM PGA1 and 13 Jg of proten per.2 ml of ncubaton volume. (B) Dependence on enzyme proten usng 3MM PGA1 and 2 mn ncubaton tme.

4 Bochemstry: Oen et al. Proc. Nat. Acad. Sc. USA 73 (1976) 119 B I 12 1 la In z 8 6 U, J. IȦ.~S~S.1.5- I o1 2 3 pg proten/ml ncubaton NAD (1M) FIG. 2. omparson of NAD dependence of PGDH of rabbt kdney paplla and bovne lung. (A) NAD dependence of rabbt kdney paplla PGDH usng 3 AM PGA1 and 13,ug of proten per.2 ml of ncubaton volume. (B) NAD dependence of bovne lung PGDH usng ether 2.8 MM PGE1 or 3. AM PGA1 and 2.3 Mg of proten per.2 ml of ncubaton volume. _e - PGA1 was dependent upon both tme and proten concentraton as shown n Fg. 1 and dd not take place n the presence of heat-nactvated enzyme. The enzyme exhbted a ph and temperature optmum of 7.5 and 37, respectvely. A comparson of propertes of the paplla enzyme wth the PGDHs prepared smlarly from the rabbt kdney cortex and bovne lung revealed some strkng dfferences as shown n Table 1. The apparent Mchaels constant (Km) for PGA1 wth the PGDH from the rabbt kdney paplla (15 AM) was the same as that derved from the kdney cortex enzyme (17,gM). Ths value contrasts wth the Km for PGA1 usng the bovne lung enzyme whch s 1 MtM. Interesngly, the Km value for PGE1 s about the same for the kdney cortex PGDH (2 MM) as observed for ths prostaglandn wth the bovne lung enzyme (1,M). ofactor requrements Added NAD dd not enhance the converson of PGA1 (3 MM) to 15-keto PGA1 by rabbt kdney paplla PGDH usng the sotope method. Smlarly, the oxdaton of PGA1 by the Table 2. NAD stmulaton of PGDH actvty at a hgher level of substrate PGA1 Enzyme added -2 mn 2-6 mn (pg of proten) -NAD +NAD -NAD +NAD (.27)a (.141)a Determnatons were performed as descrbed n Materals and Methods (5 nm A method). A zero reacton blank (6 mn ncubaton wthout PGA1) was subtracted from all values. All values are averages of duplcate determnatons except for ( )a whch are sngle determnatons. rabbt kdney cortex PGDH dd not requre added NAD; however, converson of PGE1 to 15-keto-PGE1 by ths enzyme dd requre added NAD. In contrast, PGDH of bovne lung orgn requred NAD for metabolsm of PGA1 as well as PGE1 (Fg. 2). An effect of added NAD on paplla PGDH was observed at a hgher level of PGA1 (118,M) employng the alkalne A method of measurement at 5 nm. An obvous enhancement of the ntal rate of formaton of 15- keto-pgai, as well as the amount formed n 6 mn, s evdent wth added NAD (Table 2). NADP dd not enhance the metabolsm of PGA1 by the paplla or cortex enzyme nor dd t allow the metabolsm of PGE1 by the paplla. The reduced cofactors, NADH and NADPH dd not nhbt PGA1 metabolsm by ether paplla or cortex PGDH; however, NADH nhbted the NAD-dependent converson of PGAI to 15-keto-PGAI by bovne lung PGDH. Effect of nhbtors The data n Table 1 reveal that despte the nablty of prostaglandns other than those of the A-type to act as substrates for the paplla enzyme, they are wthout excepton compettve nhbtors of the metabolsm of PGA1; PGB1 s partcularly effectve n ths regard. In addton, the ablty of a number of duretc agents to nhbt the metabolsm of PGA1, wth the paplla enzyme compared to that derved from the bovne lung (Table 3), reveals an nterestng dfference. Although the order of potency of the ndvdual compounds s seen to follow the same pattern n both nstances, the nature of the nhbton aganst the paplla enzyme s seen to be consstently compettve whereas that aganst the lung enzyme s noncompettve n nature. The fact that mersalyl acd s noncompettve n both nstances may relate to the avdty of heavy metal compounds for sulfhydryl groups. DISUSSION Although there are some dfferences n the Km and Vmax values for the ndvdual prostaglandns when they serve as

5 111 Bochemstry: Oen et al. Proc. Nat. Acad. Sc. USA 73 (1976) Table 3. Ethacrync acd A comparson of the effects of duretc agents and analogs on PGDHs derved from the rabbt kdney paplla and bovne lung usng PGA1 as substrate (sotope method) Rabbt kdney paplla PGDH Bovne lung PGDH Inhbtor Inhbtor ompound K (MM) type K (IM) type A H3-H2-- II H2 Dhydroethacrync acd cl jo- H2OOH H3-H2-H-O-j--H2-OOH H3 Dhomoetnacrync acd Ol H3-H2-- -(H2)3-OOH 11 H2 Furosemde OOH NH H2- o NH2-S 2 1 2,3-Dchloro4-(2,2-dacetylvnyl)phenoxyacetc acd (H3O)2-=O-H / O-H2-OOH Mersalyl acd -OH2 O-OH ' O-NH-H2-OH-H--.HgOH 11 O O-H3 Incubatons were performed as descrbed for paplla and lung enzyme n Table 1. = compettve nhbtor. = noncompettve nhbtor substrates for PGDH solated from varous tssues, no unque preference for ndvdual PGs was ntally reported for any of these enzyme preparatons. The recent -report, however, that some mammalan tssues contan both NAD (type I)- and NADP (type II)-dependent PGDHs whch have some preference for PGEs and PGFs, respectvely (14), ndcates that dfferences n enzyme characterstcs can be of a more fundamental nature than orgnally envsoned. The fndngs reported here, descrbng the exstence of a PGDH wth a unque specfcty for PGAs, further serve to emphasze the fact that PGDH can play a key role n regulatng tssue levels of specfc prostaglandns, rather than smply servng to catabolze prostaglandns as a group. Thus, dependng on the organ nvolved, and the physologcal functon of ndvdual PGs, PGDHs could play key regulatory roles n controllng tssue levels of specfc prostaglandns. Ths concept s consstent wth the observaton, noted earler, that PGAs survve crculaton through the bovne lung but not through the rabbt kdney. The precse mechansm by whch the rabbt paplla PGDH exerts a hgh degree of specfcty for PGAs cannot be establshed conclusvely n the absence of a purfed preparaton of ths enzyme. However, t s reasonable to conclude that the property of the PGAs that dstngushes them from other prostaglandns, namely, the presence of an unsaturated carbonyl functon n the cyclopentane rng, s central to ther nteracton wth the paplla PGDH. Recent evdence has been provded to show that unlke other prostaglandns PGAs, lke ethacrync acd, react covalently wth the sulfhydryl group of cystene and the cystene-contanng peptde glutathone (15). The bndng of PGA1 and ts dsplacement by ethacrync acd, n a cytosol preparaton of the paplla,

6 Bochemstry: Oen -et al. has been suggested to nvolve the sulfhydryl groups of PGDH. Ths concept s supported by the fact that dhydroethacrync acd, whch s ncapable of nteractng wth sulfhydryl groups, has only I' of the actvty of the parent compound as a PGDH nhbtor (Table 3). Thus, t s temptng to speculate that an nteracton of the unsaturated carbonyl functon of PGAs wth an actve sulfhydryl group n the paplla enzyme s central to the substrate specfcty of ths enzyme. However, such a suggeston remans to be confrmed by studes wth the purfed enzyme. Nevertheless, t s clear that PGA-PGDH nteracton may nvolve components of the overall PG structure n addton to the cyclopentenone rng. Ths s suggested by the fact that PGEs and PGFs, although not substrates, are compettve nhbtors of the oxdaton of PGAs by ths enzyme. Furthermore, the fact that 15-keto-PGA' s released from -the enzyme ndcates that despte the unsaturated rng carbonyl group, common both to PGA1 and ts 15-keto dervatve, the 15-hydroxyl group s an mportant factor n the affnty of ths enzyme for substrates. In addton to ts substrate specfcty, other features of the PGDH obtaned from the rabbt paplla dstngush t from that derved from the bovne lung, as well as from the cortex of the rabbt kdney. The apparent hgh degree of affnty of the PGA enzyme for NAD makes t possble to demonstrate an NAD requrement only after reducton of bound NAD by addton of a large excess of substrate. Such tenacous bndng of NAD by dehydrogenases s not wthout precedent (16). However, the precse cofactor requrement of ths PGA PGDH needs further study on a more purfed preparaton before ts exact nature can be determned. Unlke the type I and type II enzymes descrbed by Lee and Levne (14), no nhbtory acton of NADH or NADPH can be noted for ths enzyme. Most strkng, however, s the dfference n the knetc nature of the nhbton of ths enzyme by a number of duretc agents, ncludng ethacrync acd and ts analogs, when compared to the bovne lung enzyme. Although nhbton of the lung PGDH by these substances s noncompettve, nhbton aganst the paplla enzyme s consstently compettve. The excepton, mersalyl acd, a mercural compound, was noncompettve n both nstances, a property lkely attrbutable to the rreversble reacton of the heavy metal wth sulfhydryl groups n the enzyme. In the case of the kdney cortex, the NAD requrement for the metabolsm of PGE and the absence of such an acute NAD requrement for PGA metabolsm suggests that ths porton of the kdney may contan two dstnct PGDHs: one specfc for PGAs smlar to that present n the paplla, and a second capable of degradng PGEs. These observatons contrast wth reports that NAD s requred for the metabolsm of both PGEs and PGAs by the lung PGDH, and suggest that the enzyme that degrades PGEs n the kdney s ncapable of degradng PGAs. Separaton of these two enzymes s clearly Proc. Nat. Acad. Sc. USA 73 (1976) 1111 requred before these ssues can be rgorously establshed. Based upon the poor ablty of the rabbt paplla to metabolze PGE1 n the presence of NAD, ths porton of the rabbt kdney up to now has been consdered to be largely devod of a PGDH (17). Although ths present report concurs that the rabbt paplla s ncapable of convertng PGEs to ther 15-keto metaboltes, t does establsh the presence of a unque PGDH n ths porton of the kdney specfc for PGAs. Although the role of ths class of prostaglandns n kdney functon and hypertenson has been controversal, the exstence of ths new enzyme poses the possblty that PGAs may play a regulatory role of some sort n the rabbt. We have also establshed the presence of a PGA-specfc PGDH n paplla cytosol of the rat. As wth the rabbt, cytosol derved PGDH from rat kdney cortex metabolzes PGEs as well as PGAs. The Km values determned n each case were vrtually the same as those obtaned for the rabbt systems. The queston whether such an enzyme s also present n the human kdney where the paplla s less well defned remans to be establshed before supportve n vtro evdence for a role of PGA2 (medulln) n human kdney functon and hypertenson can be consdered at hand. 1. Anggard, E. & Samuelsson, B. (1964) J. Bol. hem. 239, Anggkrd, E., Green, K. & Samuelsson, B. (1965) J. Bol. hem. 24, Anggkrd, E. & Samuelsson, B. (1966) Ark. Kem 25, McGff, J.., Terragno, N. A., Strand, J.., Lee, J. B. & Longro, A. J. (1969) Nature 223, Horton, E. W. & Jones, R. L. (1969) Br. J. Pharmacol. 37, Nakano, J., Anggard, E. & Samuelsson, B. (1969) Eur. J. Bochem. 11, Raz, A. (1972) Bochem. J. 13, Attallah, A. A. & Schussler, G.. (1973) Prostaglandns 4, Attallah, A. A., Payakkapan, W. & Lee, J. B. (1974) Lfe Sc. 14, Andersen, N. H. (1969) J. Lpd Res. 1, Saeed, S. A. & Roy, A.. (1972) Bwochem. Bophys. Res. ommun. 47, Andersen, N. H. (1969) J. Lpd Res. 1, Vonkeman, H., Nugteren, D. H. & vandorp, D. A. (1969) Bochm. Bophys. Acta 187, Lee, S-. & Levne, L. (1975) J. Bol. hem. 25, Ham, E. A., Oen, H. G., Ulm, E. H. & Kuehl, F. A., Jr. (1975) Prostaglandns 1, Velck, S. F. (1955) n Methods n Enzymology, eds. olowck, S. P. & Kaplan, N.. (Academc Press, New York), p Larsson,. & Anggard, E. (1973) Eur. J. Pharmacol. 21, Matschnsk, F. M., Shanahan, D. & Ellerman, J. (1974) Anal. Bochem. 6,