TM JOURNAL or R~~.~.oEAL C~rvvrnr Vol. 216. No. 6. Xmua d Much 2b, pp. IlLIS-18%. 1971 Iwlkd ir U.S.A. The Glucagon-sensitive Adenyl Cyclase System in Plasma Membranes of Rat Liver I. PROPERTIES (Received for publirrtion, Octolw 7. 1970) STEPHEN I,. POHL, J,un BIRNBAUMER, ASD MARTIN RODBELL Frcnn Lhe Section on Ncnrbranc Heytclalion, Kational Institute of rlrthrilk and AlctaWic Dieeaece, Xalimlal Institutes of Ifealth, Bethesda, Maryland RWl.$ SUMMARY The liver parenchymal cell plasma membrane preparation devised by Seville (Biochem. Biophyr. A&, 154, 540 (1968)) contains an adenyl cyclase system which ls stimulated by glucagon and to a much lesser extent by epinephrine. The yield of adenyl cyclase can be greatly increased, at the ex- pense of some contamination by other organelles, by lncreas- lng the amount of starting material and eliminating the last step in the preparative procedure. Adenyl cyclase activity is 25-fold purified ln the membranes compared to a liver ho- mogenate. Liver membrane adenyl cyclase activity is a function of glucagon concentration over a range of lo+ to lo-' y glu- cagon and ls increased more than lo-fold by a maximally stimulating concentration of glucagon. The stimulation produced by epinephrlne is less than 10% of that produced by glucagon. Secretin, a polypeptide hormone very similar to glucagon ln its primary structure, does not stimulate liver membrane adenyl cyclase activity. Insulin changes neither the basal nor the glucagon-stimulated adenyl cyclase activity. Adenyl cyclase activities, under standard assay conditions, are proportional to time of incubation and to membrane con- centration. The enzyme requires a divalent cation, either Mg++ or Mn++, but is inhibited by Ca++. At sufficiently high concentrations, both ATP and Mg++ inhibit the enzyme. Addition of 1 my EDTA to the assay medium doubles the glucagon-stimulated activity. Fluoride ion stimulates adenyl cyclase activity but to a lesser extent than does glucagon. Glutaraldehyde, N-ethylmalelmlde, and p-chloromercuri- benzoate inhibit the adenyl cyclase system. Urea, at a con- centration of 2 Y, reduces both the maximal glucagon- and fluoride-stimulated activities, but at 0.4 M reduces only the apparent a5nity of the system for glucagon. Attempts to solubilize and purify the adenyl cyclase activity ln an active form from rat liver membranes have been un- successful. Adenyl cyclase in mammalian tissues is a membrane-bound enzyme system which catalyzes the reaction (1, 2) ATP --) cyclic AMP1 + PPj Certain polypeptide hormones and catecholamines exert nt least part of their effects by stimulating the activity of this enzyme in their target cells (3). However, the mechanism, in molecular terms, by which these hormones stimulate adenyl cyclase activity is poorly understood. In a previous communication (4) we reported t.he presence of a glucagon-sensitive adenyl cyclaw system in psrenchymal cell plasma membrane prepared from rat liver by the procedure of Neville (5). We have sulxequently found thnt the yield of adenyl cyclase can be greatly incroad, at the expense of some contamination by other organelles, by omitting part of the Neville procedure. This modified preparation has several desirable features as a starting material for studies of the mwh- anism of hormonal stimulation of adenyl cyclase. The purpo* of this paper, the fimt in a serior (&Q), is to describe pertinent geneml features of the livrr membrane prcpnn:tion and its adenyl cgclase system. EXPERXYENTAL PROCEDURE - dIaleri& Glucaion (crystalline) was supplied by Lilly. Secretin wns a gift from Dr. Victor ?ciutt (Karolinska Institutet, Stockholm). Epinephrine was purchased from Parke-Davis and wm used within 15 min of opening the vial. Highly purified glutamldr- hyde ans obtained from Lndd Research Industries, Inc., Burling- ton, Vermont. Lubrol wna obtained from Imperinl Chemicnl Industries, Providence, Rhode Island. The sources of nil othrr reagents were specified previously (10-12). MelJlods Plasma membranes were prepared from livers of male and female Sprague-Dawley rats weighing 140 to 180 g according to the procedures devised by Neville (5). The gradient for Step 13' of the procedure was a 25ml linear gradient from 26 to ls, sucrose prepared with a Beckman Cmdient Former. These membranes are referred to below as "fully purified mem- bmnes." "Partially purified membmn'es" were prepared by 1 The abbreviation used is: cyclic AhIP, cyclic adenosine 3',5'- monophosphate. * The step numbers used here are those used by Neville (5) in his description of t.he liver membrane prcparstion. 1849 1850 Glucagon-sensilive A&my1 Cyclase System. I Vol. 2-l& x0. 6 doubling the amount of starting material and the volume of each of the steps, and omitting the final rate-zonal density gmdi- ent centrifugation (Steps 12 to 15). In some cxpcriments, one-half of partially purified membrane pcllct was cnrricd through the final centrifugation and used as fully purified mcmhrnncs. All membrane preparations wem either used immcdintcly or were stored immediately in liquid nitrogen as compnct pellets (25,000 x 0). For routine use, one or two batches of membranes were suspended in 1 my KHCOI at a conccntmtion of 8.0 to 18.0 mg of membrane protein per ml and distributed as O.l- or 0.2-ml aliquota in individual glass tubes. These tubes acre stored in liquid nitrogen and thawed individually as needed in order to avoid repeated freezing and thawing of the entire batch of mem- bmnes. Adeny Cvc&ss Assay-Adenyl cyclasc activity WIN mcasurcd by the method of Krishna, W&s, and Brodie (10) as dcscribcd previously (11, 12). Unless specified otherwise, the following conditions were standard. The assay medium contained 3.2 rnbt ATP-a-nP (25 to 50 cpm per pmole), 5 mM higClr, 1 II~I EDT.4, 25 mM Tris-HCI, pH 7.6, an ATP-regenerating system consisting of 20 mu phosphocrcatine and 1 mg per ml of crcatinc phosphokinase (20 to ,50 units per mg), and 10 to 50 pg of mcm- branc protein in a volume of 56 ~1. Hormone wcrc diluted in the crcatinc phosphokinnse solution. Incubations were init iatcd by the addition of the mcmbrancs and were continued for 10 min at 30". Reactions were terminated by boiling for 3) min. Protein was measured by the Lowry procedure (13) using bovine serum albumin as standard. "dfarkcr" E'ntymes-5'-Nucleot idnsc (EC 3.1.3.5) was measured by the method of Bodansky and Schwartz (14), glucose 6-phosphatase (EC 3.1.3.9) by the method of Swanson (15) with addition of 1 m&t EDTA, alkaline phoaphatnse (EC 3. I .3.1) by the method of Heppcl (IO), and ncid lbhosphatnsc (EC 3.1.3.2) by the same method using 100 IIIM sodium acetate buffer, pH 5.3. These four enzymes were as~ycd in 0.1 ml of appropriate medium and incubations were at 30". Incubation time varied between 10 and 90 min depending on enzyme fraction and activi y measured. Activities were proportional to enzyme concentration. Reactions wem stopped by addiCon of 0.025 ml 50% trichloracetic acid. The precipitate was removed by centrifugation, and the l&crated Pi was determined on 0.05-ml aliquots by the method of Fiske and SubbaRow (17). Suc- cinate-cytochrome c reductase was measured by the method described by Fleischer and Fleischer (18). J&&on Microscopy-Liver membrane suspensions were centrifuged in 0.4ml centrifuge tubes for 15 min at 25,000 x g. The pellets contained about 1 mg of ! ,:otein and measured about 0.6 x 0.2 mm. The pelleta were fixed at 0' for 30 min in 3% glutnraldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The fixative WM removed and the pellet rinsed for 1 hour at 0' with buffer. Post-fixation wns then performed for 2 hours nt 0' in 2% osmium tetroxide in phosphate buffer followed by dehy- dmtion in cold acetone. The pellets were then embedded in epoxy resin (19) and oriented so that complete top to bottom sections could bc obtained. Sectioning was performed with a Reichert Om U 2 microtome. Sections were stained in a lead hydroxide solution (20). Electron micrographs were obtained with a Philips EhI 300 elect,ron microscope employing a 40 I( objective aperture and an accclernting voltage of 80 kv. Ezpremion of Resdlo and Terminology-h trll figures and tables "adenyl cyclase activity" refers to nanomoles of cyclic AMP formed in 10 min per mg of menlbranc protein. Bnsnl activity is the activity measured in the nlxccncc of Klucngon or fluoride ion. Glucagon-, ep:nephrine-, nncl fluoride-stinlrll:lt~~l activities are activities mr,a~urccl in thr prcwnce of thcsr corn- pounds. RESULTS bfembrancs prepared 1Jy the complete and the rnotlificcl pro- cedurc of Keville (5) arc referred to below as fully :rntl partially purified mrmbrancs, rcspectivcly. The yield of fully purified membranes is 0.5 to 0.8 mg of membrane protein per g, wrt n-right, of liver. The girld of partially purified membranes is 1.0 to 2.0 mg of membrane protein per g, wet weight, of liver. The purity of mcmbranc preparations was checked by electron micro.scopy nnd by assay of marker cnzymcs. The fully purified plwwlcr rncmbranc preparations me composed mainly of IMW- brane shcct.s (Fig. 1A) usually arrnngcd in pairs joined by various kind+ of ccl1 junctions (Fig. 1, B and C). In addition, a rcla- t.ivcly small number of vesicles are present (Fig. IA). No other organrlles wcrc recognized. The paired membrane sheets with typical intercellular junctions and the hilt mnaliculus structure (not shown; see Rcfcrmcc 21) arc uncquivocxlly idrntifirti ns originating from the plnsrnn mcmhranc of hepatic parcnchymxl cells (21). The origin of the vcsiclcs is less certain. Iiowcvrr, thr suggcsition h.as Imn IIWIC that they reprcwnt fragments of the "l~lootl front" pl:isma mcmbmnc (22), the region of the l)lxsmn mcmbrsnc not, irnmrdi:My ncljaccnt to another p:lrrn- chymal cell. The partially purified mcmhranes difh from t hr full:, purified membranes mainly in the Inrgcr number of vesic~cs rclativc to sheets (Fig. ID). In addition, occasional nlitorhondritr nncl a nignificllnt number of small, dcnsclp stained, unicimtifircl ron- taminants (Fig. 1D) wcrc seen. The mnrkcr cnzymc data, summarized in Table I, ~I~IIKIII- strntes a substantial increase in spcific activity of 5'-nuc~lcn- tidasc, a plasma membrane marker (22), in both the partialI> and fully purified membranes relative to the crude homogenate. The glucose 6-phosphatnsc and succinntc-cytochrome c rcductn* activities indicate significant microsomnl and mitochondrial contamination of the pnrti:dly purified membrane prrparntion and that this contamination is substantially rrduccd in the fully purified membranes. Purtjkation and Yield of Adenyl Cyclase in Membrane Prepa- r&as-Table II summarizes the purification and yield of basal, glucagon-, epinephrine-, and fluoride-stimulated adengl cyclase activities in partially and fully purified plasma mrm- brancs compared to a crude homogenate of liver. The gluragon- senaitivc adenyl cyclase activity is obtained in great& yield and specitlc activity in the pnrtinlly purified membmncs; a 25-fold purification is obtained in this mntrrial. The fully puriticd mcnlbrancs have both a lower yield nnd lower specific nctivity of gluc:tgon-sensitive activity. Both preparations have scvernl-fold higher specific activity than a 1500 x g pellrt of the liver homogenate. Epincphrine-sensitive ndenyl cyc1:r.s~ is present in hoth membrane preparations at, ~nuch lower nc- tivity than glucngon-sensitive activity but is also substantially enriched in comparison to the homogc:erl;rte activity. Stintulnhm of .4det$ C&we Activf!y in Mentbrnve Prepara- him7 by f~arntoncs crud I:luoride lo~~---~~lrw.:~go~~ produces by far the largest stimnlafion of the livrr mcml)r:mc adcnyl cycl:rsr activity of uny agent which WC have tested; the stimulation ISSW 0f lU:~rch 25, 1971 S. L. Pohl, L. Ilirnbuuntcr, and Al. RmA!w.ll Fra. 1. I':loctron microgrnphn of m~ml~rnno propnrntions. C, dctnil of R tight junction nhnwing two mombrnno nhcots with PMB, ldrrclmrr mrmbrnnc wltrc~ts; l', vrri(~lc. A, fldly pnrificd fu~otl nutcr lcnflcta (arrow). hlngnificntion X 300,0(w). 0, pnr- mcml~rnncs. Mngnifiwtiwl X 30,tJW. 0, higher mrtgnification tinily purified mcml~mncu. rlrrow inclicntes uniclcntified densely Rhowing junctions ktwcen two meml~rnne shrets nnd typical staining conlnminnnt. SIngnificntion X 30,000. unit membrane strncture (arruv0). Jlngnificntion X 150,000. ~nducctl by ghicngon is from 10 to 30 times the Ixm:d activity. ndrcnocorticotrol)ill, thyrotropin, luteinizing hormone, vnso- The activities rqmrtd hcrc nrc pc:rtcr than thaw in our lnvvious press/n, :tntl p:unthyroitl hormone, fdctl to stinudntc the liver colnrilullic:ltion (4) prirnnrily Irc:iuse of the ntltlition of EDTh mrmbrnnc deny1 cycln.~ sptcm. The fnilurc of sccrctin to to the :~s*:iy medium. The effect of IDTA is tlc~cribd Irlow. stimulnte is of partidrr interest since the prirnnry structures Epincphrinc produces II s1nd1 stimukttion of thr rat liver of ~IIKX~OII antI secret in arc sindnr (23). Insulin :It a con- plasrnn nieml~r:inc ndrnyl c~~l:isr :wt ivity (T:ible I I I), nn rrntration of 10 munits per ml failed to chnnp! the Ix~sal or effect which WC failrd to ~~MYW in the lndimin:u~y rspcrimrnts gluc:iaclii-~tirnnI:rtcd nclcnyl ryclrwc activities. rrportctl nirlicr (4). Howcvcr, the rpinrphrinc stirnulntion The liver mcnibrnnc! ndcnyl cyclnsc system is sensitive to low is only 1.5 to 2.5 t.inws the lelsnl nrtivity or lrss thnn 10yO of rnnrcntr:itions of ~lur:igc~n. As shown in Fig. 2, ndcnyl cyclnsc the stimulntion producd by glnnngon. nctivity is :I flirirticm of gluargon conccntrntinn over a range of A vnricty of other polgpeptide hornronca tcstrtl, secretin, lO-`O to lo-' u with hnlf-nwximd stirnukrtion orcurring at 1852 GZucugon-sensilive Adenyl Cydaae Syetem. I Vol. 246, No. 6 about 4 x 10-r Y. The maximal activity obtained with glucagon bated for 10 min in a medium containing ATP-regenerating is greater in the partially purified than in the fully purified system, the glucagon-stimulated adenyl cyclase activity is membranes. The. other parameters of the dose-response re- proportional to both time of incubation and to membrane con- lationahip of adenyl cyclase activity to glucagon concentration centration. This is also true for the fluoride-stimulated ac- are identical in the two preparations. tivity (data not shown). Without the ATP-regenerating Fluoride ion stimulates adenyl cyclase activity in the liver system, the reaction is linear only for relatively short incubation membrane preparations vable II). The fluoride concentration times and at low membrane concentrations. The liver plasma dependence of this effect is similar in the partially and fully membrane preparation contains a potent ATPn.se (24). Even purified membranes (Fig. 3). The effect of fluoride ion will'bc with the ATP-rcgcnerating system, the proportionality to ?imc treated more fully in a separate report (6). and membrane concentration in lost at membrane protein con- The liver membrane preparations may be stored in liquid centrations above 50 pg per assay (50 ~1). The reason or rcawns nitrogen for at leaat 3 months without lose of glucagon- or fluo- for this nonproportionnlity at high membrane concentrations ride-stimulated adenyl cyclase activities. is unknown, but several factors may contribute including ag- ~estav C~f&nefer Adeny1 C~c&ae A&u&+-Fig. 4 presents gregation of membranes, destruction of glucagon, inadequacy the time course of the glucagon-stimulated adenyl cyclase reac- of the ATP-regenerating system, or production of an inhibitor. tion with varying amounts of liver membrane protein assayed The pH of the standard assay medium, 7.6, is the optimum in media with and without ATP-regenerating system. It is for glucagon- and fluoride-stimulated activities in three. buffer apparent that under the standard assay conditions specified systems. Although grcnbr enzyme activities were measured under "Methods," i.e. 10 to 50 pg of membrane protein incu- at 37" and 42', the time courses were nonlinear at these tern- peratures; therefore, 30" was chosen as the stnndard incubation TABLE I temperature. Addition of 1 mx EDTA to the amay medium Aclivilier o/ marker enzycr in liver humogenalcs and in parlially and jully purQied membranes TABLE III Rat livers were ground in a Dounce homogenizer ae described by Seville (5). An aliquot of thin crude homogenate was re- Hormone specificity Of adenyl cychse in liver membranee ground in a ground glass homogenizer to facilitate pipetting. The Partially and fully purified plasma membranes were assnycd for remainder of the homogenate WM used to prepare partially and adenyl cyclsae activit.y in media containing nest imrdant, cpineph- fully puriticd membranes en described nnder "Methndn." rine, secretin, glucagon, or 10 mx SaF. llormones, when pres- -- ent, were at 20 rg per ml. Activities nre expre.ssed as the mean * Spaltic utlvily in half the range oftriplicate determinations ~ -- - ------.---I...--~ __ ENyttlc Crude 3 I I pE:k' Full Adrnyl cyclaac activity pur ed iIT Addition homogcnr tc Pl-- plMta membranea membranes Partially purified plasma membranes Fully purified plaza membratws --, i d S'-Nucleotidase . . . . . . . . . . . . 0.032 Cluccse &pboaphatase. . . . . . . . 0.938 Alkaline phosphatsse.. . . . . . . . . 0.0041 Acid phoaphataae . . . . . . . . . . . . . 0.019 Surrinrte-cytochmme c reductase 0.025 - 0.535 0.932 None 0.30 f 0.06 0.021 0.012 Epinephrine 0.42 f 0.02 0.015 0.010 Secretin 0.34 f 0.02 0.008 o.Go6 Glucagon 3.33 f 0.M 0.028 o.oG3 NaF 1.92 f 0.0-l ._--___ - .-.. -- --..-.- - ---_-...- _ .._ TABLE II Pnrificalion of adenyl cyche O ?o?? ??????? ? ?? *nolrsllO mir/m# )rsf&t 0.10 f 0.01 0.28 f 0.04 0.22 * 0.04 2.79 f 0.02 l.fil f 0.M --. -.- ___ Adenyl cyclaae activities of the crude homogenate and the partially and fully purified membrane preparations described in Table 1 were meaeured under standard assay conditions except that 1 rn3( cyclic AMP and 10 mw theophylline were added to the medium. When present, glucagon and epinephrine concentrations were 20 fig per ml and NaF was 10 my. as nsnnmales of cyclic AMP formed in 10 min per mx of nrotein. Adenyl cyclase activity is cxi*ressed Homogenate. . . . . . . . . . . . . . . . . . Partially purified plasma mem- branea. . . . . . . . . . . . . . . . . . . . . . Fully purified plasma membranes . Basal aetivily subtracted. `The glucrgon-stimulated activities in this experiment arc somewhat greater thnn those in other esperimcnts described here pas- sibly because it was performed several months later during which time the supplier of the ATP -P-**I' made a sigtrilicnnt change in the preparative procedure. ._ ..--.----LA- -. in sdenyl Q&K Basal due to sdditiea of Total pmtria #hyl WC'- utivity Chlcagon Epi- ncpbrble h'lF -1 w 17,090 0.034 -1-j 0.244 0.0X o*lg4 117 0.207 G.130 0.500 2.558 21 0.112 4.1GG 0.273 2.951 Iesuo of March 25,197l S. L. P&l, L. Bimbaunwr, and Al. Rodbell Fxo. 2 (&it). EYfect of glucagon concentration on adenyl cyclase activity in partially purified (0) and fully purified (0) plasma membranes. Fxo. 3 (emtar). Etlect of fluoride ion concentrrrtion on adenyl cyclase activity in partially purified (0) and fully purified plasma membranes (0). Fro. 4' (rfgU). Time course of the glucagon-stimulated adenyl caused a doubling of the glucagon-stimulated activity and lesser increases in the basal and fluoride-stimulated activities (Table Iv). The effects of varying ATP and magnesium concentrations on adeuyl cyclase activity are shown in Figs. 6 and 6, reapec- tively. Bite the substrata concentration Cannot be optimiwd for both glucagon- and fluoride-stimulated activities, 3.2 rrm was chosen arbitrarily for standard amay conditions because it gives near optimal activities for both stimulants. In the presence of 3.2 mx ATP, 5 my Mk+ givea optimum basal, glucagon-, and fluoride-stimulated activities. Manganous ion may be substituted for magnesium (0) but calcium cannot and, at oonoentrations above 10-a Y, is inhibitory in the presence of magnesium. A ayolio phosphodieaterase inhibitor, theophylline or caffeine, was not included in the standard assay medium because of our earlier finding (4) that the cyclic phosphodieaterase activity of the liver membrane preparation is so low as not to interfere with the adenyl cyclaes assay under routine assay conditions with both partially and fully purified membranes. "Soluble" E zpeGaun&A variety of methods were employed to alter the membranes in such a way that the adenyl oyclam activity would remain in the supernstant for at least 1 hour in a centrifugal field exceeding loO,OCKl x 0. These in- eluded sonic oscillation, succinylation, and exposure to high sslt concentrationa, urea, and detergents. AU of them treat menta either abolished the adenyl oyclase activity or failed to aokbiib the enzyme. Using the Luhrol procedure devised by Isvey (25), approximately 15% of the fluoride-stimulated activity was obtained in a high speed supernatant. However, this activity was insensitive to glucagon. Efleets of Glukmakkh~de, Sulfhyctryl Reaqals, and Ursa- As shown in Table V, glutaraldehyde treatment completely inac- tivatea the adenyl cyclase system of the rat liver membrane preparation. Adenyl cyolase activity is also inhibited by treat ment of the membranes with N-ethyhnaleimide and p-chloro- mercuribensoate, agents which react with sulfhydryl groups (26). ' In other experiments, the extent of inhibition by sulf- 2 4 6 8 IO I2 IS lb cyclsse reaction. Partially purified membranes were incubated for varying times in ndenyl cyclase assay medium containing 10 rg per ml of glucagon prepared with (A) and without (B) the ATP- regenerating system. Numbera on curuc~ indicate micrograms of membrane protein used for each study. Assay conditions were otherwise the same as described under "Methods." 0 and o , serve only to distinguish adjacent curvea. TABLE IV EJecta of EDTA on adenyl eyclase activity Partially purified membranes were incubated in standard aesay medium with or without 1 mx EDTA. When present, glucagon was 10 r~ per ml and NaF WM 10 mu. Addith I* None Glucagon NaF FIO. 6 (&If). EfTect of varying ATP concentrbtion on basal, glucagon-, and fluoridestimulated adenyl cyclaee activities. Partially purified membranes were incubated in media containing no stimulant (o), 10 rg per ml of glucagon (O), 10 my NaF (A), and varying concentrations of ATP. Other conditions were as described under "Methods " ho. 6 (right). Efiect of krying Mg++ concentrations on basal, glucagon-, sod fluoridestimulated adenyl cyclase activities. Partially purified membranes were incubated in media containing no stimulant (o), 10 rg per ml of glucagon (0), 10 myL NaF (A), and varying concentrations of M&J*. Other conditions were as deaoribed under "Methods " . 1854 Gl~im-sensilioc Adenyl Cyclme Sydem. I Vol. 246, No. 6 TABLE V b'ffecb o$ glutaraldehydc and rulfhydryl reagenls on adeny cyclere a&oily For treatment with glutaraldehyde, partially purified mem- bra&, 28 pg of membrane protein, were incubated in 0.02 ml of a medium containing 50 mu crcodylatc-nitrate buf?er, pH 7.4, and 4.wc sucmee with or without 1% glutaraldehyde. After 10 min at 30o, adenyl cyclase emay reagents were added in a volume of 0.03 ml and adcnyl cyclase activity was determined as described under `Methods." For treatment with sulfhydryl reagents, fully puri- fied membranes, 18 pg of membrane protein, were incubated in 0.02 ml of a medium containing 25 mx Trin-HCI, pH 7.4, and no addition, 1 mu N-ethylmaleimide, or 0.05 my p-chloromercuri- bemoate. After 16 min at 22', adcnyl cyclase assay reagents were added in a 1 olume of 0.03 ml. EDTA was omitted from thestand- ard assay medium, and P-mercaptoethanol was included at a final concentration of 1 mx. When present, glucagon ~~88 10 pg per ml and NaF WM 10 myI. Control . . . . . . ..*..................... 0.78 4.80 2.99 Glutaraldohyde . . . . . . . . . . , . . , . . . , . . 0.02 0.01 0 04 Control. . . . . . . . . . . . . . . . . . . . . . . . ., ,. . 0.0s 2.41 3.21 N-Ethylmnleimide. . . . . . . . . . . . , . . , . , . 0.03 1.09 2.29 p-Chloromercuribenroate.. . . . . . . . . . . . 0.00 I .29 1.49 0-O -UREA I I I lo-' IO-' CLUCAGON IM 1 WO. 7. Effect OS the addition of 0.4 Y urea to the standard adenyl cyclase assay medium on the dose-response relationship between glucagon and adenyl cyclase activity. hydryl reagenta was found to be nearly complete with lone;er incubation timen, and addition of 2-mcrcaptoethanol or di- thiothreitol was found to block the effect of these agents. Incubation of the membranes in 2 II urea for 10 min at 30" reduces both the glucsgon- and fluoride-stimulated activities by 60 to 30%. Addition of 0.4 M urea to tbe adenyl cyclase assay medium decreases the apparent affinity of the system for glucagon (Fig. 7). Other effeck of urea on the adenyl cyclase system will be described in a later paper (7). DDlCUS8ION Adenyl cyclase activity is widely distributed in mammalian tissues (1, 3) and is controlled by a variety of polypeptide hor- mones and catecholamines (3). Despite the centrnl position of the enzyme system iu hormone action, little is knowu of the mechanism by which hormones control ita activity. It is mrm- brane-bound and lo.* its response to hormones when attempts are made to solubilize and purify it.9 components. For this reason, we have studied adenyl cycl~~se systems in intact mem- branes and have employed indirect means of characterizing the component8 (12,27,2X8). Previous studies of the adenyl cyclase system in fat cell ghosts indicated that at least five different hormones stimulate the activity of a single catalytic component through discrete re- ceptors (27, %), and that the affinity of an allosteric site for magnesium may be involved in the me&a&m of hormonal stimulation (12). Further study of the fat cell ghost system waq hampered by the small amounts of this material that can prac- tically be obtained and by its relative instability. The finding of a glucagon-sensitive adenyl cyclase in the plasma membrane preparation devised by Neville was initially of interest because it added further evidence for the cellular location of adenyl cyclmse in plasma membranes (4, 29-31). In addition, it can be prepared in large quantities and is stable for long periods of time in the frozen state.. Furthermore, liver plasma membrane preparation have been characteris& more extensively (5,22,32,33) than any other source of plasma mem- branes containing hormonally Rensitive adenyl cyclasc. Fully purified liver plasma membranes consist of paired membrane sheets with junctions and the bile canaliculus struc- ture which unequivocally identify the origin of these sheets as the plasma membrane of hepatic parenchymal cells. Vesicles are also present which, according to Benedetti aud Emmelot (!L?2), may be derived from the plnqma membrane because they are isopycnic with the membrane sheets and because preparations containing vesicles have low specific activities for enzyme and chemical markers of othrr organelles. This suaestion is sup- ported by the report (33) that purified liver plasma membranes contain undetectable amounts of a unique glycolipid found in other types of liver membranes. Our finding that the fully and partially purified membranes, the latter being rich in vesicles, have the same specific aotivity of 5'.nucleotidase, a plasma mcm- branc marker (34), adds to the evidence that the vesicles are derived from plasma membrane. Adenyl cyclasc activity is considerably enriched, relative to liver homogenates, in both partially and fully purified mem- branes. The similarity in the two preparations of the dose rez+ponse curves for glucagon and fluoride ion suggests that omission of the final purification step doea not introduce adenyl cyclase with diflereut properties from that in the fully purified membranes. The glucose 6.phospbatase and succinate-cyto- chrome c reductase activities indicate a considerable contamina- tion of the partially purified membranes with other organelles, but because of the much higher yield of adenyl cyclase activity we have considered the partially purified membranes preferable to the fully purified membranes for our studies. It should be emphasized that the present studies do not indi- cate, particularly with the partially purified membrane prepara- tion, that adenyl cyclase is preeent exclusively in plasma mem- branes of parenchymal cells. Them is hi&c-hemical evidence (35) that glucagon- and epinephrine-sensitive adenyl cyclase systems are present in plasma membranes of both reticuloen- dothelial and parenchymal cells. It is possible that the higher specific activity of the glucagon-stimulated adenyl cyclase in Issue of March 25,1071 S. L. Pohl, L. Bimbaumer, and ill, Rodbell 1 s5.5 partially purified membranes represents contributions of both parenchymal and rcticuloendothelial cells, Unfortunately, quantitative assessment of the contributions of glucagon-sensitive adenyl C~C~IIBB from the two cell tylzs cannot be. made from the histochemical study since glutiraldehyde, used as fixative, inactivates adenyl cyclase in isolated membranes nnd causes substantial loss of activity in blocks or sections of liver used for the. histochemical studies (35). Studies of the hormone re- sponsiveness of isolated parenchymal and reticuloendothelial cells may provide a definitive answer to this question. Glucagon, of the several hormones and other agents tested, produced the greatest stimulation of ndcnyl cyclase activity in the liver membrane preparations. In contrast to our previous report (4)` epinephrine also produced a stimulation of adenyl cyclase activity although to a much leswr degree than did glu- cagon. Bitensky el al. (36, 37) have provided evidence for two different adenyl cyclase systems in liver homogenates, one sensitive to glucagon and the other to epinephrine. Our finding that glucagon- and epinephrine-sensitive activities are not in- creased by the same factor in partially and fully purified mem- branes compared to the homogenate is consistent with the hypothesis that there are two adenyl cyclase systems present in liver. Marinetti el al. (38,39) have reported recently a much greater epinephrine ntimulation of adenyl cyclase in rat liver membranes prepared in a diierent manner than in this study. They also reported several other ob.servntions which are opposed to our findings in the liver membrane preparation and the findings of other investigators (3) in different tissues. For example, they report inhibition by fluoride, activation by calcium ion, in- hibition by insulin, and activation of adenyl cyclase by p-chloro- mercuribensoate. Finally, their preparation appears to be lOO- ta IOOO-fold less sensitive to glucagon than our preparation. Fluoride ion stimulates the liver plasma membrane adenyl cyclase system as it does all other mammalian systems (3). However, under our standard assay conditions, the activity obtained with a maximally stimulating concentration of glucagon is about two times that obtained with a maximally stimulating concentration of fluoride ion. Fluoride-stimulated activity ir not exceeded by hormonal stimulation in several other systems (3, 12, 40, 41) and the suggestion has been made that an ap- propriate concentration of fluoride ion produces maximal ex- pression of enzyme activity (1, 41). Inclusion of 1 rnr.r EDTA into the incubation medium in the prescnt study enhanced the response of liver sdenyl cyclase to glucagon. In the following report (6) it will bs seen that a number of agents selectively aktr the response of liver adenyl cyclase to fluoride ion and glucagon. The adenyl cyclase assay conditions and method of measure- ment have been adequately described in publications from this and other laboratories (4, 10-12, 25, 38, 41) and do not require extensive comment. However, it is important to note that under our stsndard assay conditions, the measured activity is pro- portional to time of incubation and to membrane concentration. The liver plasma membrane preparation contains both a potent ATPase (24) and a system which rapidly inactivates glucagon in the assay medium (7). Consequently, any significant change in the composition of the assay medium requires at least that the linearity of encyme activity with time be checked. Mammalii adenyl cyclase systems are highly complex and probably multimolecular. An understanding, in molecular terms, of the mechanism or mechanisms by which polypeptide hormones control these systems depends ulxm identification, chsracteriration, and isolation of the components of whirh rnch systems are made. 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