THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol 253, No. 9, Issue of May 10, pp 3114-3122, 1978

PrmIed m 1J S.A

Characterization of r3H]Adenosine Binding to Fat Cell Membranes*

(Received for publication, August 29, 1977, and in revised form, December 27, 1977)

CRAIG C. MALBON,$ RICHARD C. HERT, AND JOHN N. FAIN

From the Section of Physiological Chemistry, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

I >H IAdenosine binding to fat cell plasma membrane prep- arations was examined by a vacuum filtration technique. Appreciable binding of adenosine to fat cell membranes could only be demonstrated in the presence of the adenosine deaminase inhibitors, erythro-Y-(2-hydroxy-3-nonyljadenine or deoxycoformycin. The binding of adenosine to these membranes was rapid, reaching near equilibrium within 10 min at 37°C. and was reversible. Hound adenosine dissn- ciated very rapidly fnllnwing a lOO-fold dilution at 0°C or 22°C. Only 7% of equilibrium-bound adenosine remained at 2 min following IOO-fnld dilution at 22°C. Scatchard plots of equilibrium binding data were nonlinear, suggesting at least two populations of adenosine binding sites possessing differ- ent affinities for adennsine. The apparent dissociation cnn- stants. K,, , and maximum binding capacities, B,,,,, , were 9.5 x 10 Ii M and 28 pmol/mg of protein and 9.5 X 10F1 M and 1700 pmol/mg nf protein for the high and low affinity adenosine binding sites, respectively.

The high affinity sites display maximum binding at pH 7.5. above pH 7.5. or below pH 6.5 adennsine binding to the fat cell membranes declines sharply. The divalent cations calcium or magnesium, at concentrations greater than 1 mM. inhibit the binding of adennsine to the high affinity sites of the fat cell membranes. Adennsine binding is re- duced by prior exposure of the membranes to trypsin, chymntrypsin, or neuraminidase or by thermal denatura- tinn. Purine derivatives compete with adennsine for binding to the membrane site in the following potency order: ade- nine > ATP = AI)P > cyclic AMP = AMP = innsine. Inhibition studies of high affinity adennsine binding per- formed with these purine derivatives indicate this binding is to multiple components of differing specificity and not to a single, hnmngenenus class of binding sites. Thenphylline, but not dipyridamnle or p-nitrnbenzylthioguannsine. is a potent inhibitor of adennsine binding to the membranes. One micromolar thenphylline inhibits adennsine binding

* This work was supported by United States Public Health Service Research Grants AM-10149 and AM-14648 from the National Insti- tute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health. The costs of publication of this article was defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of National Research Service Award 1 F32 AM 05425- 01 from the National Institute of Arthritis, Metabolism and Diges- tive Diseases, National Institutes of Health. To whom all corre- spondence should be addressed.

14%. Highly purified fat cell plasma membranes possess the major portion of adenosine binding sites identified with the crude fat cell plasma membranes. The highly purified frar- tion likewise displayed the two pnpulatinns of adenosine binding sites identified in the crude preparation. Adenosine (100 PM) did not affect the specific binding of (-)- 1 “Hldihydroalprennln1 to fat cell plasma membranes. Nei- ther 10 PM (-)-isoprnterenol nor 10 FM(-)-alprenolol af- fected the ability nf fat cell membranes to bind I”H]adenosine. This study demonstrates that fat cell plasma membranes possess sites which bind l”Hladennsine with high affinity. The relationship between these adennsine binding sites and the influence of adenosine on cyclic AMP levels in intact fat cells and on adenylate cyclase activity is discussed.

Dole (1) first demonstrated the ability of adenosine to inhibit epinephrine-stimulated free fatty acid release from fat pads. This observation has since been amply confirmed in adipose tissue (2, 3) and isolated fat cell suspensions (4, 5). Additionally, it has been shown that adenosine and adenosine- containing compounds such as 5’-AMP, ADP, ATP, and NAD possess a capacity to inhibit glucagon- (6), ACTH-’ (7). and norepinephrine-stimulated (3, 7) lipolysis in adipose tissue. Schwabe et al. (8) demonstrated that adenosine is continu- ously released to the medium by fat cell suspensions.

Sattin and Rall (9) first reported the ability of adenosine to elevate adenosine 3’:5’-monophosphate (cyclic AMP) levels in brain slices. Shimizu and associates (10, 11) confirmed this observation. Subsequent reports have shown adenosine stim- ulation of cyclic AMP levels in platelets (12-14). isolated bone cells (15), and cultured cell lines (16-21). Adenosine stimula- tion of cyclic AMP levels in several clones of mouse neuroblas- toma cells required, however, the presence of RO-20-1724, a cyclic AMP phosphodiesterase inhibitor (18). Adenosine also stimulates adenylate cyclase activity in cell membrane prep- arations (22-271, although some of these preparations display a biphasic stimulation and inhibition of the cyclase by adeno- sine (22, 24, 25). In contrast, epinephrine- and glucagon- stimulated cyclic AMP accumulation are inhibited by adeno-

’ The abbreviations used are: ACTH, adrenocorticotrophic hor- mone; EHNA, erythro-9-(2-hydroxy%nonyl)adenine; K,,, equilib- rium dissociation constant; B,,,,, , maximum binding capacity; NBTG, p-nitrobenzylthioguanosine.

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Adenosine Binding to Fat Cell Plasma Membranes 3115

sine in isolated hepatocytes (28). Hepatic adenylate cyclase is likewise inhibited by adenosine (29-31). Micromolar concen- trations of adenosine potently inhibit the rise in cyclic AMP accumulation stimulated by lipolytic hormones in intact rat fat cells (4. 32. 33). Higher concentrations of adenosine also inhibit the activation of fat cell “ghost” adenylate cyclase by hormones (4).

Fain and associates (4. 33. 34) have demonstrated that adenosine inhibition of norepinephrine-stimulated cyclic AMP accumulation in rat fat cells is rapid, enhanced by the pres- ence of inhibitors of adenosine deaminase, readily reversed by adding purified adenosine deaminase to the medium. and antagonized by methylxanthines. Adenosine covalently linked to stachyose, although too large to penetrate the cell mem- brane. has recently been shown to be nearly equipotent as free adenosine in inhibiting cyclic AMP accumulation by both chicken and rat fat cells (34). These studies suggest 1) that adenosine may regulate the intracellular accumulation of cyclic AMP via binding to a site on the plasma membrane, and 2) that methylxanthines may exert their antagonism of adenosine action through competition with adenosine for this binding site.

This paper reports the results of studies designed to probe adenosine interactions with the fat cell membrane by utilizing tritiated adenosine and a direct binding approach. A prelimi- nary report of this study has been presented (35).

MATERIALS AND METHODS

Female, 200- to 250-g Sprague-Dawley rats (Charles River CD strain) were fed laboratory chow aa’ lib&urn. The (-)- I”Hldihydroalprenolo1 (specific activity, 32.6 Cilmmol) and I”Hladenosine, labeled at positions 2 and 8, (specific activity, 30 to 50 Ci/mmol) were obtained from New England Nuclear, Boston. MA. The purity of the tritiated adenosine was greater than 97% as determined by the supplier and thin layer chromatography in this laboratory. The following compounds were generously provided by the sources indicated: erythro-9.(2.hydroxy-3.nonyl)adenine, Dr. Donald Namm, The Wellcome Research Laboratories, Research Triangle Park, NC; deoxycoformycin (Covidarabine@), Dr. H. W. Dion, Parke, Davis and Co., Detroit, MI; N”-(phenylisopropyl)- adenosine, Dr. Harold Stork, Boehringer-Mannheim, West Ger- many; 2’,5’-dideoxyadenosine, Drug Research and Development, Chemotherapy, National Cancer Institute, Bethesda, MD. All other nucleotides, nucleosides, and reagents were obtained from Sigma Chemical Co., St. Louis, MO. Enzymes were obtained either from Sigma or Boehringer-Mannheim. New York, NY.

White fat cells were obtained by enzymatic digestion of parame- trial adipose tissue according to the procedure of Rodbell (36). Pooled adipose tissue (-100 g) from 15 to 20 rats was minced with scissors and placed in small plastic bottles. Each bottle, containing 8 to 10 g of tissue and 10 ml of Krebs-Ringer phosphate buffer containing 3% albumin and 1 mg/ml of crude collagenase (Clostridium histolyti- cum. Worthington Biochemical Corp., Lot CLS 46E168P). was incu- bated for 60 min at 37°C. The Krebs-Ringer phosphate buffer contained 128 rnM NaCl, 1.4 rnM CaCl,, 1.4 rnM MgSO,, 5.2 rnM KCl, and 10 rnM NagHPO,. The albumin buffer was made fresh daily and the pH adjusted to 7.4 with NaOH after addition of the bovine serum albumin Fraction V powder (Armour, Lots NlOlOl and P56607). At the end of the 60 min digestion, cells were filtered through one layer of nylon chiffon and washed twice with the albumin buffer.

Fat cell membranes were prepared according to Williams et al. (371 with slight modification. Isolated fat cells were washed twice with Krebs-Ringer phosphate buffer containing 3% albumin (pH 7.41, and resuspended in a 0.25 M sucrose, 1 mM EDTA, 10 rnM Tris- HCl, pH 7.5, buffer (1 ml of buffer/g wet weight of packed cells). The cells were homogenized at room temperature by six strokes of a glass Potter-Elvehjem homogenizer (Arthur Thomas Corp.) fitted with a Teflon pestle operated on a Dyna-Mix (Fisher Scientific) at setting No. 4. The homogenate was centrifuged at 15,000 x g for 15 min at 4°C. The pellet was washed by resuspension in a 50 rnM Tris-HCl, pH 8.0, at 4”C, 10 rnM MgCl, buffer (which was ice cold) and then

centrifuged again as described above. This step was repeated one additional time. The final pellet was resuspended in a 10 mM Tris- HCl, pH 7.4, 5 rnM MgCl, buffer to a final protein concentration of 2.5 mg/ml. This membrane fraction was used directly in the binding assay or frozen and maintained at -20°C until assayed. No loss of adenosine binding was noted in fat cell membranes stored in this fashion for up to 2 weeks. Highly purified fat cell plasma membranes were prepared according to the method of McKee1 and Jarett (38) with the following modification. The pellet (P-3) was resuspended and layered on 25 ml of 35% sucrose (d = 1.151, 1 mM EDTA. and 5 rnM Tris-HCl, pH 7.4, and centrifuged at 24,000 rpm for 90 min in a Beckman model L ultracentrifuge fitted with a SW 25.1 rotor. The milky band of plasma membranes at the sucrose/sample interface was collected, diluted with 4 volumes of 0.25 M sucrose, 1 mM EDTA, and 10 rnM Tris-HCl, pH 7.4, and centrifuged at 16,000 x g for 15 min. The pellet was resuspended in 10 rnM Tris-HCl, pH 7.4, 5 mivr MgCl, buffer. The amount of membrane protein was determined by method of Lowry et al. (39) using crystalline bovine serum albumin as a standard.

Binding assays were routinely performed in 10 mM Tris-HCl, pH 7.4, 5 rnM MgCl, buffer at a final volume of 0.2 ml containing 200 to 400 pg of membrane protein, 10 to 100 nM 1”Hladenosine and an adenosine deaminase inhibitor. Incubations of the radioligand, com- petitors, and membranes were initiated by the addition of mem- branes into the incubation vessel. The incubation vessels (Falcon, 17 x 100 mm, No. 2017 tubes) were incubated at the indicated temperature with constant shaking for up to 60 min. To assess binding of l”H]adenosine to the filters, duplicate tubes without membranes were incubated in all experiments under identical conditions as those with membranes. Assay tubes were routinely in triplicate for both tubes containing fat cell membranes and those containing just buffer in place of the membranes. At the end of an incubation period, 8 ml of ice cold incubation buffer was added to each tube to terminate the reaction. The contents of the tube were rapidly filtered by vacuum through a single Whatman GFiC filter (24 mm diameter) or Millipore HA filter (24 mm, 0.45 km). The filter was then washed with an additional &ml aliquot of this same buffer to minimize binding to the filter. This wash procedure requires less than 5 s to complete. The filtration assay has been shown to be comparable to equilibrium dialysis with several distinct advantages (40). The filters were placed directly into glass scintillation vials and then dried in a 50°C oven for 20 min. A 4-ml aliquot of a solution containing Triton X-100 (1 part) and 5% Omnifluor in toluene (2 parts) was added to each vial and the vial counted. Under standard assay conditions utilizing 30 nM radioligand, binding of 1:‘Hladenosine in the tubes containing membranes represented 1000 to 1200 cpm, while binding in the absence of membranes represented 200 to 300 cpm. 1”HlAdenosine bound by the membranes was calculated by subtracting the no-membrane control value from the value obtained in the presence of membranes. Binding data were then converted to units of femtomoles bound per mg of membrane protein.

The nature of the radioactivity bound by the fat cell membranes was determined as follows. Fat cell membranes (640 pg1 were incubated with 100 nM [“Hladenosine for 20 min at 4°C under standard assay conditions. Following this period, the mixture was filtered and washed on a Whatman GFiC filter. Control incubations were performed as above omitting the fat cell membranes. Filters from three identical incubations were immediately immersed in 3 ml of ice cold 1 N HClO,. The perchloric acid extracts were passed over columns of NoritiCelite, washed with water, and the adenosine and derivatives eluted with 50% aqueous pyridine according to Hepp et al. (41). The eluant of the pyridine wash was collected, lyophilized, and dissolved in 75 ~1 of water. Aliquots of this solution (5 ~1) were chromatographed in a single direction on a plastic sheet (20 x 20 cm) using polyethyleneimine-impregnated plates of cellulose MN 300 (Brinkmann CEL 300 PEI) (0.1 mM). The developing solvent was 1-butanol, glacial acetic acid, and water (50:25:251 or 1-butanol, formic acid, and water (77:13:10).

The chromatographic procedure using polyethyleneimine-cellu- lose plates and the 1-butanol:acetic acid resulted in very discrete spots and reproducible separations except for that of labeled adenine from adenosine. The location of the various compounds was verified by co-chromatography of one unlabeled purine, nucleoside, or nu- cleotide to each lane on the plate permitting identification of the compound under ultraviolet light. Generally, the chromatograph was separated in five areas. The R, values for each fraction and marker compound were as follows: 1, 0 to 0.05 (ATP and ADP. 0 to 0.04); 2, 0.05 to 0.32 (AMP, 0.05 to 0.23; cyclic AMP, 0.05 to 0.261; 3,

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3116 Adenosine Binding to Fat Cell Plasma Membranes

0.32 to 0.48 (inosine, 0.4 to 0.48); 4, 0.48 to 0.57 (hypoxanthine, 0.5 to 0.55); 5, 0.57 to 0.75 (adenosine, 0.6 to 0.66; adenine, 0.62 to 0.73).

Chromatography on the polyethyleneimine-cellulose plates using the 1-butanol:formic acid separated adenosine from adenine. The R, values for each fraction and marker compound in this system was as follows: 1, 0.09 to 0.26 (adenosine, 0.09 to 0.26); 2, 0.26 to 0.38 (adenine, 0.26 to 0.36). ATP, ADP, AMP remain at the origin in this solvent system (R, i 0.02).

The spots were scraped off the plates with a razor blade and added to a counting vial containing 0.5 ml of 1 N HCl. The contents were mixed and 4 ml of a counting solution containing 1 part of Triton X- 100 (Research Products Co.) and 2 parts of 5% Omnifluor (New England Nuclear) in toluene was then added. The vials were counted in a liquid scintillation counter for at least 10 min.

RESULTS

Initial experiments utilizing a rapid filtration assay failed to demonstrate any appreciable binding of 13H1adenosine to crude plasma membranes prepared from rat fat cells. As shown in Table I. adenosine binding to fat cell membranes could only be demonstrated when an inhibitor of adenosine deaminase was present in the incubation medium. Erythro-9- (2-hydroxy-3-nonylladenine (EHNA). a potent adenosine de- aminase inhibitor, at 1 to 100 PM increased the amount of adenosine bound by these membranes. Deoxycoformycin, an- other potent inhibitor of adenosine deaminase (42). similarly provided the condition necessary to observe 13H]adenosine binding to the membranes (Table I). Since the amount of adenosine deaminase activity of these membrane preparations varied, binding studies were routinely performed in the pres- enCe Of 100 pM EDNA.

The binding of 13H1adenosine at 10 nM radioligand increased linearly with the protein concentration of the fat cell mem- branes utilized in the assay from 60 to 450 pg of membrane protein/tube (Fig. IA). A 350- to 400-pg aliquot of membrane protein was routinely used in the assay of adenosine binding to the fat cell membranes.

The kinetics of adenosine binding to fat cell membranes at 30 nM adenosine was rapid; more than 50% of the equilibrium binding value was obtained within 60 s (Fig. 1B). Increasing

TABLE I

Binding of adenosine to fat cell plasma membranes: effects of

erythro-9-(2.hydroxy-3.nonyl)adenine and deoxycoformycin

Crude fat cell plasma membranes (350 /*g of protein) were incu- bated with 10 or 30 IIM 13Hladenosine in the presence or absence of an adenosine deaminase inhibitor in a final volume of 0.2 ml for 60 min at 4°C in the standard assay medium. At the end of the incubation, [3H]adenosine binding to the membranes was assayed in triplicate by vacuum filtration on Whatman GFiC filters. Nonspe- cific binding was determined in the absence of membranes and this value subtracted as background. The data are mean values of triulicate determination of a sinale exneriment.

Inhibitor WI-, IWI-

Concen- A,“~c~~ Adenosine tration tration bound

Experiment A Erythro-9-(2-hydroxy- 0 3-nonylladenine 1

10 100

Experiment B Erthyro-9-@hydroxy- 1 3-nonylladenine 10

Deoxycoformycin 1 10

TLM

10 10 10 10

30 89

30 179

30 30

fmollmg protein

0

49 68 79

210

A 0.2 0.4 0 10 20 30

PROTEIN (mg) TIME Cm,,,)

FIG. 1. Binding of L3Hladenosine to fat cell plasma membranes. Effects of concentration of membrane protein, time, and tempera- ture. A, fat cell plasma membranes (60 to 450 pg of protein) were incubated with 10 nM [3HIadenosine in a final volume of 0.2 ml for 10 min at 0°C in the standard assay buffer (10 mM Tris-HCl, pH 7.4, 5 rnM MgCl,, 0.1 rnM EHNA. Adenosine binding was assayed as in Table I. B, fat cell plasma membranes (350 pg of protein) were incubated for the indicated times and temperature with 30 nM [3H]adenosine at a final volume of 0.2 ml of assay medium. At the end of the incubation, adenosine binding was assayed as in Table I. Data points are the mean values of triplicate determinations of a representative experiment.

the temperature at which the incubation was performed to 37°C reduced the equilibrium adenosine binding capacity of the membranes by 30%. This reduction in the adenosine binding may be the result of increased metabolism of some critical component of the system at this temperature or perhaps the result of simple heat denaturation of the particu- late membranes. Exposing the fat cell membranes alone to 37°C for 15 min reduced the ability of these membranes to subsequently bind I 3H]adenosine following a 20-min incuba- tion at 4°C by 30 to 40% (data not shown). To avoid this complication. binding assays were routinely performed in an ice bath (O-4”) or at 22°C in a shaking water bath.

Saturation studies were next conducted, the results of these are shown in Fig. 2. Adenosine binding by fat cell membranes was concentration-dependent but could not be saturated at concentrations of adenosine as high as 1 mM. Analysis of this binding data by the method of Scatchard (43). as shown in the inset of Fig. 2. produced a nonlinear plot. A simple interpre- tation of these data suggests the existence of two populations of binding sites with differing affinities for adenosine. The higher affinity sites display an apparent dissociation constant (K,,) of 9.5 ? 3 x lo-” M (n = 3) and a maximum binding capacity (B,,,,) of 28 2 6 pmol of adenosine boundimg of membrane protein. The lower affinity adenosine binding sites display an apparent K,, of 9.5 2 2 x 10m4 M and a B,,,, of 1700 * 300 pmol of adenosine boundimg of membrane protein. The low binding affinity of this population explains the inability of 1 mM adenosine to saturate these sites and suggests these sites have little physiological relevance.

The identity of the radioactivity bound by the fat cell membranes was determined. Under the standard assay condi- tions using 100 nM 13H]adenosine. at least 80% of the bound radioactivity was identified as adenosine (Table II). Using the two solvent systems, no radioactivity in the form of inosine or adenine was detected by thin layer chromatography.

It was of interest to determine more precisely the subcellu- lar localization of the adenosine binding components of fat cells. Fat cells were isolated and divided into two equal portions. Crude fat cell plasma membranes (similar to those used in the above studies) were prepared from one of these

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Adenosine Binding to Fat Cell Plasma Membranes 3117

portions. Highly purified fat cell plasma membranes were prepared from the other portion according to the method of McKee1 and Jarett (38). Adenosine binding was then assayed in both this highly purified fraction and the standard crude fat cell plasma membrane preparation. The results of these studies are shown in Table III. Nearly 70% of the total adenosine binding capacity of the crude membrane fraction measured at 0.1 and 1 PM adenosine was retained in the highly purified plasma membranes. A somewhat higher per- centage (78%) of adenosine binding measured at 100 PM was recovered in the highly purified fraction. The highly purified fat cell plasma membranes also display two populations of adenosine binding sites with affinities similar to those dis- played in Fig. 2 (data not shown). These data demonstrate that adenosine binding components can be identified and are perhaps localized in the plasma membrane of the fat cell.

Dissociation of adenosine bound by fat cell membranes at equilibrium with 30 nM adenosine was examined following a loo-fold dilution (Fig. 3). Dissociation was very rapid when performed at 22°C or 0°C. More than 50% of the bound adenosine dissociated within 60 s following a loo-fold dilution at either temperature. Data obtained from the vacuum filtra- tion assay thus probably underestimates actual binding by approximately 10% since some dissociation is possible during the period of filtration and washing (~5 s). This same tech-

0.1 1 10 X30 loo0 CONCENTRATION OFADENOSINE (mtcrorrdar)

FIG. 2. Binding of [3H]adenosine to fat cell plasma membranes; effect of increasing adenosine concentration. Fat cell plasma mem- branes (350 pg of protein) were incubated with 100 nM [3Hladenosine and varying amounts of unlabeled adenosine to yield a final adeno- sine concentration ranging from 0.1 PM to 1.0 mm Incubations were performed in 10 mM Tris-HCl, pH 7.4, 5 rnM MgCl, containing 0.1 mM EHNA at 4°C for 20 min. Binding was assayed by vacuum filtration on a single Whatman GF/C filter. The inset to this figure is a transformation of the data according to the method of Scatchard (431. Bound adenosine (B) is expressed in picomoles/mg of membrane protein. Data points are the mean values of triplicate determinations of a representative experiment. B/F, ratio of bound to free.

nique has been used with fat cell membranes to characterize several other rapidly dissociating radioligands (40. 44). The nonlinearity of the semilogarithmic plot of adenosine dissocia- tion probably reflects dissociation from the two populations of binding sites.

The pH dependence of adenosine binding to fat cell mem- branes was also examined. Adenosine binding was maximum when performed at pH 7.5. declining sharply at pH regions above 7.5 or below 6.5 (data not shown). Binding assays were. therefore. routinely carried out at pH 7.4.

Concentrations of either calcium or magnesium below 200 PM did not appreciably affect the capacity of the fat cell membranes to bind adenosine (data not shown). Concentra- tions of either divalent cation greater than 1 mM inhibited adenosine binding, 50% inhibition being attained at 15 mM calcium or 30 mM magnesium. Fat cell membranes washed twice with 1 mM ethylenediaminetetraacetic acid (EDTA) and resuspended in 10 PM EDTA demonstrated no reduction in their capacity to bind adenosine (data not shown).

TABLE III

Binding of lzlHladenosine to crude and highly purified plasma

membranes prepared from isolated rat fat cells

Isolated rat fat cells (63 g, packed wet weight) were prepared and divided into two equal portions. Crude fat cell plasma membranes were prepared from one portion according to the method of Williams et al. (37); highly purified fat cell plasma membranes were prepared from the remaining portion by a modification of the method of McKee1 and Jarett (38) as described under “Materials and Methods.” Aliquots (150 pg of membrane protein) of each preparation were incubated with the indicated concentrations of 13H]adenosine in a final volume of 0.2 ml for 20 min at 4°C in the standard assay buffer. At the end of the incubation, 13HIadenosine binding to the mem- branes was assayed in triplicate by vacuum filtration on Millipore HA filters (25 mm, 0.45 +m). Nonspecific binding was determined in the absence of membranes and subtracted as background. Data are expressed as the mean value i SE. of three separate experiments performed on separate occasion

Plasma membrane type

mg Crude plasma mem- 22.1

branes Highly purified plasma 14.4

membranes

% of adenosine binding recovered in the highly purified plasma membranes

S.

1

i-

3HlAdenosine binding capacity assayed at a final adenosine concentration of:

68% 69% 78%

TABLE II

Adenosine binding to fat cell membranes: identification of bound radioactivity

Fat cell membranes (640 pg of membrane protein) were incubated column, washed, and eluted with 50% aqueous pyridine. The eluant with 100 nM L3H1adenosine for 20 min at 4°C under standard assay was lyophilized, redissolved in water, and applied to a polyethylene- conditions. Following the incubation, this mixture was filtered and imine-impregnated plate (20 x 20 cm) of cellulose MN 300 (Brink- washed on a Whatman GF/C filter. Filters from three identical mann GEL 300 PEI). Thin layer chromatography was performed incubations were immediately immersed in 3 ml of ice cold 1 N with 1-butanol:H,O:glacial acetic acid (50:25:251. HClO,. The perchloric acid extract was applied to a Norit/Celite

Solvent Bound radioactivity, cpm (o/o of label recovered)

ATP, ADP AMP, cyclic AMP Inosine Hypoxanthine Adenine, Adenosine

1-butanol:H,O:glacial acetic acid (50:25:25) 4 (1%) 60 (14%) 4 (1%) 18 (4%) 338 (80%)”

u Additional thin layer chromatography performed with 1-butanol:H,O:formic acid (77:10:13) as the developing solvent demonstrated that nearly 100% of the label chromatographed as adenosine with no label detected in the position of adenine.

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3118 Adenosine Binding to Fat Cell Plasma Membranes

0 IO 20

TIME (mln)

FIG. 3. Dissociation of I:‘H]adenosine from fat cell plasma mem- branes. Fat cell plasma membranes (2.1 mg) were incubated with 30 nM I:SH]adenosine for 20 min at indicated temperatures in a final volume of 0.5 ml of assay medium. At the end of this incubation period, adenosine binding was assayed in a O.l-ml aliquot by vacuum filtration on Whatman GF/C filters. Nonspecific binding was deter- mined in the absence of membranes and this value subtracted as background. The incubation mixture was then diluted more than loo-fold with assay medium of corresponding temperature and adenosine binding assayed in lo-ml aliquots at the indicated times. The data represent the mean value of triplicate determinations of a representative experiment.

The effects of various enzymatic and temperature pretreat- ments on the ability of fat cell membranes to bind adenosine are shown in Table IV. Pretreatment of the membranes with 0.1 mg/ml of trypsin or chymotrypsin for 3 min at 37°C reduced the adenosine binding capacity 40 to 70%. Ovomucoid trypsin inhibitor at 0.16 mg/ml partially reversed this trypsin effect. The inhibitor alone slightly enhanced the binding of adenosine to the membranes, perhaps by inhibiting endogenous protease activity of the membrane preparation. Neuraminidase pre- treatment also reduced the adenosine binding capacity of the fat cell membranes. while hyaluronidase was without effect. Exposing the membranes to 50°C or 100°C for 1 min reduced adenosine binding to fat cell membranes approximately 40 and 70%. respectively. These observations suggest binding of adenosine by the fat cell membranes is thermally labile and requires the integrity of a protein component(s) of the mem- brane and perhaps key neuraminic acid residues.

To test the specificity of the high affinity adenosine binding to fat cell membranes, the binding of I”H]adenosine at a concentration of 30 nM was assayed in the presence of various purine derivatives. As shown in Table V. N”-(phenylisopro- pyl)adenosine was ineffective as a competitor with adenosine for binding to the membranes, whereas the 2’.5’-dideoxyaden- osine analog was very potent. The order of effectiveness of the other purine derivative inhibitors was adenine > ATP = ADP > AMP = cyclic AMP = inosine. Increasing the concentration of 5’-AMP from 10 to 100 FM resulted in no significant increase in inhibition. Similarly, increasing the adenine concentration from 1 to 100 PM only slightly increased the inhibition of I”Hladenosine binding to the membranes. These data strongly suggest that the high affinity adenosine binding is not to a single, homogeneous class of binding sites, but more likely represents binding to multiple classes of high affinity sites of

differing specificities. GTP had no effect (data not shown). p-Nitrobenzylthioguanosine (NBTG) and dipyridamole are

potent inhibitors of adenosine uptake in a variety of systems (14. 45-47). Ebert and Schwabe (5) reported that 20 PM

dipyridamole almost completely blocked adenosine uptake in rat fat cells. Rosenblit and Levy (48) similarly reported that 30 FM dipyridamole inhibited adenosine uptake in rat fat cells 74%. As shown in Table VI. both NBTG and dipyridamole were poor inhibitors of adenosine binding to fat cell mem- branes. NBTG. at 100 pM inhibited adenosine binding 27% while dipyridamole, at 100 pM, inhibited adenosine binding only 8%.

The observed antagonism of adenosine by theophylline (33. 34) prompted our examination of the effect of theophylline on the binding of adenosine by the fat cell membranes (Table VI). Theophylline inhibited adenosine binding in a dose-dependent fashion. Binding by fat cell membranes at 30 nM adenosine was inhibited 8% by 0.1 PM theophylline and 23% by 10 PM

theophylline. By comparison, l-methyl-3-isobutyl xanthine was very ineffective as a competitor of adenosine binding to the fat cell membranes.

Carlson (49) originally reported that nicotinic acid. but not nicotinamide, was an effective inhibitor of lipolysis in rat adipose tissue. Allen and Clark (50) reported that 100 FM

nicotinic acid inhibited basal, epinephrine- . adrenocortico- tropic hormone- . and fluoride-stimulated adenylate cyclase activity of fat cell homogenates. Pereira and Holland (3) reported, in addition, that nicotinamide-adenine dinucleotide

TABLE IV

Binding of [‘3HJadenosine to fat cell plasma membranes: effect of

various enzymatic and temperature pretreatments on subsequent binding of [:JHjadenosine

Crude fat cell plasma membranes were incubated with the indi- cated enzymes for 3 min at 37°C or at the indicated temperatures for the designated times and subsequently incubated at 4°C for 20 min with 10 nM ISH]adenosine and then assayed by vacuum filtration on Whatman GF/C filters. Nonspecific binding was measured in the absence of membranes and subtracted as background. Data are expressed as the mean values of triplicate determinations from experiments performed on separate days, each with a different membrane preparation.

Experiment A Experiment B Treatment

13Hladenosine bound ISHladenosine bound fmollmg protein % fmollmgprotein %

None 124 130 Trypsin (0.1 mg/ml) 73 60 58 44 Chymotrypsin (0.1 mgi 65 52 77 59

ml) Neuraminidase (0.1 66 53 45 35

mgiml) Hyaluronidase (0.1 124 100 130 100

mg/ml) Heating at 50°C for 1 56 45 99 76

min Heating at 100°C for 1 26 21 44 34

min Incubation at 4”C, no 153 123 180 138

treatment

None Ovomucoid trypsin in-

hibitor (0.16 mgiml) Trypsin (0.1 mgiml) Both

79 93 82 104 102 110

22 28 42 45 67 85 66 71

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Adenosine Binding to Fat Cell Plasma Membranes 3119

TABLE V TABLE VI

Effects of uarious purine derivatives on binding of [z’H]adenosine to Effects of various agents on binding of /:‘H/adenosine to fat cell fat cell plasma membranes plasma membranes

Crude fat cell plasma membranes (350 pgl were incubated with 30 Crude fat cell plasma membranes (350 kg) were incubated with 30 nM tritiated adenosine, with or without the indicated concentration nM tritiated adenosine, with or without the indicated concentration of agent, in 0.2 ml of standard assay medium. The incubation was of agent, in 0.2 ml of standard assay medium. The incubation was carried out at 4°C for 20 min. Adenosine binding was assayed by carried out at 4°C for 20 min. Adenosine binding was assayed by vacuum filtration on Whatman GFlC filters. Data are expressed as vacuum filtration on Whatman GF/C filters. The data are expressed the mean values i S.E. for n separate experiments. as the mean value k S.E. from IL separate experiments.

Agent concentra- tion % Inhibition

Adenosine

N”(Phenylisopropyl)adenosine

2’,5’-Dideoxyadenosine

AMP

Cyclic AMP

ADP

ATP

Adenine

Inosine

FM

1 10

100

1

10 100

1

100

1

10 100

1

10 100

1 10

100

1 10

100

0.01

1

100

1 10

100

6.1 k 2.9 21.9 + 3.5 49.5 -c 4.8

1.4 k 1.0

3.3 k 3.0 2.0 k 1.4

10.3 I ! 5.4

53.8 2 4.1

2.8 k 1.4

15.5 k 5.8 22.2 k 5.7

7.0 f 3.6

5.5 -+ 5.5

17.4 f 7.8

6.1 +- 3.2 17.1 t 2.8

43.1 k 5.1

3.5 k 2.3 17.0 f 5.1 38.7 IL 5.2

15.4 f 1.7

40.0 +- 6.0

52 +- 4.7

3.2 f 2.0

3.3 k 2.7 18.5 k 4.4

n

9 8

9

5 3 6

3

4

8

5 9

5

4

5

6 5

6

8 6

8

5

4

5

5

3 6

(NAD) inhibited epinephrine-stimulated lipolysis of rat adi- pose tissue. As shown in Table VI, NAD inhibited the binding of adenosine to the fat cell membranes, whereas nicotinic acid was an ineffective competitor of adenosine binding, as was nicotinamide (data not shown).

The effects of several of these compounds on the low affinity adenosine binding was probed using 1 mM l”H]adenosine to test the specificity of the low affinity binding component (Table VII). 2’,5’-Dideoxyadenosine, N”-(phenylisopropyl) adenosine. and theophylline at 10 FM failed to appreciably inhibit adenosine binding to the low affinity sites. A lOO-PM concentration of adenine reduced binding by approximately 20%. However, adenine inhibition of the high affinity sites probably accounts for most of this inhibition.

We examined the possibility that adenosine may be inhibit- ing hormone-stimulated cyclic AMP accumulation via modifi- cation of the hormone/receptor interaction. P-Adrenergic hor- mone binding to fat cell membranes, as probed with (-)- l”HJdihydroalprenolo1 (37, 51). was not significantly altered by 100 PM adenosine (data not shown). Likewise, neither 10 pM

Agent concentra tion

w

1

10

100

% Inhibition n

p-Nitrobenzylthioguanosine

Dipyridamole

Theophylline

1-Methyl-3-isobutyl xanthine

Nicotinamide-adenine dinucleo- tide

Nicotinic acid

1 10

100

0.1 1

10

1

10 100

1 100

1

100

5.3 f 4.4 6

7.0 2 5.4 4

26.6 rt 6.4 6

3.1 2 3.0 6 2.4 f 2.2 5

7.5 f 2.5 6

8.1 i 1.2 5

14.2 i 2.0 5 23.9 i 3.7 5

1.6 I 1.0 3 5.3 i 3.3 3

13.0 13.9 3

8.5 T 1.3 5

33.4 i 2.9 5

1.1 AZ 1.1 3 1.8 -t 1.8 3

TABLE VII

Effects of various purine derivatives on binding of l:‘Hladenosine (1 mM) to fat cell plasma membranes

Crude fat cell plasma membranes (350 pg) were incubated with 1.0 mM tritiated adenosine, with or without the indicated concentra- tion of agent, in 0.2 ml of standard assay medium. The incubation was performed at 4°C for 20 min. Adenosine binding was assayed by vacuum filtration on Whatman GFiC filters. Data are the mean values f SE. of three separate experiments. Adenosine binding to the membranes in the control situation was 1023 pmobmg of mem- brane protein.

Agent

2’,5’-Dideoxyadenosine N”-(Phenylisopropyl)adenosine Adenine Theophylline

concentra- tion % Inhibition

PJf

100 10.7 ? 3.5 100 5.0 f 5.0 100 22.7 e 3.9

100 6.8 2 3.8

(-)-alprenolol nor 10 PM (-)-isoproterenol affected I”Hladen- osine binding to the fat cell membranes (data not shown).

DISCUSSION

Adenosine regulates adenylate cyclase activity and intracel- lular cyclic AMP levels in a wide spectrum of tissues and cell types. Specific plasma membrane receptors for adenosine have been postulated as the site of adenosine action in brain slices (9, 11, 52), platelets (14. 221, cultured human astrocytoma and glioma cells (19, 241, isolated bone cells (15). mouse neuroblas- toma cells (23), transformed human lung fibroblasts (211, and Leydig tumor cells (27). although studies aimed at direct identification of these sites have not been reported. Plasma membrane adenosine receptors were proposed by Davies (7) as

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3120 Adentsinc Binding to Fat Cell Plasma Membranw

the site of adenosine action in adipose tissue. A similar proposal was advanced by other groups on the basis of studies performed with isolated fat cells (5. 34). The present study is. 10 the best of our knowledge. the first report characterizing 1 “H ladcnosine binding to a plasma membrane preparation. The plasma membrane fractions prepared from isolated white fat cells exhibit a capacity to bind adenosine. The binding of adcnosine at nanomolar concentrations to these membranes can be assayed by the vacuum filtration technique herein described.

The kinetics of adenosine binding to fat cell plasma mem- branes was rapid. rcac.hing 50%. of the equilibrium level within 1 min at 0°C. 22°C. and 37°C. Adenosine inhibition of norcpinephrine-stimulated cyclic AMP accumulation in fat cells is very rapid; if added I min after norepinephrine. adenosinc reduced cyclic AMP accumulation during the next minute (53). Adenosine has also been shown to act rapidly in cultured neuroblastnma cells (18). isolated bone cells (1%. and platelets (14).

obtained between 10 and 100 WM. Pereira and Holland (3) rcportcd a 44% inhibition of norepinephrine-stimulated lipol- ysis in adipose tissue by I FM adenosine and a similar level of inhibition by 1 to 10 @M ATP. ADP. or 5’-AMP. Adcnosine (1 PM) produced an 80% inhibition of norepinephrine-stimulated cyclic AMP accumulation in the presence of theophylline (53). Dilute fat cell suspensions were more sensitive to inhibition of norepinephrine-stimulated cyclic AMP accumulation by aden- osine and by adenosinr-containing compounds (8). In this system. 0.1 PM adcnosine almost completely inhibited the cyclic AMP accumulation due to norepinrphrinc and adeno- sine-containing compounds displayed the following ordrr of potency: adenosinc > 5’-AMP > ADP > ATP (8). How much of the inhibition of both l:‘H ladenosine binding and hnrmonc- stimulated lipolysis and cyclic AMP accumulation by these adenosine-containing compounds is due to adcnosine released via their metabolism has not been established.

Addition of adenosine deaminase to incubated fat cells increases basal cyclic AMP accumulation and pntentiates the norepinephrinc-stimulated increase in cyclic AMP (53. 54). Near maximal effects of adenosinc dcaminasc on norepineph- rine-stimulated cyclic AMP accumulation in fat cells were achieved at approximately 20 s of incubation (33). Inhibitors of adenosinc deaminasc were required in the prosent study to detect appreciable adenosine binding to the fat cell plasma membranes. These inhibitors also potentiate adenosine inhi- bition of norepinephrine-stimulatid cyclic AMP accumulation in fat cells (33. 34). A similar observation was reported in lymphocytes. where adenosine deaminase reverses and EHNA potcntiates the stimulation of cyclic AMP levels by adenosine cm.

Additional evidence suggesting adenosine exerts its effects via a surface receptor is based on cxpcriments utilizing an adenosine derivative which is too large to penetrate ccl1 membranes (55). Adenosinc covalently linked to a water- soluble oligosaccharide. stachytrsr. with a molecular weight greater than 1500. has been shown to virtually equipotcnt to free adenosine in inhibiting cyclic AMP accumulation by rat fat cells (34). Olsson et al. (55) first demonstrated the ability of this novel adenosine compound to exert an adenosine-like. dose-dependent coronary vasodilation following intracoronary infusion into dogs.

Adenine was a very potent inhibitor of adrnnsinc binding to fat cell membranes (Table V). From a structural standpoint. this observatinn is certainly not unexpected. Dolr (I) reported early that unlike adcnosine. adenine enhanced epincphrine- stimulated lipolysis in adipose tissue. Davies (7) subsequently reported a 71% stimulation of norepinephrine-induced lipoly- sis in adipose tissue by 0.4 rnM adenine. In fat cells. significant elevation of theophylline-induced lipoiysis was demonstrated with 1 pM adenine (5). In contrast to the situation in fat cells. cyclic AMP levels of cultured mouse neuroblastoma cells were stimulated by adenosinr and inhibited by adeninc (18). Z- Chloroadenosine stimulation of adenglatc cyclasc in mouse neuroblastoma preparations was actually antagonized by ad- enine (23). Likewise. bone cell adenylate cyclase is stimulatid by adenosine and inhibited by adenine (25). Adcnine at both 18 and 73 ~IVI. however. had no effect on norrpinephrinc- stimulated adenylate cyclase activity of fat cell ghosts (4).

Dipyridamole is a potent inhibitor of the uptake of I:‘Hladcnosine into rat fat cells (5. 48). Kbert and Schwabe (5) observed that dipyridamole (10 P.M) potentiated the ability of adenosinr to inhibit lipolysis in fat cells rather than rcducc it. although it almost completely blocked the uptake of adenosine into the cells. These investigators concluded that the plasma membrane was probably the sitr of this action of adenosine in the fat cell. Dipyridamolr (40 to 100 ELM) likewise failed to block the reduction by adenosine of norcpinephrine-stimulated cyclic AMP accumulation (53). Neither dipyridamole nor NBTG. another potent inhibitor of adenosine uptake (14. 46). were effective inhibitors of adenosine binding to fat cell membranes.

Fain et al (4. 56) reported that 6 to 10 PM 2’.6’-dideoxyaden- osine inhibited nnrPpincphrine-stimulated cyclic AMP accu- mulation in fat cells approximately 50%. Fifty pcrccnt inhibi- tion of norepinephrine-stimulated adenylate cyclasr activity in fat cell ghosts was obtained with 5 p.~ 2’.5’-dideoxyadcno- sine (4). This adenosinc analog was a potent inhibitor of 1:‘HJadenosine binding to fat cell plasma membranes (Table V). 2’.5’-Didcoxyadcnosine has also been shown to be a potent inhibitor of both glucagon-stimulated adenylate cyclase activ- ity in rat liver membranes (28. 31) and glucagon- and cpineph- rine-stimulated cyclic AMP accumulation in intact hrpato- cytes (28).

N”-(Phenylisopropyl)adenosine. which cannot bc dcami- nated by adenosinc dcaminase, was shown by Fain (53) to be the most potent adenosine analog tested with respect to inhibition of cyclic AMP accumulation in intact fat cells. Cyclic AMP acrumulatiun and lipolysis due to 0.1 PM nortpi- nephrine in the presence of adenosinc deaminase was almost completely blocked by 0.01 P*M N”-(phenylisoprr)pyI)adcnosinr (S6). However, this adenosine analog. even at 73 PM. was virtually without eflbct on adenylate cyclase activity of fat cell ghosts (4). N’,-(PhenyIisttpropyl)adenosinc failed to inhibit adcnosino binding to the fat cell membranes (Table VI).

The binding of lYHladenosinr to fat cell plasma membranes Why N”-(phenylisopmpyl)adenosine. the most potent inhib- was inhibited by ATP. ADP. and 5’-AMP, Dole (I) originally itor of norepincphrine-stimulated lipolysis and cyclic AMP reported that addition of ATP. I?‘-AMP. or adenosine (at 0.8 to accumulation in the intact cells is so ineffective as an inhibitor 4 mM) to incubated adipose tissue inhibited the lipolytic action of both adenylate cyolase and adenosinc binding remains of epinephrine. Kappeler (6) demonstrated that adenosinc. 5’- obscure. Perhaps the protocols used to prepare the fat cell AMP. ADP. and ATP inhibited glucagon-stimulated lipolysis ghosts and membranes alter. in a critical fashion. the charac- in adipose tissue; half-maximal inhibition by adenosine was ter of the site increasing its afftnity for 2’.5’-didcoxyadenosine

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Adenosine Binding to Fat Cell Plasma Membranes 3121

while simultaneously reducing its affinity for N”-(phenyliso- propyl)adenosine. Blume and Foster (23) reported the selective loss of 2-chloroadenosine-stimulatable adenylate cyclase activ- ity of neuroblastoma cells following fractionation, although basal cyclase activity did not change. Penit et al. (26) similarly noted that purified membrane fractions prepared from neuro- blastoma cells displayed a marked reduction in adenylate cyclase responsiveness to the stimulatory action of adenosine. Alternatively, N”-(phenylisopropyl)adenosine and 2’.5’-di- deoxyadenosine could exert their influence on cyclic AMP accumulation in fat cells via separate distinct sites (56).

Another common observation concerning adenosine action and mammalian cell cyclic AMP metabolism is that regardless of whether adenosine stimulates (9, 12, 14, 18, 19) or inhibits (33, 34) the accumulation of cyclic AMP, methylxanthines ap- pear to antagonize its action in these systems. In rat fat cells, theophylline inhibits uptake of tritiated adenosine and op- poses the action of adenosine (5. 48, 56). The present studies demonstrate that theophylline inhibits adenosine binding to fat cell membranes. Significant inhibition of adenosine bind- ing was detected with 0.1 PM theophylline. a concentration of theophylline too low to inhibit cyclic AMP phosphodiesterase (57).

Fain et al. (56) found l-methyl-3-isobutyl xanthine to be more potent than theophylline in inhibiting adenosine uptake in the intact fat cell. The present study shows that theophyl- line inhibits adenosine binding to fat cell membranes. In contrast, 1-methyl-3-isobutyl xanthine failed to inhibit adeno- sine binding even at 100 PM. These data suggest that inhibi- tors of adenosine uptake of intact fat cells. such as dipyrida- mole and methylxanthines. are generally poor inhibitors of adenosine binding. Although theophylline does inhibit [“HI- adenosine binding, this effect is probably not related to the effect of theophylline on adenosine uptake. Unlike adenosine uptake, inhibition of adenosine binding by theophylline ap- pears to be specific for this methylxanthine. McKenzie et al. (58) recently similarly reported that the ability of adenosine to relax intestinal smooth muscle is antagonized by theophylline but not 1-methyl-3-isobutyl xanthine.

The present study demonstrates that highly purified plasma membranes prepared from fat cells do possess adenosine binding sites. This fat cell plasma membrane preparation was previously shown to be enriched in both adenylate cyclase activity (38) and P-adrenergic receptors (37). Since adenosine antagonizes the action of catecholamines on lipolysis and cyclic AMP accumulation in intact fat cells. it was of interest to explore the possibility that adenosine might, inhibit or modify catecholamine binding to its putative /?-adrenergic receptor. Adenosine possessed no significant ability to alter catecholamine interaction with its receptor as probed with (-1-l “Hldihydroalprenolol. p-Adrenergic agonists and antago- nists had no effect on 13H]adenosine binding to the fat cell membranes.

It is interesting to speculate on the function of these adenosine binding components identified in the fat cell plasma membrane preparations. The inability of dipyridamole to effectively compete for these binding sites with adenosine would argue against these sites being involved in adenosine uptake. The required presence of EHNA or deoxycoformycin to demonstrate adenosine binding would similarly rule out adenosine deaminase. Several characteristics of the binding sites suggest these sites may be involved in the regulation of adenylate cyclase activity. Adenosine has been shown to be an inhibitor of fat cell cyclic AMP phosphodiesterase activity (4.

7). The action of adenosine in fat cells is to inhibit, rather than potentiate, cyclic AMP accumulation arguing against this enzyme being the binding site. In addition. I-methyl-3-isobu- tyl xanthine. one of the most potent phosphodiesterase inhib- itors, failed to compete with adenosine for these sites.

Perhaps some of these adenosine binding components are regulatory sites on the adenylate cyclase complex which modulate the activity of this enzyme. This scheme is only speculative, however, and the precise identity and function of these adenosine binding components remains to be estab- lished. Rosenblit and Levy (48) have recently approached this same problem by using a photoreactive derivative of adeno- sine, 8-azidol 2-JH]adenosine. Photolysis of this derivative with intact fat cells led to its incorporation into several protein components of the membrane (48). However. the specificity of this labeling technique was not addressed in this report (48).

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