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Isolation and characterization of a liver plasma membrane fraction enriched in glucagon-sensitive adenylate cyclase
ARCHIVES
OF
Isolation
BIOCHEMISTRY
AND
174, 291-297
BIOPHYSICS
(1976)
and Characterization of a Liver Plasma Membrane Enriched in Glucagon-Sensitive Adenylate Cyclasel
NORBERT Memorial
I. SWISLOCKI,” Sloan-Kettering
JOAN
Cancer
Center,
Received
TIERNEY, 1275
York
September
AND
Avenue.
EDWARD Neu
York,
Fraction
S. ESSNER” New
York
10021
23, 1975
Partially purified liver plasma membranes were fractionated further on sucrose layers. Three membrane populations, numbered Peaks 1, 2, and 3, were isolated at densities of 1.23, 1.16, and 1.03, respectively. Peaks 1 and 2 were enriched to a similar degree in 5’-nucleotidase activity, a plasma membrane marker, relative to membranes in Peak 3. Electron micrographs indicated that Peak 1 possessed desmosomes and bile canaliculi, while Peak 2 contained large vesicles as well as smaller vesicular structures attached to membranes. The latter have been attributed to hepatocyte sinusoidal surfaces. All three membrane fractions contained adenylate cyclase activity with the highest specific activity found in Peak 2. The enzyme in all three peaks was F sensitive with higher sensitivity in Peaks 1 and 2. Glucagon sensitivity of adenylate cyclase in Peak 2 membranes was four times that of Peak 1. Only Peak 2 membranes were sensitive to epinephrine. The Peak 2 membranes were three times more sensitive to glucagon than the partially purified membranes from which they were derived. These findings indicate that, while both bile canalicular and sinusoidal faces of hepatocytes possess adenylate cyclase, the sinusoidal fraction is more sensitive to glucagon. Soluhilized adenylate cyclase of the Peak 2 membranes, obtained as the 165,OOOg supernate of membranes treated with Lubrol-PX, was sensitive to stimulation by guanyl nucleotide analogs. Guanyl nucleotide sensitivity thus resides in the catalytic site and is not dependGrit on membrane integrity. All three membrane fractions possessed similar activities of nucleotide phosphohydrolase activity.
Liver fractions enriched with plasma membranes have been widely used to study hormone-membrane interactions with particular attention being directed towards the adenylate cyclase system. Studies in many laboratories have characterized the binding of hormones to isolated liver membranes (l-3), membrane conformational changes after exposure of membranes to hormone (4, 5), adenylate cyclase activation by hormones (3, 6, 7) and guanyl nucleotides (8, 9), and modulation of several of these phenomena by guanyl nucleotides (9, 10).
For the most part the Neville procedure (11) has been used as the method of isolation of liver plasma membranes (l-10). However, adenylate cyclase in the fully purified membranes, enriched in 5’-nucleotidase and consisting of bile canalicular surfaces, was less sensitive to glucagon than the less purified membrane float preparation (6). Many investigators (1, 3, 6-lo), therefore, have utilized the less purified but more hormone-sensitive product obtained in the step (step 11) just prior to isolation of the final membrane preparation (11). During the course of investigations of binding of guanyl nucleotides to the partially purified liver plasma membranes we had occasion to modify the final step of the Neville procedure ( 11) in order to separate three membrane fractions which appeared (12) upon sucrose gradient centrifugation
’ This investigation was supported in part by Research Grants No. CA-08748, CA-16889, CA-15773, CA-17213, and AM-17210 of the National Institutes of Health. -1 To whom requests for reprints should be made. ‘( Current address: School of Medicine, Wayne State University, Detroit, Mich. 48201. 291 Copyright All rights
$2 1976 by Academic Press, Inc. of reproduction in any form reserved.
292
SWISLOCKI,
TIERNEY
as originally described (11). In the original procedure (ll), the heaviest membrane fraction which contains bile canalicular surfaces was harvested and used as the liver plasma membrane preparation, while the lighter two fractions were not utilized. We have separated the three membrane fractions obtained in the gradient by fractionation of the partially purified liver membrane on sucrose layers and investigated several properties of the isolated membrane fractions. All the membrane fractions contained adenylate cyclase activity. As will be shown, the fraction of intermediate density consisting of blood sinusoidal surfaces, was more sensitive to glucagon than the heavier bile canalicular membranes. METHODS Membranes from livers of hypophysectomized rats (Charles River), 145-165 g, were prepared as described in an earlier communication from this laboratory (Ref. (121, Fig. 2). Critical to the isolation of the three membrane fractions was the use of an appropriate Dounce homogenizer. Homogenizers with suitable clearances were selected so that the pestle drop-time in water was 2.0-2.5 s over the 20cm distance of full travel across the shearing surface. Briefly, 0.5 rnM CaCl, was added (13) to the homogenizing medium of 1 mM NaHCO,, to increase yield of membranes, and the procedure of Neville (11) was used to step 11. There, the partially purified membranes, here designated float, were skimmed off the preparative sucrose medium (ll), washed with 1 mM NaHCO,, containing 0.5 mM CaCl,, and centrifuged at 35,000g for 20 min. The pellet was resuspended in 18 ml of the buffer, 6 ml of which was applied to each of the SW 25.1 tubes tilled with sucrose layers made as follows: 50% sucrose, 4.1 ml; 37%, 10 ml; lo%, 10 ml. Centrifugation was at 2000 rpm for 1 h at 5°C in a Beckman Model L-2 ultracentrifuge with the brake off. Three membrane fractions were obtained, at the sucrose interfaces of densities 1.23, 1.16, and 1.03, and harvested by dropwise collection after puncturing the tube. The membrane fractions were characterized by electron microscopy and enzyme assays. For electron microscopy the membranes were washed free of sucrose and pelleted at 35,000g for 20 min. Small pellets of the fractions were fixed for 1 h in cold, phosphate-buffered 1% osmium tetroxide, dehydrated in graded alcohols, and embedded in Spurr’s resin. Thin sections were prepared on a diamond knife, stained with lead citrate, and photographed
AND
ESSNER
with a Siemens 101 electron microscope. Adenylate cyclase was assayed by the method of Krishna et al. (141, and 5’-nucleotidase was assayed according to Song and Bodansky (15). Guanyl nucleotide hydrolysis was determined as described earlier (12, 161. Protein was determined by the Lowry procedure (171. When membranes were solubilized with detergent they were washed with 1 mM NaHCOZI containing 0.5 mM CaCl,, and 1 mg of membrane protein was resuspended per 1 ml of 0.2 M Tris-HCl, pH 7.5, containing 0.25 M sucrose, 1 mM EDTA, and 40 mM Lubrol-PX (molecular weight, 600). This mixture was centrifuged at 165,OOOg for 1 h at 5°C and the supernates containing solubilized adenylate cyclase were removed. RESULTS
As indicated in Table I when partially purified liver membranes, obtained as the float in step 11 of the Neville procedure, were washed by resuspension in buffer and centrifugation at 35,000g for 20 min, 20% of the float protein was not pelleted. The loss was due to protein that remained in the wash supernate. Further fractionation of the washed float was accomplished on sucrose layers, by which procedure three populations of membranes were obtained. The membranes of intermediate density, Peak 2, contained half of the recovered protein with the remainder divided equally between Peaks 1 and 3. The specific activity of 5’-nucleotidase, used as a plasma membrane marker, was similar in Peaks 1 and 2, while Peak 3 membranes TABLE PROTEIN
AND
5’-NUCLEOTIDASE
LIVER
MEMBRANE
Fraction Float Washed float Peak 1 Peak 2 Peak 3
I DISTRIBUTION
IN
FRACTIONS~
Protein (mg)
5’-Nucleotidase
56.1 45.1 7.6 15.3 7.7
0.053 0.727 0.653 0.227
Density
1.23 1.16 1.03
o Membranes were prepared from 20 g of rat liver by the Neville procedure which was carried to step 11 (11). Subsequently, sucrose layers were substituted (Methods) for the sucrose gradient as utilized in the last step of the original procedure. 5’-Nucleotidase activity is in micromoles per milligram of protein per minute.
ADENYLATE
CYCLASE
possessed much lower marker enzyme activity. Electron micrographs of membranes in Peaks 1 and 2 are shown in Fig. 1. Both fractions contain numerous membranous elements. Peak 1 membranes contain bile canaliculi and desmosome-like structures arranged along membranes which are apposed to each other. Peak 2 membranes, on the other hand, showed many separate membrane large vesicles and, notably, structures which contained numerous small vesicles that were probably derived from the sinusoidal faces of hepatocytes. Peak 3 (not shown) contained membranous elements which could not be identified. Adenylate cyclase activity was examined in the three membrane fractions, and the enzyme present in Peak 2 membranes exhibited the highest specific activity (Table II). This has been consistently observed in many membrane preparations although the absolute activity has varied from preparation to preparation. Adenylate cyclase present in all three membrane fractions was sensitive to stimulation by F-. With 10 mM F-, adenylate cyclase in Peaks 1 and 2 was stimulated about sevenfold whereas enzyme activity in Peak 3 membranes was increased but twofold. The three membrane fractions were likewise examined for sensitivity to glucagon and epinephrine (Table III), since both hormones had been demonstrated to increase adenylate cyclase activity in partially purified liver membranes (I, 3, 7). Glucagon stimulated Peak 1 enzyme twofold, Peak 2 enzyme eightfold, and Peak 3 enzyme fivefold. Epinephrine increased enzyme activity by 47% and only in Peak 2 membranes, the only fraction upon which an effect of epinephrine, although small relative to glucagon, was observed. Insulin did not alter adenylate cyclase activity in any of the three membrane fractions. In view of the relative enrichment of adenylate cyclase in Peak 2 and the high sensitivity of Peak 2 membranes to glucagon this membrane fraction and the partially purified membranes from which it was derived were examined for dose-response sensitivity to glucagon. As seen in
FROM
293
LIVER
Fig. 2, adenylate cyclase activity in Peak 2 membranes increased over the range of lo-!’ to 10-j M glucagon. At the high hormone concentration a maximal ninefold stimulation was observed. Higher concentrations of glucagon exerted no further increase in enzyme activity. The partially purified membranes (Fig. 3) were stimulated over threefold by the same concentration of glucagon. When compared to the partially purified membranes, the Peak 2 membranes were three times more sensitive to glucagon. We had previously demonstrated (16) that adenylate cyclase present in solubilized partially purified membranes exhibited sensitivity to guanyl nucleotides that was similar to the membranes from which the enzyme was obtained. Examination of the solubilized membrane fractions indicated (Table IV) that adenylate cyclases present in 165,OOOg supernates of membranes in Peaks 1 and 2 that had been solubilized with Lubrol-PX were equally sensitive to the quanyl nucleotide analogs GMP-PNP and GMP-PCP4 which doubled enzyme activity. The enzyme activity of Peak 3 membranes was inhibited by guanyl nucleotide analogs. As had been observed with the enzyme in the partially purified membranes (16), GMP-PNP was the most potent guanyl nucleotide in stimulating adenylate cyclase activity of solubilized Peak 2 membranes. GTP, however, which stimulated the enzyme in the solubilized float (16), inhibited the activity present in the three solubilized membrane fractions. As we reported earlier (12, 16), partially purified membranes and detergent-solubilized preparations thereof possess nucleotide phosphohydrolases. As seen in Table V, nucleotide hydrolysis of wide specificity is carried out to approximately the same degree in all three membrane fractions. GTP, ITP, and GMP-PCP were good substrates; the GTP analog was slightly more resistant to hydrolysis. Solubilization of the membranes with detergent did not alter the specificity of hydrolysis. ’ Abbreviations used: GMP-PCP, thylene diphosphonate; GMP-PNP, dophosphate.
5’-guanylylme5’-guanylylimi-
294
SWISLOCKI,
TIERNEY
AND
ESSNER
FIG. 1. (A), Electron micrograph of membranes in Peak 1. Numerous desmosome-like structures are evident. Arrows indicate two bile canaliculi. tron micrograph of membranes in Peak 2. Large vesicular and membranous dent, some of which contain many attached, small vesicles (arrow). x6400.
Sensitivity of the adenylate cyclase in Peak 2 membranes to Lubrol-PX was examined since this detergent was reported by B&r and Kulshrestha (18) to increase
membranes and x4800. (B), Elecelements are evi-
the activity of the enzyme in membranes of Ehrlich ascites cells. As seen in Table VI Lubrol-PX at lo-” to 10e4 M had no effect on the enzyme activity. The detergent at
ADENYLATE
CYCLASE TABLE
ADENYLATE
CYCLASE
DISTRIBUTION
AND _____
FLUORIDE
FROM II SENSITIVITY
IN
Membrane
Additions 1
None 10 mM
LIVER
MEMBRANE
peaks
FRACTIONS” ______
_.
_---
2
3
______-.-_~-___-231.3 t 28.1 1670.0 2 21.7
69.5 f 2.4 457.4 2 40.1
NaF
295
LIVER
195.9 438.0
+ 31.0 k 27.0 ---
a Membrane fractions were prepared and washed as described in the footnote to Table I. Adenylate cyclase activity is expressed in picomoles of cyclic AMP per milligram of protein per 10 min and was determined in triplicate with 37 kg of membrane protein in 0.32 rnM ATP in 0.1 ml of 50 mM Tris-HCl, pH 7.4, with 2 rnM EDTA and 10 mM MgC12, with 2-5 &i of I w:‘~P]ATP (16). TABLE HORMONE
SENSITIVITY
OF LIVER
(1 Membrane respectively.
MEMBRANE
FRACTIONS”
Membrane
Hormone
None Glucagon Epinephrine Insulin
III
~-___ 83.6 188.9 68.3 53.5
1 t i i +
_.--.-__-__ 15.5 14.4 10.6 16.6
FIG. 2. Dose-response of Peak 2 membranes to glucagon. Peak 2 membranes were isolated as described in Methods. These membranes were resuspended in homogenizing medium, washed free of sucrose prior to assay, and assayed as indicated in the footnote to Table II. ATP concentrations were 3.2 mM.
2
125.3 f 992.7 i 177.2 i 135.2 2
fractions were prepared and assayed as described Hormone concentrations were 1 pg/O. 1 ml of medium.
---
peaks 3 15.1 82.7 13.3 20.5
69.5 376.7 77.8 82.4
in the
footnotes
? i i ?z
to Tables
13.1 7.2 16.8 21.9 ____ I and
II,
FIG. 3. Dose-response of partially purified liver plasma membranes to glucagon. Membranes were prepared by the Neville procedure carried to Step 11 (111, washed free of sucrose, and resuspended in homogenizing medium prior to assay.
DISCUSSION
lo-” M decreased enzyme activity by 72%. The prostaglandin antagonist 7-oxa-13prostynoic acid which has detergent-like properties in human erythrocytes (19) increased enzyme activity at lo-” M but at the other concentrations examined had an inhibitory effect.
The membranes isolated in the Peak 2 fraction possess the properties of blood sinusoidal surfaces of hepatocytes. They have been enriched in 5’-nucleotidase activity, a marker enzyme for plasma membranes, and appear on electron microscopy as large free vesicles and as small vesicles attached
296
SWISLOCKI,
TIERNEY TABLE
GUANYL
NUCLEOTIDE
SENSITIVITY
Nucleotide
0~165,000g
AND
ESSNER
IV
SUPERNATES LUBROL-PX"
OF MEMBRANE
Float
Membrane 1
None GMP-PNP GMP-PCP GTP GDP
124.1 248.9 239.6 156.8 80.6
? i k r e
FRACTIONS
16.0 11.9 34.7 15.7 3.2
48.2 89.5 36.9 34.7 52.3
SOLUBILIZED
peaks 2
+ 1.7 i 10.7 k 13.9 i 6.4 k 19.9
53.9 127.3 93.6 28.1 52.3
WITH
-c 2 k r 2
3 8.4 11.2 13.2 15.4 14.0
30.4 17.6 15.9 4.7 8.7
+ 5.1 i 12.4 + 7.1 2 5.0 2 5.7
u Membrane fractions were prepared and assayed as described in the footnotes to Tables I and II. Guanyl nucleotide concentrations were 0.1 mM. Membranes were solubilized by resuspending the membrane pellets in 0.2 M Tris-HCl, pH 7.5, containing 40 mM Lubrol-PX, 0.25 M sucrose, 1 mM EDTA, and were centrifuged at 165,OOOg for 1 h. The supernates were used as the source of the enzyme. TABLE
V
TABLE
NUCLEOTIDE HYDROLYSIS BY MEMBRANES AND BY 165,OOOg SUPERNATES OF MEMBRANES SOLUBILIZED WITH LUBROL-PX"
Peak
1
Peak
2
Peak
3
VI
OF PEAK 2 MEMBRANES AND 7-OXA-%PROSTYNOIC
Concentration
Lubrol-PX
GTP
ITP
GMPPCP
202.8 189.5 214.9 179.5 171.4 72.8
192.2 158.7 212.4 138.5 146.5 48.2
141.7 123.8 158.0 123.0 109.9 88.1
n Nucleotide hydrolysis was determined as described earlier (12, 16) by incubation of 50 /*g of protein in 0.1 ml of 50 mM Tris-HCl and 2 mM MgCl,, pH 7.4. Nucleotide concentrations were 0.1 mM to which 0.1 FCi of ‘%-labeled nucleotide was added. After incubation for 10 min at 3o”C, the reaction mixtures were boiled for 3 min and applied to PEI-cellulose sheets for chromatographic separation of hydrolytic products in LiCl (PEI, polyethyleneimine). Appropriate blanks and standards were run with each assay. Lanes corresponding to reactions were cut into l-cm segments and radioactivity thereon measured by liquid scintillation spectrophotometry. Results are expressed in picomoles of nucleotide hydrolyzed per microgram of protein per 10 min. to membrane sheets. A noteworthy feature of this fraction is the absence of bile canaliculi which were found in the denser Peak 1 membranes. Significantly, the Peak 2 membranes were enriched in adenylate cyclase activity that was three times more sensitive to glucagon than the partially purified membranes from which they were
lo-” 1 o-:, 10 4 lo- :I None
182.0 174.3 209.6 57.1 204.8
2 * k f k
TO LUBROL-PX ACID" 7-Oxa-13-prostynoic acid
(M)
Substrates
Peak 1 Solubilized Peak 2 Solubilized Peak 3 Solubilized
SENSITIVITY
10.6 19.1 13.6 8.9 40.3
173.8 135.5 323.6 134.5
+ * -+ i-
29.9 17.9 57.2 50.8
’ Peak membranes were prepared and assayed as described in the footnote to Table II. Lubrol-PX and 7-oxa-13-prostynoic acid were added just prior to assay for adenylate cyclase.
derived. The blood sinusoidal fraction was also three times more sensitive to glucagon than the bile canalicular surfaces from which they were separated. The Peak 2 membranes, as isolated and characterized here, have several features in common with a membrane fraction obtained by Wisher and Evans (20) who reported, after these studies were completed, the isolation of several types of hepatocyte membranes of which one fraction, called the microsomal-light membranes (M-L), possessed properties that were attributed to blood sinusoidal surfaces. Both of these membrane preparations (Peak 2 and the M-L fraction of Wisher and Evans) had a density of 1.16, appeared as vesicles on tissue disruption, were the predominant membrane fraction obtained, were more sensitive to glucagon than other fractions, and, significantly, possessed adenylate cyclase activity that was similarly stimu-
ADENYLATE
CYCLASE
lated (ninefold) by maximal concentrations of glucagon. It should be expected that cell surface receptors in liver for blood-borne regulators like glucagon would be located on the vascular side of the hepatocyte. Thus the lower glucagon sensitivity of the membrane fraction with bile canalicular surfaces (Peak 1) relative to the blood sinusoidal membranes (Peak 2) would be expected. These findings explain, in part, the observations of Pohl et al. (6) that fully purified liver plasma membranes isolated by the Neville procedure (ll), which contain bile canaliculi, were less sensitive to glucagon than the parent partially purified membranes. On the basis of the present data it can be seen that the partially purified membranes contained the glucagon-sensitive membranes which, however, were discarded in the final purification step (11). The early observation (6) that full membrane purification by the Neville procedure resulted in a decline in glucagon sensitivity led investigators to utilize the partially purified membrane preparation to study adenylate cyclase activity as a consequence of glucagon-membrane interactions (1, 3, 6-10). As reported here, a simple further fractionation of the partially purified membranes yields a membrane fraction which is more appropriate to the study of hormone-membrane interactions. Adenylate cyclase activity in membranes isolated from many sources is modulated by guanyl nucleotides (see Ref. (16)). In a previous communication (16) we presented evidence that solubilized adenylate cyclase obtained as a 165,000g supernate of detergent-treated membranes possessed the same properties of activation by guanyl nucleotides as did the parent partially purified membranes. On the basis of those findings we suggested that the guanyl nucleotide site resided on the soluble adenylate cyclase and that the structural integrity of the liver plasma membrane was not an obligatory requirement for the guanyl nucleotide effects on adenylate cyclase activity. These findings have been presently extended to adenylate
FROM
297
LIVER
cyclase solubilized from either bile canaliculi or blood sinusoidal membranes. REFERENCES 1. RODBELL, M., BIRNBAUMER, 1861-1871. 2. JOHNSON, A., JR.
mun.
L.
C. B., BLECHER, (1972)
M.,
AND Biophys.
Biochem.
N.
GIORCIO,
Res.
Com-
46. 1035-1041.
3. BIRNBAUMER, 300, 129-158. 4. RUBIN, BERG,
H. M. J., POHL, S. L., AND (1971) J. Biol. Chem. 246,
KRANS,
L.
M.
S.,
M.
(1973)
(1973)
Biochzm.
Acta
Biophys.
N. I., AND SONENBiochem. Biophys. 157,
SWISLOCKI,
Arch.
243-251. N.
5. SWISLOCKI,
I.,
M.,
SONENBERG,
POSTEL-VI-
NAY, M-C., GOIDL, J. A., ROBERTS, DERSON, G., PRIDDLE, M., AND
J. E., HENTIERNEY, J.
(1973) in Advances in Human Growth Hormone Research: A Symposium, Publ. No. NIH-74-612, p. 588, U.S. Government Printing Office, Baltimore. 6.
POHL, S. L., BIRNBAUMER, (1971) J. Bid. Chem.
L.,
AND
RODBELL,
M.
246, 184991856. A. M.. PERRENOUD, M. J. (1973) Eur. J. Biochrm.
13.
F., CHAMBAUT, L., AND HANOUNE, 38, 185-192. RODBELL, M., BIRNBAUMER, L.. POHL, S. L., AND KRANS, H. M. J. (1971) J. Bid. Chem. 246, 1877-1882. RODBELL, M., LIN, M. C., AND SALOMON, Y. (1974) J. Bid. Chern. 249, 59-65. RODBELL, M., KRANS, H. M. J., POHL, S. L., AND BIRNBAUMER, L. (1971) J. Bid Chem. 246, 187221876. NEVILLE, D. M., JR. (1968) Biochem. Biophys. Acta 154, 540-552. SWISLOCKI. N. I., SCHEINBERG, S., AND SONENBERG, M. (1973) Biocheru. Biophys. Res. Cornmun. 52. 313-319. RAY, T. K. (1970)Biochim. Biophys. Actn 196, l-
14.
KRISHNA,
15.
(1968) -J. Pharmncol. Exp. SONG. C. S., AND BODANSKY,
7.
8.
9.
10.
11. 12.
LERAY.
9.
G.,
WEISS,
B.,
AND
BRODIE,
B.
163, (1967)
379-385. J. Blol.
Thu.
B.
0. 242, 694-699. SWISLOCKI. N. I.. AND TIERNEY, J. (1975) Arch. Biochem. Biophys. 168, 455-462. LOWRY, 0. H., ROSEBROUCH. N. J., FARR, A. L., AND RANDALL, R. J. (195115. Bid. Chem. 193, Chem.
16. 17.
18.
265-275. BAR, H. P., AND
J. B&hem. 19.
20.
SWISLOCKI, s. (1974) WISHER, them.
M. J.
KULSHRESTHA,
S. (1975)
53. 472-474. N. I., TIERNEY,
J., AND
Prostngzandin.s
7. 401-410.
H., 116.
AND EVANS, 375-388.
W.
Canad.
RITTERSTEIN. H.
(1975)
Bio-
OF
Isolation
BIOCHEMISTRY
AND
174, 291-297
BIOPHYSICS
(1976)
and Characterization of a Liver Plasma Membrane Enriched in Glucagon-Sensitive Adenylate Cyclasel
NORBERT Memorial
I. SWISLOCKI,” Sloan-Kettering
JOAN
Cancer
Center,
Received
TIERNEY, 1275
York
September
AND
Avenue.
EDWARD Neu
York,
Fraction
S. ESSNER” New
York
10021
23, 1975
Partially purified liver plasma membranes were fractionated further on sucrose layers. Three membrane populations, numbered Peaks 1, 2, and 3, were isolated at densities of 1.23, 1.16, and 1.03, respectively. Peaks 1 and 2 were enriched to a similar degree in 5’-nucleotidase activity, a plasma membrane marker, relative to membranes in Peak 3. Electron micrographs indicated that Peak 1 possessed desmosomes and bile canaliculi, while Peak 2 contained large vesicles as well as smaller vesicular structures attached to membranes. The latter have been attributed to hepatocyte sinusoidal surfaces. All three membrane fractions contained adenylate cyclase activity with the highest specific activity found in Peak 2. The enzyme in all three peaks was F sensitive with higher sensitivity in Peaks 1 and 2. Glucagon sensitivity of adenylate cyclase in Peak 2 membranes was four times that of Peak 1. Only Peak 2 membranes were sensitive to epinephrine. The Peak 2 membranes were three times more sensitive to glucagon than the partially purified membranes from which they were derived. These findings indicate that, while both bile canalicular and sinusoidal faces of hepatocytes possess adenylate cyclase, the sinusoidal fraction is more sensitive to glucagon. Soluhilized adenylate cyclase of the Peak 2 membranes, obtained as the 165,OOOg supernate of membranes treated with Lubrol-PX, was sensitive to stimulation by guanyl nucleotide analogs. Guanyl nucleotide sensitivity thus resides in the catalytic site and is not dependGrit on membrane integrity. All three membrane fractions possessed similar activities of nucleotide phosphohydrolase activity.
Liver fractions enriched with plasma membranes have been widely used to study hormone-membrane interactions with particular attention being directed towards the adenylate cyclase system. Studies in many laboratories have characterized the binding of hormones to isolated liver membranes (l-3), membrane conformational changes after exposure of membranes to hormone (4, 5), adenylate cyclase activation by hormones (3, 6, 7) and guanyl nucleotides (8, 9), and modulation of several of these phenomena by guanyl nucleotides (9, 10).
For the most part the Neville procedure (11) has been used as the method of isolation of liver plasma membranes (l-10). However, adenylate cyclase in the fully purified membranes, enriched in 5’-nucleotidase and consisting of bile canalicular surfaces, was less sensitive to glucagon than the less purified membrane float preparation (6). Many investigators (1, 3, 6-lo), therefore, have utilized the less purified but more hormone-sensitive product obtained in the step (step 11) just prior to isolation of the final membrane preparation (11). During the course of investigations of binding of guanyl nucleotides to the partially purified liver plasma membranes we had occasion to modify the final step of the Neville procedure ( 11) in order to separate three membrane fractions which appeared (12) upon sucrose gradient centrifugation
’ This investigation was supported in part by Research Grants No. CA-08748, CA-16889, CA-15773, CA-17213, and AM-17210 of the National Institutes of Health. -1 To whom requests for reprints should be made. ‘( Current address: School of Medicine, Wayne State University, Detroit, Mich. 48201. 291 Copyright All rights
$2 1976 by Academic Press, Inc. of reproduction in any form reserved.
292
SWISLOCKI,
TIERNEY
as originally described (11). In the original procedure (ll), the heaviest membrane fraction which contains bile canalicular surfaces was harvested and used as the liver plasma membrane preparation, while the lighter two fractions were not utilized. We have separated the three membrane fractions obtained in the gradient by fractionation of the partially purified liver membrane on sucrose layers and investigated several properties of the isolated membrane fractions. All the membrane fractions contained adenylate cyclase activity. As will be shown, the fraction of intermediate density consisting of blood sinusoidal surfaces, was more sensitive to glucagon than the heavier bile canalicular membranes. METHODS Membranes from livers of hypophysectomized rats (Charles River), 145-165 g, were prepared as described in an earlier communication from this laboratory (Ref. (121, Fig. 2). Critical to the isolation of the three membrane fractions was the use of an appropriate Dounce homogenizer. Homogenizers with suitable clearances were selected so that the pestle drop-time in water was 2.0-2.5 s over the 20cm distance of full travel across the shearing surface. Briefly, 0.5 rnM CaCl, was added (13) to the homogenizing medium of 1 mM NaHCO,, to increase yield of membranes, and the procedure of Neville (11) was used to step 11. There, the partially purified membranes, here designated float, were skimmed off the preparative sucrose medium (ll), washed with 1 mM NaHCO,, containing 0.5 mM CaCl,, and centrifuged at 35,000g for 20 min. The pellet was resuspended in 18 ml of the buffer, 6 ml of which was applied to each of the SW 25.1 tubes tilled with sucrose layers made as follows: 50% sucrose, 4.1 ml; 37%, 10 ml; lo%, 10 ml. Centrifugation was at 2000 rpm for 1 h at 5°C in a Beckman Model L-2 ultracentrifuge with the brake off. Three membrane fractions were obtained, at the sucrose interfaces of densities 1.23, 1.16, and 1.03, and harvested by dropwise collection after puncturing the tube. The membrane fractions were characterized by electron microscopy and enzyme assays. For electron microscopy the membranes were washed free of sucrose and pelleted at 35,000g for 20 min. Small pellets of the fractions were fixed for 1 h in cold, phosphate-buffered 1% osmium tetroxide, dehydrated in graded alcohols, and embedded in Spurr’s resin. Thin sections were prepared on a diamond knife, stained with lead citrate, and photographed
AND
ESSNER
with a Siemens 101 electron microscope. Adenylate cyclase was assayed by the method of Krishna et al. (141, and 5’-nucleotidase was assayed according to Song and Bodansky (15). Guanyl nucleotide hydrolysis was determined as described earlier (12, 161. Protein was determined by the Lowry procedure (171. When membranes were solubilized with detergent they were washed with 1 mM NaHCOZI containing 0.5 mM CaCl,, and 1 mg of membrane protein was resuspended per 1 ml of 0.2 M Tris-HCl, pH 7.5, containing 0.25 M sucrose, 1 mM EDTA, and 40 mM Lubrol-PX (molecular weight, 600). This mixture was centrifuged at 165,OOOg for 1 h at 5°C and the supernates containing solubilized adenylate cyclase were removed. RESULTS
As indicated in Table I when partially purified liver membranes, obtained as the float in step 11 of the Neville procedure, were washed by resuspension in buffer and centrifugation at 35,000g for 20 min, 20% of the float protein was not pelleted. The loss was due to protein that remained in the wash supernate. Further fractionation of the washed float was accomplished on sucrose layers, by which procedure three populations of membranes were obtained. The membranes of intermediate density, Peak 2, contained half of the recovered protein with the remainder divided equally between Peaks 1 and 3. The specific activity of 5’-nucleotidase, used as a plasma membrane marker, was similar in Peaks 1 and 2, while Peak 3 membranes TABLE PROTEIN
AND
5’-NUCLEOTIDASE
LIVER
MEMBRANE
Fraction Float Washed float Peak 1 Peak 2 Peak 3
I DISTRIBUTION
IN
FRACTIONS~
Protein (mg)
5’-Nucleotidase
56.1 45.1 7.6 15.3 7.7
0.053 0.727 0.653 0.227
Density
1.23 1.16 1.03
o Membranes were prepared from 20 g of rat liver by the Neville procedure which was carried to step 11 (11). Subsequently, sucrose layers were substituted (Methods) for the sucrose gradient as utilized in the last step of the original procedure. 5’-Nucleotidase activity is in micromoles per milligram of protein per minute.
ADENYLATE
CYCLASE
possessed much lower marker enzyme activity. Electron micrographs of membranes in Peaks 1 and 2 are shown in Fig. 1. Both fractions contain numerous membranous elements. Peak 1 membranes contain bile canaliculi and desmosome-like structures arranged along membranes which are apposed to each other. Peak 2 membranes, on the other hand, showed many separate membrane large vesicles and, notably, structures which contained numerous small vesicles that were probably derived from the sinusoidal faces of hepatocytes. Peak 3 (not shown) contained membranous elements which could not be identified. Adenylate cyclase activity was examined in the three membrane fractions, and the enzyme present in Peak 2 membranes exhibited the highest specific activity (Table II). This has been consistently observed in many membrane preparations although the absolute activity has varied from preparation to preparation. Adenylate cyclase present in all three membrane fractions was sensitive to stimulation by F-. With 10 mM F-, adenylate cyclase in Peaks 1 and 2 was stimulated about sevenfold whereas enzyme activity in Peak 3 membranes was increased but twofold. The three membrane fractions were likewise examined for sensitivity to glucagon and epinephrine (Table III), since both hormones had been demonstrated to increase adenylate cyclase activity in partially purified liver membranes (I, 3, 7). Glucagon stimulated Peak 1 enzyme twofold, Peak 2 enzyme eightfold, and Peak 3 enzyme fivefold. Epinephrine increased enzyme activity by 47% and only in Peak 2 membranes, the only fraction upon which an effect of epinephrine, although small relative to glucagon, was observed. Insulin did not alter adenylate cyclase activity in any of the three membrane fractions. In view of the relative enrichment of adenylate cyclase in Peak 2 and the high sensitivity of Peak 2 membranes to glucagon this membrane fraction and the partially purified membranes from which it was derived were examined for dose-response sensitivity to glucagon. As seen in
FROM
293
LIVER
Fig. 2, adenylate cyclase activity in Peak 2 membranes increased over the range of lo-!’ to 10-j M glucagon. At the high hormone concentration a maximal ninefold stimulation was observed. Higher concentrations of glucagon exerted no further increase in enzyme activity. The partially purified membranes (Fig. 3) were stimulated over threefold by the same concentration of glucagon. When compared to the partially purified membranes, the Peak 2 membranes were three times more sensitive to glucagon. We had previously demonstrated (16) that adenylate cyclase present in solubilized partially purified membranes exhibited sensitivity to guanyl nucleotides that was similar to the membranes from which the enzyme was obtained. Examination of the solubilized membrane fractions indicated (Table IV) that adenylate cyclases present in 165,OOOg supernates of membranes in Peaks 1 and 2 that had been solubilized with Lubrol-PX were equally sensitive to the quanyl nucleotide analogs GMP-PNP and GMP-PCP4 which doubled enzyme activity. The enzyme activity of Peak 3 membranes was inhibited by guanyl nucleotide analogs. As had been observed with the enzyme in the partially purified membranes (16), GMP-PNP was the most potent guanyl nucleotide in stimulating adenylate cyclase activity of solubilized Peak 2 membranes. GTP, however, which stimulated the enzyme in the solubilized float (16), inhibited the activity present in the three solubilized membrane fractions. As we reported earlier (12, 16), partially purified membranes and detergent-solubilized preparations thereof possess nucleotide phosphohydrolases. As seen in Table V, nucleotide hydrolysis of wide specificity is carried out to approximately the same degree in all three membrane fractions. GTP, ITP, and GMP-PCP were good substrates; the GTP analog was slightly more resistant to hydrolysis. Solubilization of the membranes with detergent did not alter the specificity of hydrolysis. ’ Abbreviations used: GMP-PCP, thylene diphosphonate; GMP-PNP, dophosphate.
5’-guanylylme5’-guanylylimi-
294
SWISLOCKI,
TIERNEY
AND
ESSNER
FIG. 1. (A), Electron micrograph of membranes in Peak 1. Numerous desmosome-like structures are evident. Arrows indicate two bile canaliculi. tron micrograph of membranes in Peak 2. Large vesicular and membranous dent, some of which contain many attached, small vesicles (arrow). x6400.
Sensitivity of the adenylate cyclase in Peak 2 membranes to Lubrol-PX was examined since this detergent was reported by B&r and Kulshrestha (18) to increase
membranes and x4800. (B), Elecelements are evi-
the activity of the enzyme in membranes of Ehrlich ascites cells. As seen in Table VI Lubrol-PX at lo-” to 10e4 M had no effect on the enzyme activity. The detergent at
ADENYLATE
CYCLASE TABLE
ADENYLATE
CYCLASE
DISTRIBUTION
AND _____
FLUORIDE
FROM II SENSITIVITY
IN
Membrane
Additions 1
None 10 mM
LIVER
MEMBRANE
peaks
FRACTIONS” ______
_.
_---
2
3
______-.-_~-___-231.3 t 28.1 1670.0 2 21.7
69.5 f 2.4 457.4 2 40.1
NaF
295
LIVER
195.9 438.0
+ 31.0 k 27.0 ---
a Membrane fractions were prepared and washed as described in the footnote to Table I. Adenylate cyclase activity is expressed in picomoles of cyclic AMP per milligram of protein per 10 min and was determined in triplicate with 37 kg of membrane protein in 0.32 rnM ATP in 0.1 ml of 50 mM Tris-HCl, pH 7.4, with 2 rnM EDTA and 10 mM MgC12, with 2-5 &i of I w:‘~P]ATP (16). TABLE HORMONE
SENSITIVITY
OF LIVER
(1 Membrane respectively.
MEMBRANE
FRACTIONS”
Membrane
Hormone
None Glucagon Epinephrine Insulin
III
~-___ 83.6 188.9 68.3 53.5
1 t i i +
_.--.-__-__ 15.5 14.4 10.6 16.6
FIG. 2. Dose-response of Peak 2 membranes to glucagon. Peak 2 membranes were isolated as described in Methods. These membranes were resuspended in homogenizing medium, washed free of sucrose prior to assay, and assayed as indicated in the footnote to Table II. ATP concentrations were 3.2 mM.
2
125.3 f 992.7 i 177.2 i 135.2 2
fractions were prepared and assayed as described Hormone concentrations were 1 pg/O. 1 ml of medium.
---
peaks 3 15.1 82.7 13.3 20.5
69.5 376.7 77.8 82.4
in the
footnotes
? i i ?z
to Tables
13.1 7.2 16.8 21.9 ____ I and
II,
FIG. 3. Dose-response of partially purified liver plasma membranes to glucagon. Membranes were prepared by the Neville procedure carried to Step 11 (111, washed free of sucrose, and resuspended in homogenizing medium prior to assay.
DISCUSSION
lo-” M decreased enzyme activity by 72%. The prostaglandin antagonist 7-oxa-13prostynoic acid which has detergent-like properties in human erythrocytes (19) increased enzyme activity at lo-” M but at the other concentrations examined had an inhibitory effect.
The membranes isolated in the Peak 2 fraction possess the properties of blood sinusoidal surfaces of hepatocytes. They have been enriched in 5’-nucleotidase activity, a marker enzyme for plasma membranes, and appear on electron microscopy as large free vesicles and as small vesicles attached
296
SWISLOCKI,
TIERNEY TABLE
GUANYL
NUCLEOTIDE
SENSITIVITY
Nucleotide
0~165,000g
AND
ESSNER
IV
SUPERNATES LUBROL-PX"
OF MEMBRANE
Float
Membrane 1
None GMP-PNP GMP-PCP GTP GDP
124.1 248.9 239.6 156.8 80.6
? i k r e
FRACTIONS
16.0 11.9 34.7 15.7 3.2
48.2 89.5 36.9 34.7 52.3
SOLUBILIZED
peaks 2
+ 1.7 i 10.7 k 13.9 i 6.4 k 19.9
53.9 127.3 93.6 28.1 52.3
WITH
-c 2 k r 2
3 8.4 11.2 13.2 15.4 14.0
30.4 17.6 15.9 4.7 8.7
+ 5.1 i 12.4 + 7.1 2 5.0 2 5.7
u Membrane fractions were prepared and assayed as described in the footnotes to Tables I and II. Guanyl nucleotide concentrations were 0.1 mM. Membranes were solubilized by resuspending the membrane pellets in 0.2 M Tris-HCl, pH 7.5, containing 40 mM Lubrol-PX, 0.25 M sucrose, 1 mM EDTA, and were centrifuged at 165,OOOg for 1 h. The supernates were used as the source of the enzyme. TABLE
V
TABLE
NUCLEOTIDE HYDROLYSIS BY MEMBRANES AND BY 165,OOOg SUPERNATES OF MEMBRANES SOLUBILIZED WITH LUBROL-PX"
Peak
1
Peak
2
Peak
3
VI
OF PEAK 2 MEMBRANES AND 7-OXA-%PROSTYNOIC
Concentration
Lubrol-PX
GTP
ITP
GMPPCP
202.8 189.5 214.9 179.5 171.4 72.8
192.2 158.7 212.4 138.5 146.5 48.2
141.7 123.8 158.0 123.0 109.9 88.1
n Nucleotide hydrolysis was determined as described earlier (12, 16) by incubation of 50 /*g of protein in 0.1 ml of 50 mM Tris-HCl and 2 mM MgCl,, pH 7.4. Nucleotide concentrations were 0.1 mM to which 0.1 FCi of ‘%-labeled nucleotide was added. After incubation for 10 min at 3o”C, the reaction mixtures were boiled for 3 min and applied to PEI-cellulose sheets for chromatographic separation of hydrolytic products in LiCl (PEI, polyethyleneimine). Appropriate blanks and standards were run with each assay. Lanes corresponding to reactions were cut into l-cm segments and radioactivity thereon measured by liquid scintillation spectrophotometry. Results are expressed in picomoles of nucleotide hydrolyzed per microgram of protein per 10 min. to membrane sheets. A noteworthy feature of this fraction is the absence of bile canaliculi which were found in the denser Peak 1 membranes. Significantly, the Peak 2 membranes were enriched in adenylate cyclase activity that was three times more sensitive to glucagon than the partially purified membranes from which they were
lo-” 1 o-:, 10 4 lo- :I None
182.0 174.3 209.6 57.1 204.8
2 * k f k
TO LUBROL-PX ACID" 7-Oxa-13-prostynoic acid
(M)
Substrates
Peak 1 Solubilized Peak 2 Solubilized Peak 3 Solubilized
SENSITIVITY
10.6 19.1 13.6 8.9 40.3
173.8 135.5 323.6 134.5
+ * -+ i-
29.9 17.9 57.2 50.8
’ Peak membranes were prepared and assayed as described in the footnote to Table II. Lubrol-PX and 7-oxa-13-prostynoic acid were added just prior to assay for adenylate cyclase.
derived. The blood sinusoidal fraction was also three times more sensitive to glucagon than the bile canalicular surfaces from which they were separated. The Peak 2 membranes, as isolated and characterized here, have several features in common with a membrane fraction obtained by Wisher and Evans (20) who reported, after these studies were completed, the isolation of several types of hepatocyte membranes of which one fraction, called the microsomal-light membranes (M-L), possessed properties that were attributed to blood sinusoidal surfaces. Both of these membrane preparations (Peak 2 and the M-L fraction of Wisher and Evans) had a density of 1.16, appeared as vesicles on tissue disruption, were the predominant membrane fraction obtained, were more sensitive to glucagon than other fractions, and, significantly, possessed adenylate cyclase activity that was similarly stimu-
ADENYLATE
CYCLASE
lated (ninefold) by maximal concentrations of glucagon. It should be expected that cell surface receptors in liver for blood-borne regulators like glucagon would be located on the vascular side of the hepatocyte. Thus the lower glucagon sensitivity of the membrane fraction with bile canalicular surfaces (Peak 1) relative to the blood sinusoidal membranes (Peak 2) would be expected. These findings explain, in part, the observations of Pohl et al. (6) that fully purified liver plasma membranes isolated by the Neville procedure (ll), which contain bile canaliculi, were less sensitive to glucagon than the parent partially purified membranes. On the basis of the present data it can be seen that the partially purified membranes contained the glucagon-sensitive membranes which, however, were discarded in the final purification step (11). The early observation (6) that full membrane purification by the Neville procedure resulted in a decline in glucagon sensitivity led investigators to utilize the partially purified membrane preparation to study adenylate cyclase activity as a consequence of glucagon-membrane interactions (1, 3, 6-10). As reported here, a simple further fractionation of the partially purified membranes yields a membrane fraction which is more appropriate to the study of hormone-membrane interactions. Adenylate cyclase activity in membranes isolated from many sources is modulated by guanyl nucleotides (see Ref. (16)). In a previous communication (16) we presented evidence that solubilized adenylate cyclase obtained as a 165,000g supernate of detergent-treated membranes possessed the same properties of activation by guanyl nucleotides as did the parent partially purified membranes. On the basis of those findings we suggested that the guanyl nucleotide site resided on the soluble adenylate cyclase and that the structural integrity of the liver plasma membrane was not an obligatory requirement for the guanyl nucleotide effects on adenylate cyclase activity. These findings have been presently extended to adenylate
FROM
297
LIVER
cyclase solubilized from either bile canaliculi or blood sinusoidal membranes. REFERENCES 1. RODBELL, M., BIRNBAUMER, 1861-1871. 2. JOHNSON, A., JR.
mun.
L.
C. B., BLECHER, (1972)
M.,
AND Biophys.
Biochem.
N.
GIORCIO,
Res.
Com-
46. 1035-1041.
3. BIRNBAUMER, 300, 129-158. 4. RUBIN, BERG,
H. M. J., POHL, S. L., AND (1971) J. Biol. Chem. 246,
KRANS,
L.
M.
S.,
M.
(1973)
(1973)
Biochzm.
Acta
Biophys.
N. I., AND SONENBiochem. Biophys. 157,
SWISLOCKI,
Arch.
243-251. N.
5. SWISLOCKI,
I.,
M.,
SONENBERG,
POSTEL-VI-
NAY, M-C., GOIDL, J. A., ROBERTS, DERSON, G., PRIDDLE, M., AND
J. E., HENTIERNEY, J.
(1973) in Advances in Human Growth Hormone Research: A Symposium, Publ. No. NIH-74-612, p. 588, U.S. Government Printing Office, Baltimore. 6.
POHL, S. L., BIRNBAUMER, (1971) J. Bid. Chem.
L.,
AND
RODBELL,
M.
246, 184991856. A. M.. PERRENOUD, M. J. (1973) Eur. J. Biochrm.
13.
F., CHAMBAUT, L., AND HANOUNE, 38, 185-192. RODBELL, M., BIRNBAUMER, L.. POHL, S. L., AND KRANS, H. M. J. (1971) J. Bid. Chem. 246, 1877-1882. RODBELL, M., LIN, M. C., AND SALOMON, Y. (1974) J. Bid. Chern. 249, 59-65. RODBELL, M., KRANS, H. M. J., POHL, S. L., AND BIRNBAUMER, L. (1971) J. Bid Chem. 246, 187221876. NEVILLE, D. M., JR. (1968) Biochem. Biophys. Acta 154, 540-552. SWISLOCKI. N. I., SCHEINBERG, S., AND SONENBERG, M. (1973) Biocheru. Biophys. Res. Cornmun. 52. 313-319. RAY, T. K. (1970)Biochim. Biophys. Actn 196, l-
14.
KRISHNA,
15.
(1968) -J. Pharmncol. Exp. SONG. C. S., AND BODANSKY,
7.
8.
9.
10.
11. 12.
LERAY.
9.
G.,
WEISS,
B.,
AND
BRODIE,
B.
163, (1967)
379-385. J. Blol.
Thu.
B.
0. 242, 694-699. SWISLOCKI. N. I.. AND TIERNEY, J. (1975) Arch. Biochem. Biophys. 168, 455-462. LOWRY, 0. H., ROSEBROUCH. N. J., FARR, A. L., AND RANDALL, R. J. (195115. Bid. Chem. 193, Chem.
16. 17.
18.
265-275. BAR, H. P., AND
J. B&hem. 19.
20.
SWISLOCKI, s. (1974) WISHER, them.
M. J.
KULSHRESTHA,
S. (1975)
53. 472-474. N. I., TIERNEY,
J., AND
Prostngzandin.s
7. 401-410.
H., 116.
AND EVANS, 375-388.
W.
Canad.
RITTERSTEIN. H.
(1975)
Bio-