ABCHIVES
OF BIOCHEMISTRY
The Binding
AND
BIOPHYSICS
175,
221-228 (19761
of Cyclic Nucleotides to Membranes of the Endo@asmic Reticulum of Normal and Neoplastic Rat Liver-l
RAMESHWAR
K. SHARMA*, CHARLES A. MCLAUGHLIN3, AND HENRY C. PITOT
Departments of Oncology and Pathology, The McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706 Received October 28, 1975 Studies are presented which demonstrate that the smooth and rough endoplasmic membranes of normal and neoplastic rat liver possess binding sites for cyclic nucleotides exhibiting a high degree of specificity. In contrast to normal liver, which has only a single binding site for cyclic AMP on membranes of the endoplasmic reticulum, cyclic AMP binding to the intracellular membranes of hepatoma 5123% and 7777 exhibits two apparent binding sites. The binding constant for cyclic AMP of one site on the tumor membranes is comparable to that of the normal liver, whereas the value of the second intrinsic association constant is 4- to IO-fold greater than liver. The possibility that the presence of the second cyclic AMP binding site might be a function of the rapid growth of the tumors was unlikely since membrane preparations from neonatal rats showed a single affinity association constant which was similar to that of normal liver. In addition, membranes of the endoplasmic reticulum of the Morris hepatomas 5123C and 7777 exhibit a binding site for cyclic GMP which is absent from the intracellular membranes of liver.
Since the discovery of 3’,5’-cyclic AMP in vertebrate tissues by Sutherland and his associates (1) the occurrence and metabolic functions of this and other cyclic nucleotides have been shown to be both widespread and essential for the normal functioning of prokaryotic (2,3) and eukaryotic (4-6) cells. Within the past decade studies from several laboratories (7-10) have implicated cyclic nucleotides as potentially important in the neoplastic transforma-
tion as well as in the maintenance of the malignant state itself. These studies have involved not only the intracellular concentrations of cyclic nucleotides including cyclic AMP5 (ll), cyclic GMP (12), and more recently cyclic CMP (131, but also measurements of adenylate (14) and guanylate (15) cyclases in neoplastic tissues. With the elucidation of the specific role of cyclic AMP in activating intracellular protein kinases, studies of both cyclic nucleotide-dependent and -independent protein kinases within neoplastic cells have been forthcoming (161. The correlation of measurements of cyclic nucleotide levels, cyclases and ki-
i This work was supported in part by grants from the National Cancer Institute (CA-071751 and the American Cancer Society (E-588). 2 Visiting Professor and Special Fellow of the National Cancer Institute (CA-55861). Present address; 5 Abbreviations used: Cyclic AMP, cyclic adenoDepartment of Biochemistry, University of Tennessee Health Center, Memphis, Tennessee 38163. sine 3’,5’-monophosphate; cyclic GMP, cyclic guano3 Postdoctoral Fellow in Biochemical Pathology of sine 3’,5’-monophosphate; cyclic IMP, cyclic inosine the National Institute of General Medical Science 3’,5’-monophosphate; cyclic CMP, cyclic cytidine cyclic UMP, cyclic uridine (GM-47855). Present address: National Institute of 3’5’-monophosphate; 3’,5’-monophosphate; STKM buffer, 0.25 M sucrose Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana 59840. in 0.05 M Tris-HCl, pH 7.4, 0.025 M KCl, and 0.01 M 4 To whom reprint requests should be addressed. MgCI,. 221 Copyright 8 1976 by Academic press, Inc. All rights of reproduction in any forin reserved.
222
SHARMA,
MCLAUGHLIN
nase activities with other biochemical characteristics of the neoplastic cell has been accomplished to the greatest extent with cells transformed in vitro (7,8). Studies concerned with levels of cyclic nucleotides and the effects of exogenous cyclic AMP on morphology and membrane structure (17) as well as proliferation (18) have been reported. On the other hand, investigations related directly to the functional receptor of cyclic AMP and cyclic GMP, i.e., the regulatory subunit of protein kinase (19) in neoplastic cells, have been relatively infrequent. The studies described in this paper may be related to this latter subject and demonstrate that specific differences in the cyclic nucleotide receptor proteins localized within intracellular membranes have been found between normal and neoplastic hepatic tissues. MATERIALS
AND METHODS
Materials. Cyclic [VH]AMP and cyclic [S3H]GMP were purchased from Amersham Searle, Chicago. Nonradioactive nucleotides were obtained from Sigma Chemical Company. All the other chemicals were reagent grade and were obtained commercially. Animal preparations. Male albino Sprague-Dawley (Badger Rat Company, Madison, Wisconsin) and Buffalo strain (Simonsen Laboratories, Great Bear Lake, Minnesota) rats were used in the investigations reported in this paper. The Morris hepatocellular carcinomas were transplanted into But&lo strain rats with sterile technique. The tumors were harvested from the hind legs, where they had been inoculated 4 to 8 weeks earlier. All animals were maintained on a Purina laboratory chow diet fed ad Zibitum. The rats, both normal and tumor-bearing were kept in rooms equipped for automatic light and dark exposure of 12-h periods each. Animals were sacrificed by decapitation, and the livers, as well as the tumors where present, were removed as rapidly as possible and immersed in icecold STKM buffer (0.25 M sucrose in 0.05 M TrisHCl, pH 7.4, 0.025 M KCl, and 0.001 M MgCl,). Muscle, connective tissue, and necrotic debris were dissected from the viable tumor tissue prior to homogenization. All tissues were homogenized in STKM buffer and the smooth and rough endoplasmic reticulum from both liver and hepatoma were isolated as previously described (20). A discontinuous gradient containing 20 ml of the postmitochondrial fraction of liver or tumor in 1.3 M STKM (1.3 M sucrose used in place of 0.25 M sucrose) layered over 8 ml of 2.0 M STKM and itself overlaid with 5 ml of
AND PITOT
STKM was prepared and centrifuged in a Beckman 60 Ti rotor at 214,OOOgfor 3 h. The smooth endoplasmic reticulum at the 0.44-1.3 M sucrose interface as well as the rough endoplasmic reticulum at the l.l2.0 M sucrose interface were collected, diluted in STKM, and repelleted in a Beckman 30 rotor at 78,500g for 30 min. The pellets were rinsed and homogenized and the suspension was layered on 0.44 M STKM and sedimented again in a 30 rotor at 7500%. The membrane pellets were then rinsed, homogenized in 20 mM Tris-HCl, pH 7.4, containing 5 mM P-mercaptoethanol, and used immediately or stored at -60°C. Electron microscopic examination of the membrane fractions by techniques previously described (20) confirmed the morphologic characterization of each of the fractions. Cyclic nucleotide binding assays. The assay for cyclic AMP-binding and cyclic GMP-binding protein was essentially that described by Gilman (21). The incubation mixture at 0°C contained 50 mM Naacetate, pH 4.0, 20 mM EDTA, and 0.73 pmol of cyclic [3H]AMP (sp act 27.5 Ci/mmol) or 0.3 pmol of cyclic [3H]GMP (sp act 15 Cilmmol) in a final volume of 0.1 ml (22, 23). The reaction was started by the addition of 100 pg of membrane preparation. At the end of the incubation, 2 ml of a 20 mM phosphate buffer, pH 6.0, was added and the diluted reaction mixture was immediately filtered through a Millipore disk (2.5-cm diameter with 0.45~pm pore size). Each tube was rinsed once with 2 ml of phosphate buffer and 2 ml of 10 mM MgCl,. The filter was dried at 90°C and then counted in 10 ml of Scintisol-complete (Isolab, Inc., Akron, Ohio). A tube containing the complete reaction mixture, but without any membrane protein, served as a blank, and all data were corrected for this value. In those experiments designed to study the competition between various cyclic nucleotides and their binding to membranes of the endoplasmic reticulum, the same assay was utilized with the addition of an excess amount of nonradioactive nucleotides as competitors; the concentrations of these are given in each figure. The plots in Figs. l-6 are representative experiments taken from four to seven separate analyses. RESULTS
Previous studies from this laboratory (24) demonstrated the characteristics of the binding of cyclic AMP to membranes of the smooth endoplasmic reticulum of hepatic tissue. Figure 1 is a Scatchard plot of the binding of cyclic AMP to membranes of the rough endoplasmic reticulum isolated from rat liver. As can be seen from the figure, the plot of binding affinity demonstrates a single binding site with a disso-
CYCLIC w g
m;::f
NUCLEOTIDE
BINDING
I 010
t
o;.
o;;\
,
0.30
040 B/F
0.50
060
0.70
FIG. 1. Binding afBnity of cyclic AMP to the receptor protein of the rough endoplasmic reticulum of the liver. The amount of cyclic AMP is plotted as a function of the ratio of the concentration of bound to free cyclic nucleotide. The concentration of free cyclic 13H1AMP was obtained by subtracting the cyclic 13H1AMP bound from the total cyclic [3H]AMP concentration initially added to the incubation mixture. Duplicate assays were performed with 100 pg of binding protein in the incubation medium, as mentioned in the text. To evaluate K the molar concentration was determined from the total amount of ligand bound in 0.1 ml of incubation mixture. A single binding fraction follows the equation, amount bound = number of sites - K, (bound/ free).
ciation constant of 0.16 x lo-* M. This intrinsic dissociation constant is quite comparable to that reported by us previously for the binding affinity of cyclic AMP to membranes of the hepatic smooth endoplasmic reticulum (24). Therefore, the intrinsic association constant of cyclic AMP for both the smooth and rough endoplasmic reticulum is quite comparable to that seen for regulatory proteins which bind cyclic AMP under other circumstances and in other parts of the cell (25). However, when one determines the concentration of binding sites of cyclic AMP on each of these membrane fractions, a significant difference is seen (Table I). The concentration of binding sites of the smooth endoplasmic reticulum is about 55% more than the number of sites for this cyclic nucleotide on the rough endoplasmic reticulum. The values (average 2 SEM of seven determinations) for smooth and rough membranes shown in Table I differ quite significantly from each other (P < 0.005). In preliminary investigations we have also found that the concentration
TO ENDOPLASMIC
223
RETICULUM
of binding sites for cyclic AMP on a subfraction of the smooth endoplasmic reticulum, probably the Golgi fraction (201, is also significantly lower than the concentration of cyclic AMP binding sites seen on the smooth endoplasmic reticulum. A very important aspect of the binding sites for cyclic AMP on the smooth and rough endoplasmic reticulum is their specificity. The data in Tables II and III clearly demonstrate that of all the cyclic nucleotides and other nucleotides tested (Table II) only cyclic IMP as well as cyclic AMP itself could compete successfully for the TABLE
I
BINDING OF CYCLIC AMP TO THE RECEITOR PROTEINS OF SMOOTH AND ROUGH ENDOPLASMIC RETICULUM OF LIVERY
Membrane Smooth Rough SERRER
fraction
Binding
of CAMP (pmol/ me9 1.330 + 0.035
0.853
ratio
* 0.079 1.55
a Duplicate assays were performed on seven sepa-
rate samples with 100 pg of binding protein in the incubation medium which contained 0.73 pmol cyclic 13HlAMP (44,000 dpm) in 0.1 ml of 50 mM sodium acetate buffer (pH 4.0). See text for further details. TABLE
II
THE EFFECT OF NUCLEOTIDES ON THE BINDING CYCLIC t3H]AMP TO THE ROUGH END~PLASMIC RETICULUM OF LIVERY
Nucleotides added None Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic AMP GTP ADP ATP
Nucleotide concentration (fold excess)
OF
l$&;P bound (46) 100
AMP GMP GMP GMP IMP CMP UMP
500 250 500
33 120 117
1000 136 1000 30 1000 118 1000 108 1000 135 1000 129 1000 119 1000 129 ’ The reaction mixture contained 50 mM sodium acetate buffer (pH 4.01, 0.73 pmol cyclic r3HlAMP (44,000 dpm), 100 pg of membrane preparation, and unlabeled nucleotide as indicated. The values presented are representative of at least four experiments.
224
SHARMA,
MCLAUGHLIN
TABLE III EFFECT OF VARIOUS CONCENTRATIONS OF CYCLIC GMP ON THE BINDING OF CYCLIC [3H]AMP T O THE SIUOOTH END~FXASMIC RETICULUM OF LIVE@ NucleoNucleotide Cyclic tides concentration 13H]AMP added (fold excess) bound (%) None 100 Cyclic AMP 200 11 Cyclic AMP 400 9 Cyclic AMP 800 6 Cyclic GMP 200 99 Cyclic GMP 400 67 Cyclic GMP 800 72 (2The conditions and numbers of the experiments are similar to those depicted in Table II.
binding of the cyclic nucleotide to the membranes of the rough endoplasmic reticulum. Those values greater than 100% indicate that no competition for the binding of cyclic 13HlAMP had occurred. Equally important is the fact that cyclic GMP even in lOOO-fold excess over the labeled cyclic AMP had no competing effect on the binding of this cyclic nucleotide to membranes of the rough endoplasmic reticulum. In Table III a related study of the competition of cyclic AMP and cyclic GMP for the binding of radioactive cyclic AMP to membranes of the smooth endoplasmic reticulum is seen. In this instance some competition for the cyclic AMP binding site at very high levels of cyclic GMP was elicited although at the present time we do not feel that these values represent significant competition for the binding sites but rather that cyclic GMP does not compete with cyclic AMP for binding to the endoplasmic reticulum. Furthermore, in other studies, utilizing the cyclic nucleotides shown in Table II, no significant competition for the binding of cyclic 13HlAMP to membranes of the smooth endoplasmic reticulum was exhibited by other nucleotides with the exception of cyclic IMP, which did compete to the same levels seen for the rough endoplasmic reticulum in Table II. Therefore, in both the smooth and rough endoplasmic reticulum the binding sites for cyclic nucleotides exhibit a high degree of specificity for cyclic AMP. In order to determine whether the cyclic AMP binding sites of the endoplasmic re-
AND PITGT
d & CYCLIC
2 4 AMP (pmoler)
FIG. 2. Binding affinity of CAMP to the receptor protein of rough endoplasmic reticulum preparations which have been treated with varying concentrations of KC1 + 1 mre puromycin. Duplicate assays were performed with 100 kg of binding protein in the incubation medium with varying concentrations of exogenously added nonradioactive cyclic AMP in 0.1 ml of 50 mM sodium acetate buffer (pH 4.0).
ticulum were loosely or firmly bound to the surface of this organelle, the experiment demonstrated in Fig. 2 was performed. In this instance the association of cyclic 13HlAMP to membranes of the endoplasmic reticulum was studied as a function of the concentration of exogenously added nonradioactive cyclic AMP. The competition curve noted is not affected by extremely high ionic strength which is capable of dissociating many protein molecules which are loosely bound to the surface of the endoplasmic reticulum. Puromycin was also added in these experiments in order to release some growing polypeptide chains and ribosomal subunits present on the surface of the endoplasmic reticulum (26). As can be seen from the figure none of these treatments affected the binding of cyclic AMP to membranes of the smooth endoplasmic reticulum. Similar results were obtained when the rough endoplasmic reticulum fraction was substituted for the smooth fraction. This therefore indicates that the binding sites on the membrane are relatively tightly associated with the membrane structure and cannot be removed by high salt treatment. Cyclic nucleotide binding to hepatoma membranes of the endoplasmic reticulum.
Figures 3 and 4 show the Scatchard plot of the binding of cyclic AMP to the smooth endoplasmic reticulum of Morris hepato-
CYCLIC
NUCLEOTIDE
BINDING
TO ENDOPLASMIC
225
RETICULUM
FIG. 3. Binding affinity of cyclic AMP to the receptor protein of the smooth endoplasmic reticulum of the Morris hepatoma 5123C. The conditions of the experiments are identical to those depicted in Fig. 1. s : P 0 x 4 s m
*
i ;j
1.5
(I) (il)
IO
0.5
\
,k
0 = s
0
I
\
\
\
Kd=1.16X16% K,,=3.03X
Id%,
\
2
4
5
6
S3F
FIG. 4. Binding affinity of cyclic AMP to the receptor protein of the smooth endoplasmic reticulum of the Morris hepatoma 7777. The conditions of the experiments are identical to those depicted in Fig. 1.
mas 5123C and 7777. As can be seen in contrast to the characteristics of the cyclic AMP binding to smooth and rough endoplasmic reticulum in liver, two binding affinities are apparent, each site exhibiting a significantly different affinity for the cyclic nucleotide. The dissociation constant for the steeper portions of the curves shown in Figs. 3 and 4 are 1.04 and 1.16 x 1O-8 M, respectively. The values for the shallow lines are 0.23 and 0.03 x lo-* M, respectively, for the binding of cyclic AMP to the smooth endoplasmic reticulum of these two neoplasms. In each case, the slopes of the two portions of the curve differ by a factor between 4 and 40. The values seen for CAMP binding affinity to the rough endoplasmic reticulum of the liver is comparable to the smaller value in each of the neoplasms (Fig. 1). The specificity of the cyclic AMP binding site of the smooth endoplasmic reticulum of hepatoma 5123C can be seen in
Table IV. The competition characteristics are quite similar to those exhibited for the smooth and rough endoplasmic reticulum of normal liver as demonstrated in Tables II and III. Only cyclic IMP competes significantly with the membrane binding sites of the tumor. Very high levels of cyclic GMP, cyclic UMP, and cyclic CMP again exhibit a very small degree of competition, which we do not feel is significant and in fact may be due to contaminants within these preparations. However, further studies of the specificity of the binding site for cyclic nucleotides of the smooth endoplasmic reticulum of liver and hepatoma led to the findings depicted in Fig. 5. In this figure the binding of cyclic [3H]GMP TABLE
IV
THE EFFECT
OF NUCLWTIDES ON THE BINDING CYCLIC lSHIAMP TO THE SMOOTH END~~LASMIC RETICULUM OF HEPA-IOMA 51230 NE?-
added
None Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic AMP GTP ADP ATP
Nucleotide concentration (fold excess)
-
OF
&gP bound (81
100 20 11 7 96 97 79 16 76 80 91 91 100 101 a The conditions and numbers of the experiments are similar to those depicted in Table II. AMP AMP AMP GMP GMP GMP IMP UMP CMP
250 596 1096 250 500 1000 500 500 500 500 500 596 500
226 “6 x z05
6
SHARMA,
MCLAUGHLIN
I
bf
;5
50 CYCLIC
I I1 20.0 I50 250 GMP (pmoles) IO.0
FIG. 5. Binding affinity of cyclic GMP to the receptor protein of the smooth endoplasmic reticulum of the liver and Morris hepatoma 5123C. Duplicate assays were performed with 100 pg of membrane preparation, 3 pmol of lSH]cyclic GMP, and various amounts of cyclic GMP in 0.1 ml of 50 mM sodium acetate buffer (pH 4.0).
to the smooth endoplasmic reticulum of liver and hepatoma 5123C in the presence of various concentrations of nonradioactive cyclic GMP is depicted. As can be seen, there is essentially no significant binding of the radioactive cyclic GMP itself or diluted in the presence of cold carrier to the liver membrane preparation. On the other hand, quite significant binding of this labeled cyclic nucleotide occurs to the smooth endoplasmic reticulum of hepatoma 5123C which is successfully competed for by the addition of nonradioactive cyclic GMP. These data, taken in the context of those presented in Table IV, argue that the smooth endoplasmic reticulum of this neoplasm exhibits binding sites for cyclic GMP which may be separate from the cyclic AMP binding sites seen both in the smooth endoplasmic reticulum of liver and hepatoma (Figs. 1 and 3, Tables II and IV). Further studies have also demonstrated that the smooth endoplasmic reticulum of hepatoma 7777 exhibits the cyclic GMP binding site just as do those membranes of the hepatoma 5123C. Furthermore, the specificity of the cyclic AMP binding sites of the smooth endoplasmic reticulum in hepatomas 7800 and 7777 cannot be significantly competed for by high levels of cyclic GMP (data not shown). Cyclic AMP binding of membranes of neonatal liver. As has been discussed by
several authors previously (27, 28), the rate of growth of neoplastic tissue may be responsible for some of the peculiar bio-
AND PITGT
chemical characteristics found in such tissues when they are compared with their tissue of origin. In addition, the liver offers a further significant problem in that normal adult liver is predominantly tetraploid (29) whereas most of the neoplasms studied are diploid or near diploid (30). Therefore, we felt it important to determine the characteristics of cyclic AMP binding to membranes of neonatal rat liver which exhibits a rate of growth comparable to the neoplasms in which the binding of cyclic AMP has been investigated in this paper and also is diploid in karyotype (29). In Fig. 6, a Scatchard plot of the binding affinity of cyclic AMP to the receptor proteins of the smooth endoplasmic reticulum of a neonatal rat 7 days of age is seen. The dissociation constant from this curve is 0.16 x lOUs M, or essentially identical with that value seen for the endoplasmic reticulum in liver. Therefore, it would appear that the peculiar binding characteristics of cyclic AMP to the endoplasmic reticulum of several hepatomas investigated in this study are not directly related to the rate of growth or the karyotypic changes seen in these neoplasms. We do not yet know whether cGMP binds to membranes from neonatal liver. DISCUSSION
The major action of cyclic AMP and, more recently, cyclic GMP in binding to regulatory sites of protein kinases in eukaryotic cells is now well known from a
FIG. 6. Binding affinity of cyclic AMP to the receptor protein of the smooth endoplasmic reticulum of the liver from neonate rats. The conditions of the experiments are identical to those depicted in Fig. 1.
CYCLIC
NUCLEOTIDE
BINDING
number of studies. By means of an investigation of the affinity of cyclic nucleotides for individual proteins as well as subcellular organelles within cells, one may infer until proven otherwise the localization of the regulatory subunits of protein kinases which mediate the cyclic nucleotide activation of these enzymes within the cell. The studies presented in this paper are directed toward the localization and specificity of cyclic nucleotide binding proteins within the subcellular organelles of both normal and neoplastic hepatic tissue. Although there have not been many investigations on the localization of the protein kinase regulatory subunit or cyclic nucleotide binding proteins in neoplasms, some investigations of the proteins of the cytosol of hepatomas in uiuo and in vitro have been described. MacKenzie and Stellwagen (31) have described the absence of a cyclic AMP-binding fraction in an established hepatoma cell line (HTC cells) although these cells do exhibit several forms of protein kinase. Granner (32) has extended these investigations to include two other hepatoma-derived cultured cell lines. He demonstrated that the binding of cyclic AMP to soluble cytosol proteins is highest in liver and then decreases in the three neoplasms he studied. Golberg et al. (33) demonstrated that in relatively slowly growing hepatocellular carcinomas including one in this study, the Morris 7800, the binding of cyclic AMP was significantly lower in the “cytoplasm” of the tumor when compared with liver. While these authors did not strictly define the “cytoplasm” preparations used, the reference cited indicates that the preparations consisted of soluble cytosolic proteins. Furthermore, no kinetic evidence regarding the specificity and nature of the binding sites for cyclic nucleotides was presented. On the other hand, two rapidly growing neoplasms exhibited cyclic AMP binding capacity equal to that of liver. The binding of cyclic GMP was also studied and was found to exhibit the same characteristics in the rapidly growing tumors but showed some variation in the slowly growing neoplasms. The binding of cyclic nucleotides to membranes of the endoplasmic reticulum
TO
ENDOPLASMIC
RETICULUM
227
suggests that these intracellular organelles may exhibit protein kinase activity. A recent paper by Jergil and Ohlsson (34) described both the phosphorylation and dephosphorylation of protein by the smooth and rough endoplasmic reticulum of rat liver. These authors determined the binding of cyclic AMP per milligram of protein of the smooth and rough endoplasmic reticulum and showed the concentration of binding sites to the former to be more than twice that of the latter. This value is somewhat higher than described in this study (Table I) but in general is comparable to the findings reported herein. At this time it is not possible to relate the findings described in this paper to the regulation of genetic expression in normal hepatic cells and its abnormalities seen in hepatomas (35). On the other hand, there is considerable evidence accumulating to show that cyclic AMP binding proteins occur within membrane components of the cell (36,371 and in fact may be involved in the regulation of surface phenomena of both the normal and neoplastic cell (38). The mediation of cell surface changes by cyclic nucleotide-regulated functions such as protein kinase is clearly one of a number of possible mechanisms mediating at least some of these effects. Preliminary investigations in this laboratory have demonstrated both endogenous and exogenous phosphorylation by membranes of the endoplasmic reticulum, the products of this phosphorylation within the membrane being different in the normal as compared with the neoplastic hepatic cell. Furthermore, the potential role of the membrane of the endoplasmic reticulum in the regulation of genetic expression as postulated earlier (39) suggests a possible common thread of explanation for the findings described in this paper and the known functions of cyclic nucleotides in the regulation of genetic expression. REFERENCES 1. SUTHERLAND, E. W., AND RALL, T. W. (1958) J. Biol. Chem. 232, 1077-1092. 2. PASTAN, I., AND PERLMAN, R. L. (1966) Proc. Nat. Acad. Sci. USA 61, 1336-1342.
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3. CHALCOTT, P. H., MONTAGUE, W., AND Posp GATE, J. R. (1972) J. Gen. Microbial. 73, 197200. 4. RASMUSSEN, H. (1970) Science 170, 404-412. 5. LIDDLE, G. W., AND HARDMAN, J. G. (1971) New Engl. J. Med. 285, 560-566. 6. HARDMAN, J. G., ROBISON, G. A., AND SUTHERLAND, E. W. (1971)Ann. Rev. Physiol. 33,311336. 7. PEERY, C. V., JOHNSON, G. S., AND PASTAN, I. (1971) J. Biol. Chem. 246, 5785-5790. 8. SHEPPARD, J. R. (lS72)Nuture New Biol. 236,1416. 9. CHO-CHUNG, Y. S., AND GULLINO, P. M. (1974) Science 183, 87-88. 10. BOYD, H., LOUIS, C. J., AND MARTIN, T. J. (1974) Cancer Res. 34, 1720-1725. 11. CHAYOTH, R., EPSTEIN, S. M., AND FIELD, J. B. (1973) Cancer Res. 33, 1970-1974. 12. RUDLAND, P. S., SEELEY, M., AND SEIFERT, W. (1974) Nature (London) 251, 417-419. 13. BLOCH, A. (1974) Biochem. Biophys. Res. Commun. 58,652-659. 14. BRUSH, J. S., SUTLIFF, L. S., AND SHARMA, R. K. (1974) Cancer Res. 34, 1495-1502. 15. KIMURA, H., AND MURAD, F. (1975) PFOC. Nut. Acud. Sci. USA 72, 1965-1969. 16. PERKINS, J. P., MACINTYRE, E. H., RILEY, W. D., AND CLARK, R. B. (1971) Life Sci. 10, 10691080. 17. KORINEK, J., SPELSBERG, T. C., AND MITCHELL, W. M. (1973) Nature (London) 246,455-458. 18. OWEN, J., JOHNSON, G. S., AND PASTAN, I. (1972) J. Biol. Chem. 247, 7082-7087. 19. GILL, G. N., AND GARREN, L. D. (1971) PFOC. Nut. Acad. Sci. USA 68, 786-790. 20. MOYER, G. H., MURRAY, R. K., KHAIRALLAH, L. H., SUSS, R., AND PITOT, H. C. (1970) Lab. Invest. 23, 108-118. 21. GILMAN, A. G. (1970) Proc. Nut. Acud. Sci. USA
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67, 305-312. 22. CHEUNG, W. Y., AND PATRICK, S. M. (1974)Znt. J. B&hem. 5, 331-342. 23. SHARMA, R. K., AHMED, N. K., SUTLIFF, L. S., AND BRUSH, J. S. (1974) FEBS Lett. 45, 107110. 24. SHARMA, R. K., MCLAUGHLIN, C. A., AND P~nrr, H. C. (1975) Cancer Letters, in press. 25. WALSH, D. A., BROSTROM, C. O., BROSTROM, M. A., CHEN, L., CORBIN, J. D., REIMAN, E., SODERLING, T. R., AND KRJZBS, E. G. (1972) Adv. Cyclic Nucl. Res. 1, 33-45. 26. ADELMAN, M. R., SABATINI, D. D., AND BLOBEL, G. (1973) J. Cell Biol. 56, 206-229. 27. PITOT, H. C. (1963) Cancer Res. 23,1474-1482. 28. POTTER, V. R. (1969) Cunud. Cancer Cont. 8, S30. 29. DOLJANSKI, F. (1960)Znt. Rev. Cytol. 10,217-241. 30. NOWELL, P. C., MORRIS, H. P., AND POTTER, V. R. (1967) Cancer Res. 27, 1565-1579. 31. MACKENZIE, C. W., AND STELLWAGEN, R. H. (1974) J. Biol. Chem. 249, 5755-5762. 32. GBANNER, D. K. (1974)Arch. Biochem. Biophys. 165, 359-368. 33. GOLDBERG, M. L., BURKE, C. C., ANDMORRIS, H. P. (1975) Biochem. Biophys. Res. Comm. 62, 320-327. 34. JERGIL, B., AND OHLSSON, R. (1974) EUF. J. Biothem. 46, 13-25. 35. PITOT, H. C. (in press) Cancer, A Comprehensive Treatise (F. Becker, ed.), Vol. III, Plenum, New York. 36. RUBIN, C. S., ERLICHMAN, J., AND ROSEN, 0. M. (1972) J. BioZ. Chem. 247, 6135-6139. 37. CASNELLIE, J. E., AND GREENCARD, P. (1974) PFOC. Nat. Acad. Sci. USA 71, 1891-1895. 38. WILLINGHAM, M. C., AND PASTAN, I. (1974) J. Cell BioZ. 63, 288-294. 39. SHIRES, T. K., PITOT, H. C., AND KAUFFMANN, S. A. (1974) Biomembmnes 5, 81-145.
OF BIOCHEMISTRY
The Binding
AND
BIOPHYSICS
175,
221-228 (19761
of Cyclic Nucleotides to Membranes of the Endo@asmic Reticulum of Normal and Neoplastic Rat Liver-l
RAMESHWAR
K. SHARMA*, CHARLES A. MCLAUGHLIN3, AND HENRY C. PITOT
Departments of Oncology and Pathology, The McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706 Received October 28, 1975 Studies are presented which demonstrate that the smooth and rough endoplasmic membranes of normal and neoplastic rat liver possess binding sites for cyclic nucleotides exhibiting a high degree of specificity. In contrast to normal liver, which has only a single binding site for cyclic AMP on membranes of the endoplasmic reticulum, cyclic AMP binding to the intracellular membranes of hepatoma 5123% and 7777 exhibits two apparent binding sites. The binding constant for cyclic AMP of one site on the tumor membranes is comparable to that of the normal liver, whereas the value of the second intrinsic association constant is 4- to IO-fold greater than liver. The possibility that the presence of the second cyclic AMP binding site might be a function of the rapid growth of the tumors was unlikely since membrane preparations from neonatal rats showed a single affinity association constant which was similar to that of normal liver. In addition, membranes of the endoplasmic reticulum of the Morris hepatomas 5123C and 7777 exhibit a binding site for cyclic GMP which is absent from the intracellular membranes of liver.
Since the discovery of 3’,5’-cyclic AMP in vertebrate tissues by Sutherland and his associates (1) the occurrence and metabolic functions of this and other cyclic nucleotides have been shown to be both widespread and essential for the normal functioning of prokaryotic (2,3) and eukaryotic (4-6) cells. Within the past decade studies from several laboratories (7-10) have implicated cyclic nucleotides as potentially important in the neoplastic transforma-
tion as well as in the maintenance of the malignant state itself. These studies have involved not only the intracellular concentrations of cyclic nucleotides including cyclic AMP5 (ll), cyclic GMP (12), and more recently cyclic CMP (131, but also measurements of adenylate (14) and guanylate (15) cyclases in neoplastic tissues. With the elucidation of the specific role of cyclic AMP in activating intracellular protein kinases, studies of both cyclic nucleotide-dependent and -independent protein kinases within neoplastic cells have been forthcoming (161. The correlation of measurements of cyclic nucleotide levels, cyclases and ki-
i This work was supported in part by grants from the National Cancer Institute (CA-071751 and the American Cancer Society (E-588). 2 Visiting Professor and Special Fellow of the National Cancer Institute (CA-55861). Present address; 5 Abbreviations used: Cyclic AMP, cyclic adenoDepartment of Biochemistry, University of Tennessee Health Center, Memphis, Tennessee 38163. sine 3’,5’-monophosphate; cyclic GMP, cyclic guano3 Postdoctoral Fellow in Biochemical Pathology of sine 3’,5’-monophosphate; cyclic IMP, cyclic inosine the National Institute of General Medical Science 3’,5’-monophosphate; cyclic CMP, cyclic cytidine cyclic UMP, cyclic uridine (GM-47855). Present address: National Institute of 3’5’-monophosphate; 3’,5’-monophosphate; STKM buffer, 0.25 M sucrose Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Montana 59840. in 0.05 M Tris-HCl, pH 7.4, 0.025 M KCl, and 0.01 M 4 To whom reprint requests should be addressed. MgCI,. 221 Copyright 8 1976 by Academic press, Inc. All rights of reproduction in any forin reserved.
222
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nase activities with other biochemical characteristics of the neoplastic cell has been accomplished to the greatest extent with cells transformed in vitro (7,8). Studies concerned with levels of cyclic nucleotides and the effects of exogenous cyclic AMP on morphology and membrane structure (17) as well as proliferation (18) have been reported. On the other hand, investigations related directly to the functional receptor of cyclic AMP and cyclic GMP, i.e., the regulatory subunit of protein kinase (19) in neoplastic cells, have been relatively infrequent. The studies described in this paper may be related to this latter subject and demonstrate that specific differences in the cyclic nucleotide receptor proteins localized within intracellular membranes have been found between normal and neoplastic hepatic tissues. MATERIALS
AND METHODS
Materials. Cyclic [VH]AMP and cyclic [S3H]GMP were purchased from Amersham Searle, Chicago. Nonradioactive nucleotides were obtained from Sigma Chemical Company. All the other chemicals were reagent grade and were obtained commercially. Animal preparations. Male albino Sprague-Dawley (Badger Rat Company, Madison, Wisconsin) and Buffalo strain (Simonsen Laboratories, Great Bear Lake, Minnesota) rats were used in the investigations reported in this paper. The Morris hepatocellular carcinomas were transplanted into But&lo strain rats with sterile technique. The tumors were harvested from the hind legs, where they had been inoculated 4 to 8 weeks earlier. All animals were maintained on a Purina laboratory chow diet fed ad Zibitum. The rats, both normal and tumor-bearing were kept in rooms equipped for automatic light and dark exposure of 12-h periods each. Animals were sacrificed by decapitation, and the livers, as well as the tumors where present, were removed as rapidly as possible and immersed in icecold STKM buffer (0.25 M sucrose in 0.05 M TrisHCl, pH 7.4, 0.025 M KCl, and 0.001 M MgCl,). Muscle, connective tissue, and necrotic debris were dissected from the viable tumor tissue prior to homogenization. All tissues were homogenized in STKM buffer and the smooth and rough endoplasmic reticulum from both liver and hepatoma were isolated as previously described (20). A discontinuous gradient containing 20 ml of the postmitochondrial fraction of liver or tumor in 1.3 M STKM (1.3 M sucrose used in place of 0.25 M sucrose) layered over 8 ml of 2.0 M STKM and itself overlaid with 5 ml of
AND PITOT
STKM was prepared and centrifuged in a Beckman 60 Ti rotor at 214,OOOgfor 3 h. The smooth endoplasmic reticulum at the 0.44-1.3 M sucrose interface as well as the rough endoplasmic reticulum at the l.l2.0 M sucrose interface were collected, diluted in STKM, and repelleted in a Beckman 30 rotor at 78,500g for 30 min. The pellets were rinsed and homogenized and the suspension was layered on 0.44 M STKM and sedimented again in a 30 rotor at 7500%. The membrane pellets were then rinsed, homogenized in 20 mM Tris-HCl, pH 7.4, containing 5 mM P-mercaptoethanol, and used immediately or stored at -60°C. Electron microscopic examination of the membrane fractions by techniques previously described (20) confirmed the morphologic characterization of each of the fractions. Cyclic nucleotide binding assays. The assay for cyclic AMP-binding and cyclic GMP-binding protein was essentially that described by Gilman (21). The incubation mixture at 0°C contained 50 mM Naacetate, pH 4.0, 20 mM EDTA, and 0.73 pmol of cyclic [3H]AMP (sp act 27.5 Ci/mmol) or 0.3 pmol of cyclic [3H]GMP (sp act 15 Cilmmol) in a final volume of 0.1 ml (22, 23). The reaction was started by the addition of 100 pg of membrane preparation. At the end of the incubation, 2 ml of a 20 mM phosphate buffer, pH 6.0, was added and the diluted reaction mixture was immediately filtered through a Millipore disk (2.5-cm diameter with 0.45~pm pore size). Each tube was rinsed once with 2 ml of phosphate buffer and 2 ml of 10 mM MgCl,. The filter was dried at 90°C and then counted in 10 ml of Scintisol-complete (Isolab, Inc., Akron, Ohio). A tube containing the complete reaction mixture, but without any membrane protein, served as a blank, and all data were corrected for this value. In those experiments designed to study the competition between various cyclic nucleotides and their binding to membranes of the endoplasmic reticulum, the same assay was utilized with the addition of an excess amount of nonradioactive nucleotides as competitors; the concentrations of these are given in each figure. The plots in Figs. l-6 are representative experiments taken from four to seven separate analyses. RESULTS
Previous studies from this laboratory (24) demonstrated the characteristics of the binding of cyclic AMP to membranes of the smooth endoplasmic reticulum of hepatic tissue. Figure 1 is a Scatchard plot of the binding of cyclic AMP to membranes of the rough endoplasmic reticulum isolated from rat liver. As can be seen from the figure, the plot of binding affinity demonstrates a single binding site with a disso-
CYCLIC w g
m;::f
NUCLEOTIDE
BINDING
I 010
t
o;.
o;;\
,
0.30
040 B/F
0.50
060
0.70
FIG. 1. Binding afBnity of cyclic AMP to the receptor protein of the rough endoplasmic reticulum of the liver. The amount of cyclic AMP is plotted as a function of the ratio of the concentration of bound to free cyclic nucleotide. The concentration of free cyclic 13H1AMP was obtained by subtracting the cyclic 13H1AMP bound from the total cyclic [3H]AMP concentration initially added to the incubation mixture. Duplicate assays were performed with 100 pg of binding protein in the incubation medium, as mentioned in the text. To evaluate K the molar concentration was determined from the total amount of ligand bound in 0.1 ml of incubation mixture. A single binding fraction follows the equation, amount bound = number of sites - K, (bound/ free).
ciation constant of 0.16 x lo-* M. This intrinsic dissociation constant is quite comparable to that reported by us previously for the binding affinity of cyclic AMP to membranes of the hepatic smooth endoplasmic reticulum (24). Therefore, the intrinsic association constant of cyclic AMP for both the smooth and rough endoplasmic reticulum is quite comparable to that seen for regulatory proteins which bind cyclic AMP under other circumstances and in other parts of the cell (25). However, when one determines the concentration of binding sites of cyclic AMP on each of these membrane fractions, a significant difference is seen (Table I). The concentration of binding sites of the smooth endoplasmic reticulum is about 55% more than the number of sites for this cyclic nucleotide on the rough endoplasmic reticulum. The values (average 2 SEM of seven determinations) for smooth and rough membranes shown in Table I differ quite significantly from each other (P < 0.005). In preliminary investigations we have also found that the concentration
TO ENDOPLASMIC
223
RETICULUM
of binding sites for cyclic AMP on a subfraction of the smooth endoplasmic reticulum, probably the Golgi fraction (201, is also significantly lower than the concentration of cyclic AMP binding sites seen on the smooth endoplasmic reticulum. A very important aspect of the binding sites for cyclic AMP on the smooth and rough endoplasmic reticulum is their specificity. The data in Tables II and III clearly demonstrate that of all the cyclic nucleotides and other nucleotides tested (Table II) only cyclic IMP as well as cyclic AMP itself could compete successfully for the TABLE
I
BINDING OF CYCLIC AMP TO THE RECEITOR PROTEINS OF SMOOTH AND ROUGH ENDOPLASMIC RETICULUM OF LIVERY
Membrane Smooth Rough SERRER
fraction
Binding
of CAMP (pmol/ me9 1.330 + 0.035
0.853
ratio
* 0.079 1.55
a Duplicate assays were performed on seven sepa-
rate samples with 100 pg of binding protein in the incubation medium which contained 0.73 pmol cyclic 13HlAMP (44,000 dpm) in 0.1 ml of 50 mM sodium acetate buffer (pH 4.0). See text for further details. TABLE
II
THE EFFECT OF NUCLEOTIDES ON THE BINDING CYCLIC t3H]AMP TO THE ROUGH END~PLASMIC RETICULUM OF LIVERY
Nucleotides added None Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic AMP GTP ADP ATP
Nucleotide concentration (fold excess)
OF
l$&;P bound (46) 100
AMP GMP GMP GMP IMP CMP UMP
500 250 500
33 120 117
1000 136 1000 30 1000 118 1000 108 1000 135 1000 129 1000 119 1000 129 ’ The reaction mixture contained 50 mM sodium acetate buffer (pH 4.01, 0.73 pmol cyclic r3HlAMP (44,000 dpm), 100 pg of membrane preparation, and unlabeled nucleotide as indicated. The values presented are representative of at least four experiments.
224
SHARMA,
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TABLE III EFFECT OF VARIOUS CONCENTRATIONS OF CYCLIC GMP ON THE BINDING OF CYCLIC [3H]AMP T O THE SIUOOTH END~FXASMIC RETICULUM OF LIVE@ NucleoNucleotide Cyclic tides concentration 13H]AMP added (fold excess) bound (%) None 100 Cyclic AMP 200 11 Cyclic AMP 400 9 Cyclic AMP 800 6 Cyclic GMP 200 99 Cyclic GMP 400 67 Cyclic GMP 800 72 (2The conditions and numbers of the experiments are similar to those depicted in Table II.
binding of the cyclic nucleotide to the membranes of the rough endoplasmic reticulum. Those values greater than 100% indicate that no competition for the binding of cyclic 13HlAMP had occurred. Equally important is the fact that cyclic GMP even in lOOO-fold excess over the labeled cyclic AMP had no competing effect on the binding of this cyclic nucleotide to membranes of the rough endoplasmic reticulum. In Table III a related study of the competition of cyclic AMP and cyclic GMP for the binding of radioactive cyclic AMP to membranes of the smooth endoplasmic reticulum is seen. In this instance some competition for the cyclic AMP binding site at very high levels of cyclic GMP was elicited although at the present time we do not feel that these values represent significant competition for the binding sites but rather that cyclic GMP does not compete with cyclic AMP for binding to the endoplasmic reticulum. Furthermore, in other studies, utilizing the cyclic nucleotides shown in Table II, no significant competition for the binding of cyclic 13HlAMP to membranes of the smooth endoplasmic reticulum was exhibited by other nucleotides with the exception of cyclic IMP, which did compete to the same levels seen for the rough endoplasmic reticulum in Table II. Therefore, in both the smooth and rough endoplasmic reticulum the binding sites for cyclic nucleotides exhibit a high degree of specificity for cyclic AMP. In order to determine whether the cyclic AMP binding sites of the endoplasmic re-
AND PITGT
d & CYCLIC
2 4 AMP (pmoler)
FIG. 2. Binding affinity of CAMP to the receptor protein of rough endoplasmic reticulum preparations which have been treated with varying concentrations of KC1 + 1 mre puromycin. Duplicate assays were performed with 100 kg of binding protein in the incubation medium with varying concentrations of exogenously added nonradioactive cyclic AMP in 0.1 ml of 50 mM sodium acetate buffer (pH 4.0).
ticulum were loosely or firmly bound to the surface of this organelle, the experiment demonstrated in Fig. 2 was performed. In this instance the association of cyclic 13HlAMP to membranes of the endoplasmic reticulum was studied as a function of the concentration of exogenously added nonradioactive cyclic AMP. The competition curve noted is not affected by extremely high ionic strength which is capable of dissociating many protein molecules which are loosely bound to the surface of the endoplasmic reticulum. Puromycin was also added in these experiments in order to release some growing polypeptide chains and ribosomal subunits present on the surface of the endoplasmic reticulum (26). As can be seen from the figure none of these treatments affected the binding of cyclic AMP to membranes of the smooth endoplasmic reticulum. Similar results were obtained when the rough endoplasmic reticulum fraction was substituted for the smooth fraction. This therefore indicates that the binding sites on the membrane are relatively tightly associated with the membrane structure and cannot be removed by high salt treatment. Cyclic nucleotide binding to hepatoma membranes of the endoplasmic reticulum.
Figures 3 and 4 show the Scatchard plot of the binding of cyclic AMP to the smooth endoplasmic reticulum of Morris hepato-
CYCLIC
NUCLEOTIDE
BINDING
TO ENDOPLASMIC
225
RETICULUM
FIG. 3. Binding affinity of cyclic AMP to the receptor protein of the smooth endoplasmic reticulum of the Morris hepatoma 5123C. The conditions of the experiments are identical to those depicted in Fig. 1. s : P 0 x 4 s m
*
i ;j
1.5
(I) (il)
IO
0.5
\
,k
0 = s
0
I
\
\
\
Kd=1.16X16% K,,=3.03X
Id%,
\
2
4
5
6
S3F
FIG. 4. Binding affinity of cyclic AMP to the receptor protein of the smooth endoplasmic reticulum of the Morris hepatoma 7777. The conditions of the experiments are identical to those depicted in Fig. 1.
mas 5123C and 7777. As can be seen in contrast to the characteristics of the cyclic AMP binding to smooth and rough endoplasmic reticulum in liver, two binding affinities are apparent, each site exhibiting a significantly different affinity for the cyclic nucleotide. The dissociation constant for the steeper portions of the curves shown in Figs. 3 and 4 are 1.04 and 1.16 x 1O-8 M, respectively. The values for the shallow lines are 0.23 and 0.03 x lo-* M, respectively, for the binding of cyclic AMP to the smooth endoplasmic reticulum of these two neoplasms. In each case, the slopes of the two portions of the curve differ by a factor between 4 and 40. The values seen for CAMP binding affinity to the rough endoplasmic reticulum of the liver is comparable to the smaller value in each of the neoplasms (Fig. 1). The specificity of the cyclic AMP binding site of the smooth endoplasmic reticulum of hepatoma 5123C can be seen in
Table IV. The competition characteristics are quite similar to those exhibited for the smooth and rough endoplasmic reticulum of normal liver as demonstrated in Tables II and III. Only cyclic IMP competes significantly with the membrane binding sites of the tumor. Very high levels of cyclic GMP, cyclic UMP, and cyclic CMP again exhibit a very small degree of competition, which we do not feel is significant and in fact may be due to contaminants within these preparations. However, further studies of the specificity of the binding site for cyclic nucleotides of the smooth endoplasmic reticulum of liver and hepatoma led to the findings depicted in Fig. 5. In this figure the binding of cyclic [3H]GMP TABLE
IV
THE EFFECT
OF NUCLWTIDES ON THE BINDING CYCLIC lSHIAMP TO THE SMOOTH END~~LASMIC RETICULUM OF HEPA-IOMA 51230 NE?-
added
None Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic Cyclic AMP GTP ADP ATP
Nucleotide concentration (fold excess)
-
OF
&gP bound (81
100 20 11 7 96 97 79 16 76 80 91 91 100 101 a The conditions and numbers of the experiments are similar to those depicted in Table II. AMP AMP AMP GMP GMP GMP IMP UMP CMP
250 596 1096 250 500 1000 500 500 500 500 500 596 500
226 “6 x z05
6
SHARMA,
MCLAUGHLIN
I
bf
;5
50 CYCLIC
I I1 20.0 I50 250 GMP (pmoles) IO.0
FIG. 5. Binding affinity of cyclic GMP to the receptor protein of the smooth endoplasmic reticulum of the liver and Morris hepatoma 5123C. Duplicate assays were performed with 100 pg of membrane preparation, 3 pmol of lSH]cyclic GMP, and various amounts of cyclic GMP in 0.1 ml of 50 mM sodium acetate buffer (pH 4.0).
to the smooth endoplasmic reticulum of liver and hepatoma 5123C in the presence of various concentrations of nonradioactive cyclic GMP is depicted. As can be seen, there is essentially no significant binding of the radioactive cyclic GMP itself or diluted in the presence of cold carrier to the liver membrane preparation. On the other hand, quite significant binding of this labeled cyclic nucleotide occurs to the smooth endoplasmic reticulum of hepatoma 5123C which is successfully competed for by the addition of nonradioactive cyclic GMP. These data, taken in the context of those presented in Table IV, argue that the smooth endoplasmic reticulum of this neoplasm exhibits binding sites for cyclic GMP which may be separate from the cyclic AMP binding sites seen both in the smooth endoplasmic reticulum of liver and hepatoma (Figs. 1 and 3, Tables II and IV). Further studies have also demonstrated that the smooth endoplasmic reticulum of hepatoma 7777 exhibits the cyclic GMP binding site just as do those membranes of the hepatoma 5123C. Furthermore, the specificity of the cyclic AMP binding sites of the smooth endoplasmic reticulum in hepatomas 7800 and 7777 cannot be significantly competed for by high levels of cyclic GMP (data not shown). Cyclic AMP binding of membranes of neonatal liver. As has been discussed by
several authors previously (27, 28), the rate of growth of neoplastic tissue may be responsible for some of the peculiar bio-
AND PITGT
chemical characteristics found in such tissues when they are compared with their tissue of origin. In addition, the liver offers a further significant problem in that normal adult liver is predominantly tetraploid (29) whereas most of the neoplasms studied are diploid or near diploid (30). Therefore, we felt it important to determine the characteristics of cyclic AMP binding to membranes of neonatal rat liver which exhibits a rate of growth comparable to the neoplasms in which the binding of cyclic AMP has been investigated in this paper and also is diploid in karyotype (29). In Fig. 6, a Scatchard plot of the binding affinity of cyclic AMP to the receptor proteins of the smooth endoplasmic reticulum of a neonatal rat 7 days of age is seen. The dissociation constant from this curve is 0.16 x lOUs M, or essentially identical with that value seen for the endoplasmic reticulum in liver. Therefore, it would appear that the peculiar binding characteristics of cyclic AMP to the endoplasmic reticulum of several hepatomas investigated in this study are not directly related to the rate of growth or the karyotypic changes seen in these neoplasms. We do not yet know whether cGMP binds to membranes from neonatal liver. DISCUSSION
The major action of cyclic AMP and, more recently, cyclic GMP in binding to regulatory sites of protein kinases in eukaryotic cells is now well known from a
FIG. 6. Binding affinity of cyclic AMP to the receptor protein of the smooth endoplasmic reticulum of the liver from neonate rats. The conditions of the experiments are identical to those depicted in Fig. 1.
CYCLIC
NUCLEOTIDE
BINDING
number of studies. By means of an investigation of the affinity of cyclic nucleotides for individual proteins as well as subcellular organelles within cells, one may infer until proven otherwise the localization of the regulatory subunits of protein kinases which mediate the cyclic nucleotide activation of these enzymes within the cell. The studies presented in this paper are directed toward the localization and specificity of cyclic nucleotide binding proteins within the subcellular organelles of both normal and neoplastic hepatic tissue. Although there have not been many investigations on the localization of the protein kinase regulatory subunit or cyclic nucleotide binding proteins in neoplasms, some investigations of the proteins of the cytosol of hepatomas in uiuo and in vitro have been described. MacKenzie and Stellwagen (31) have described the absence of a cyclic AMP-binding fraction in an established hepatoma cell line (HTC cells) although these cells do exhibit several forms of protein kinase. Granner (32) has extended these investigations to include two other hepatoma-derived cultured cell lines. He demonstrated that the binding of cyclic AMP to soluble cytosol proteins is highest in liver and then decreases in the three neoplasms he studied. Golberg et al. (33) demonstrated that in relatively slowly growing hepatocellular carcinomas including one in this study, the Morris 7800, the binding of cyclic AMP was significantly lower in the “cytoplasm” of the tumor when compared with liver. While these authors did not strictly define the “cytoplasm” preparations used, the reference cited indicates that the preparations consisted of soluble cytosolic proteins. Furthermore, no kinetic evidence regarding the specificity and nature of the binding sites for cyclic nucleotides was presented. On the other hand, two rapidly growing neoplasms exhibited cyclic AMP binding capacity equal to that of liver. The binding of cyclic GMP was also studied and was found to exhibit the same characteristics in the rapidly growing tumors but showed some variation in the slowly growing neoplasms. The binding of cyclic nucleotides to membranes of the endoplasmic reticulum
TO
ENDOPLASMIC
RETICULUM
227
suggests that these intracellular organelles may exhibit protein kinase activity. A recent paper by Jergil and Ohlsson (34) described both the phosphorylation and dephosphorylation of protein by the smooth and rough endoplasmic reticulum of rat liver. These authors determined the binding of cyclic AMP per milligram of protein of the smooth and rough endoplasmic reticulum and showed the concentration of binding sites to the former to be more than twice that of the latter. This value is somewhat higher than described in this study (Table I) but in general is comparable to the findings reported herein. At this time it is not possible to relate the findings described in this paper to the regulation of genetic expression in normal hepatic cells and its abnormalities seen in hepatomas (35). On the other hand, there is considerable evidence accumulating to show that cyclic AMP binding proteins occur within membrane components of the cell (36,371 and in fact may be involved in the regulation of surface phenomena of both the normal and neoplastic cell (38). The mediation of cell surface changes by cyclic nucleotide-regulated functions such as protein kinase is clearly one of a number of possible mechanisms mediating at least some of these effects. Preliminary investigations in this laboratory have demonstrated both endogenous and exogenous phosphorylation by membranes of the endoplasmic reticulum, the products of this phosphorylation within the membrane being different in the normal as compared with the neoplastic hepatic cell. Furthermore, the potential role of the membrane of the endoplasmic reticulum in the regulation of genetic expression as postulated earlier (39) suggests a possible common thread of explanation for the findings described in this paper and the known functions of cyclic nucleotides in the regulation of genetic expression. REFERENCES 1. SUTHERLAND, E. W., AND RALL, T. W. (1958) J. Biol. Chem. 232, 1077-1092. 2. PASTAN, I., AND PERLMAN, R. L. (1966) Proc. Nat. Acad. Sci. USA 61, 1336-1342.
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