Calcium-dependent regulatory protein of cyclic nucleotide

Proc. Natl. Acad. Sci. USA

Vol. 73, No. 8, pp. 2711-2715, August 1976 Biochemistry

Calcium-dependent regulatory protein of cyclic nucleotide metabolism in normal and transformed chicken embryo fibroblasts (Rous sarcoma virus/troponin C-like protein/3':5'-cyclic-nucleotide phosphodiesterase)

D. MARTIN WATTERSON, LINDA J. VAN ELDIK, RALPH E. SMITH, AND THOMAS C. VANAMAN* Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710

Communicated by Robert L. Hill, June 1, 1976

ABSTRACT The concentration of a calcium-binding protein modulator of 3':5'-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17; 3':5'-cyclic-nucleotide 5'-nucleotidohydrolase) activity is increased in chicken embryo fibroblasts upon transformation by Rous sarcoma virus. This modulator protein from fibroblasts, which has roughly the same molecular size, charge, and functional properties as that isolated from chicken brain, comprises approximately 1.32% of the soluble protein in homogenates of fibroblasts infected and transformed by Rous sarcoma virus. In comparison, the modulator comprises approximately 0.30% of the soluble protein in homogenates of normal fibroblasts from confluent cultures and 0.36% of the soluble protein in homogenates of fibroblasts infected with a transformation-defective mutant of Rous sarcoma virus. Modulator levels in normal fibroblasts at subconfluent cell densities are 0.42-0.76% of the homogenate soluble protein, i.e., between that found in confluent normal fibroblasts and in fibroblasts transformed by Rous sarcoma virus. These observations suggest that the levels of the modulator protein are elevated under conditions in which chicken embryo fibroblasts are undergoing rapid growth and have decreased adenosine 3':5'-cyclic monophosphate levels.

Cyclic nucleotides play an important role in the regulation of cell growth and differentiation. Studies with cultured fibroblasts have shown that cellular morphology, motility, and growth, adhesion to the substratum, and macromolecular synthesis are influenced by cyclic nucleotides (for reviews see refs. 1 and 2). Cyclic nucleotides have also been shown to be involved in the regulation of several cell properties that are altered upon transformation by oncogenic viruses (1, 3). However, the mechanism of transformation is not known. At least two facts must be considered in any proposed mechanism of viral transformation. First, numerous cell properties are altered by transformation (1). Second, tumor viruses, which contain relatively few genes, readily transform cells (4). These observations suggest that there may be several discrete regulatory sites of the cell that are altered by the transforming virus. The metabolism of cyclic nucleotides, which has been shown to be altered upon viral transformation (3), could be one such regulatory site. It therefore becomes crucial to examine those processes that might affect cyclic nucleotide metabolism in normal and transformed cells. One of the more extensively characterized viral transformation systems is that of chicken embryo fibroblasts (CEF) transformed by Rous sarcoma virus (RSV), an avian RNA tumor virus. CEF transformed by RSV have lower amounts of cyclic AMP than untransformed CEF (5). Artificial elevation of intracellular cyclic AMP levels by addition of cyclic AMP analogues, adenylate cyclase [EC 4.6.1.1; ATP pyrophosphateAbbreviations: CEF, chick embryo fibroblasts; RSV, Rous sarcoma virus; Pr-RSV-C(nd), Prague strain of RSV, subgroup C; Pr-RSV-C(td), transformation defective strain of Pr-RSV-C; phosphodiesterase, 3': 5'-cyclic-nucleotide phosphodiesterase; NaDodSO4, sodium dodecyl sulfate. * From whom reprints should be requested. 2711

lyase (cyclizing)] activators, or cyclic AMP phosphodiesterase (phosphodiesterase) inhibitors causes several abnormal properties associated with transformed CEF to resemble those of normal CEF (5). These studies suggest that it is the inability of CEF to maintain normal cyclic AMP levels that gives them many of the properties characteristic of transformed cells. The levels of cyclic AMP in the cell can be controlled by at least three processes: (i) synthesis by adenylate cyclase; (ii) degradation by phosphodiesterase; and (iii) loss or transport of cyclic AMP from the cell (1, 6). Regulation of cyclic nucleotide levels by adenylate cyclase and phosphodiesterase is complex. An important feature of this regulation is the demonstration that both adenylate cyclase and phosphodiesterase are stimulated by calcium through the action of a ubiquitous calcium-binding protein (7-10), termed modulator proteint. We have previously reported that modulator protein is found in neurosecretory tissues of several vertebrate species in relatively high levels compared to nonsecretory tissue and that modulator protein is structurally and functionally similar to muscle troponin C, the calcium-binding regulatory subunit of vertebrate skeletal muscle (13, 14). We now report that the transformation of CEF by an RNA tumor virus (Prague-C strain of RSV) is associated with an increase in modulator protein concentration and have further shown the applicability of quantitative gel electrophoresis in measuring the levels of modulator protein in cell homogenate supernatants.

MATERIALS AND METHODS CEF, prepared from 11-day-old SPAFAS (Norwich, Conn.) embryos as previously described (15), were grown in medium F10 (Gibco) supplemented with tryptose phosphate broth [10% (vol/vol); Difco], calf serum [5% (vol/vol); Gibco], and antibiotics [penicillin (50 units/ml; Gibco), streptomycin (50 ,g/ml; Gibco), and fungizone (2 ,ug/ml; Squibb)]. Cells from confluent secondary cultures were harvested from roller bottles or 150 X 25 mm petri dishes (Falcon Plastics, Oxnard, Calif.) using rubber policemen. For cell density experiments, primary CEF were trypsinized, counted using a hemocytometer, and seeded in 100 X 20 mm petri dishes at 3.5, 7.0, and 14 X 106 cells per dish. The secondary cultures were harvested after 24-48 hr. All cells were pelleted by centrifugation at 1500 rpm (International t This activity has been referred to as a calcium-dependent regulator

of cyclic nucleotide metabolism and as a phosphodiesterase activator because of the observed effects of the protein on preparation of phosphodiesterase and adenylate cyclase. We have previously referred to this protein as a troponin C-like protein because of its structural similarity to skeletal muscle troponin C. The modulator protein will also function as a calcium-dependent stimulator of the ATPase activity of reconstituted skeletal muscle actomysin (G. Amphlett, T. C. Vanaman, and S. V. Perry, in preparation). Because of its potential multiple regulatory activities, we have chosen to refer to this protein as a modulator protein.

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A B

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Proc. Natl. Acad. Sci. USA 73 (1976)

D

A B

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FIG. 1. Gels and gel scans from discontinuous polyacrylamide gel electrophoretic analysis of CEF homogenate supernatants using 12.5% (wt/vol) acrylamide. Gel A: CEF transformed with Pr-RSVC(nd), 500 yg of protein. Gel B: CEF infected with Pr-RSV-C(td), 500 ,ug of protein. Gel C: confluent normal CEF, 500 ,gg of protein. Gel D: 5 ,g of chicken brain modulator protein. Arrow, modulator protein.

PR-2) for 10 min at 40 and rinsed twice with cold, phosphatebuffered saline (0.140 M NaCl, 0.002 M KH2PO4, 0.008 M Na2HPO4, pH 7.0). The cell pellets were homogenized in an Omnimixer or disrupted with a Branson Sonifier (three 15-sec bursts) in 2 volumes of homogenization buffer (0.100 M sodium acetate, 0.001 M 2-rnercaptoethanol, 0.001 M EDTA, pH 7.4). The Prague strain of Rous sarcoma virus, subgroup C [PrRSV-C(nd)] was used for viral transformation experiments. A transformation-defective mutant (td) of Pr-RSV-C (16), obtained from Dr. P. K. Vogt, was used for virus infection experiments. Virus production was assayed by the presence of RNA-dependent DNA polymerase activity in the culture medium (17). Virus was prepared for polypeptide analysis by purification as described by Cheung et al. (18), but with successive equilibrium and velocity sedimentation using 15-60% (wt/vol) and 15-40% (wt/vol) sucrose gradients, respectively. N,N'-Methylenebisacrylamide (recrystallized from acetone), acrylamide (recrystallized from chloroform), 2-mercaptoethanol, and N,N,N',N'-tetramethylethylenediamine were obtained from Eastman Organic Chemicals. All other chemicals were reagent grade and used without further purification. Protein concentration was determined by the method of Lowry et al. (19). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (NaDodSO4) was done by the procedure of Bolognesi and Bauer (20). Modulator protein was detected in various preparations by discontinuous polyacrylamide gel electrophoresis by the procedure of Watterson et al. (13). Modulator protein was quantified in homogenates by comparison of integrated areas of modulator protein peaks in gel scans of homogenates with those of brain modulator protein standard by described procedures (13), and by determination of the percentage of total Coomassie blue stain contained in modulator protein bands using a Quick Scan (Helena Laboratories) interfaced with a Digital PDP 11/10 computer.

Stimulation of "activator depleted" phosphodiesterase by modulator protein was assayed by described procedures (13). Chicken and bovine brain modulator protein, prepared by described procedures (13), stimulated "activator depleted" bovine brain phosphodiesterase in an identical manner. Modulator protein from CEF cell homogenates was concentrated and separated from fibroblast phosphodiesterase by the initial purification steps in the procedure of Watterson et al. (13), as modified by Awramik et al.t for the isolation of chicken brain modulator protein. Briefly, cell homogenates were centrifuged at 10,000 X g for 1 hr in an SS-34 rotor, the supernatant fluid was decanted, and solid ammonium sulfate (351 g/liter) was added to bring the solution to 55% (wt/vol) saturation. The resulting solution was adjusted to pH 7.0 with 1 M NH40H, stirred for 30 min at 40, then centrifuged for 30 min at 10,000 X g. The supernatant fraction was adjusted to pH 4.0 with 0.5 M sulfuric acid, stirred for 30 min at 40, and centrifuged at 10,000 X g for 30 min. The supernatant fluid was discarded. The pellet was resuspended in 0.01 M ammonium bicarbonate and dissolved by adjusting the slurry to pH 7.0 with 1 M Tris base. The protein was dialyzed against 0.01 M ammonium bicarbonate, shell frozen, and lyophilized. This pellet represents 93-100% of the modulator protein present in brain homogenate supernatants (13). RESULTS

Modulator protein in transformed and confluent normal CEF Modulator protein is a relatively small acidic protein that migrates rapidly on high percentage acrylamide gels run with discontinuous buffer systems (13). We have used discontinuous polyacrylamide gel electrophoresis for the detection and quantification of modulator protein in supernatant fractions from crude homogenates of CEF. As seen in Fig. 1, supernatants from homogenates of CEF transformed by Rous sarcoma virus appear to contain more modulator protein than homogenate supernatants of normal CEF. Equal amounts of protein (500 ,gg) from each homogenate supernatant fraction were loaded on the acrylamide gels. The modulator protein present in each homogenate supernatant was quantitated by comparison of the integrated areas of modulator protein peaks in the scans of gels containing known amounts of purified chicken brain activator protein to those obtained for known aliquots of supernatants of fibroblast cell homogenates. Staining intensity is linear with brain modulator protein concentration up to 25 ,ug per gel. Using this method, the amount of modulator protein detected per 500 ,tg of protein in homogenate supernatants from normal CEF, CEF infected with Pr-RSV-C(td), and CEF infected and transformed with Pr-RSV-C(nd) were 1.5 ,ug, 1.8,ug, and 6.6 ,ug, respectively. Summarized in Table 1, modulator protein constitutes approximately 0.30% of the soluble protein in homogenates of normal CEF, 0.36% of the soluble protein in homogenates of CEF infected with Pr-RSV-C(td), and 1.32% of the soluble protein in homogenates of CEF infected and transformed with Pr-RSC-C(nd) when calculated as percent of total protein determined by the method of Lowry et al. (19). If modulator protein is quantified as percent of total Coomassie blue stain per gel, then the amounts of modulator protein per 500-,g samples of normal CEF, CEF infected with Pr-RSVC(td), and CEF infected and transformed with Pr-RSV-C(nd) are 0.59%, 0.82%, and 2.42%, respectively. * J. L. Awramik, D. M. Watterson, P. M. Keller, W. G. Harrelson, and T. C. Vanaman, in preparation.

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Proc. Natl. Acad. Sci. USA 73 (1976)

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Table 1. Modulator protein in normal CEF, CEF infected with Pr-RSV-C(td), and CEF infected and transformed by Pr-RSV-C(nd) Sample Confluent normal Pr-RSV-C(td)-infected Pr-RSV-C(nd)-transformed Subconfluent normal 3.5 x 106 normal cells/dish 7.0 x 106 normal cells/dish 14 x 106 normal cells/dish

pg Modulator protein detected*

Fold difference

% Total proteint

% Total Coomassie blue staint

1.5 ± 0.7 1.8 ± 0.3 6.6 ± 0.4

1.0 1.2 4.4

0.30 0.36 1.32

0.59 0.82 2.42

2.6 2.1 3.8

1.7 1.4 2.5

0.52 0.42 0.76

1.07 1.01 1.69

* Determined by discontinuous polyacrylamide gel electrophoresis (see Materials and Methods). Values given are the mean and standard deviation for duplicate biological samples. Each sample homogenate supernatant was run on duplicate gels; each gel was scanned twice. t Determined by method of Lowry et al. (19). t Determined by gel scan as described in Materials and Methods.

Properties of modulator protein from transformed CEF The Coomassie blue staining material in homogenate supernatants of transformed CEF that comigrates with chicken brain modulator protein standard during discontinuous polyacrylamide gel electrophoresis was further analyzed by NaDodSO4-polyacrylamide gel electrophoresis. A 1-cm section, corresponding to the modulator protein band in a duplicate stained gel, was cut from two unstained polyacrylamide gels and eluted with 2 volumes of buffer (0.05 M phosphate, 0.001 M EDTA, 0.001 M 2-mercaptoethanol, pH 7.2) at 370 for 8 hr. The eluted fraction was dialyzed against deionized water and frozen, and the volume was reduced by Iyophilization. The sample was then prepared for electrophoretic analysis by the addition of sample buffer [1% (wt/vol) NaDodSO4, 1% (wt/vol) 2-mercaptoethanol] and boiling for 2 min. The material in the eluted fraction comigrates with purified chicken brain modulator protein during NaDodSO4-polyacrylamide gel electrophoresis and does not comigrate with virion polypeptides (Fig. 2). Virion polypeptides, when prepared and analyzed using the same conditions used for CEF, do not migrate in the gels shown in Fig. 1. The ability of a modulator protein fraction, prepared from homogenate supernatants of transformed CEF by the procedures described in Materials and Methods, to stimulate actiA B C

vator-depleted phosphodiesterase is shown in Fig. 3. This activator titration curve is identical to that of purified modulator protein of chicken brain and to those previously published for modulator protein of bovine brain and heart (10, 12). The modulator protein fraction (pH 4.0 pellet) contained 82 mg of protein and was prepared from a homogenate supernatant containing 417 mg of protein. Analysis of the pH 4.0 pellet using

0.90 0.80

0.70 0.60

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0

.0

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FIG. 2. NaDodSO4-polyacrylamide gel electrophoretic analysis of modulator protein and virion polypeptides in slab gel apparatus using 10% (wt/vol) acrylamide. Analysis was performed as described in text. Slot A: modulator protein of chicken brain, 10 Ag of protein. Slot B: fraction eluted from discontinuous gels of transformed CEF homogenates. Slot C: NaDodSO4-disrupted Pr-RSV-C(nd), 25 pug of

protein.

FIG. 3. Activation of "activator depleted" cyclic nucleotide phosphodiesterase by purified modulator protein of chicken brain (A) and by the modulator protein fraction from transformed CEF (W). Assays were performed as described in the text. Reaction mixtures of 0.5 ml contained 40 mM Tris.HCl (pH 8.0), 10 mM MnCl2, 0.1 mM CaCl2, 2 mM cyclic AMP, 50 jig of bovine brain phosphodiesterase, and the indicated amount of modulator protein. Figures plotted are the mean values for phosphate determined in triplicate analyses. Error bars indicate the standard deviation from the mean. The amount of modulator protein in the fraction from CEF was determined by quantitative polyacrylamide gel electrophoresis of 30 ,g of protein from the redissolved pH 4.0 pellet.

Biochemistry: Watterson et al.

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A

*

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Proc. Nati. Acad. Sci. USA 73 (1976)

determined by the method of Lowry et al. (19), and 1% of the total Coomassie blue stain per gel.

D

.o

A B C

D a

+

(

FIG. 4. Gels and gel scans from discontinuous polyacrylamide gel electrophoretic analysis of subconfluent homogenate supernatants using 12.5% (wt/vol) acrylamide. Gel A: CEF at 3.5 X 106 cells per dish, 500 gg of protein. Gel B: CEF at 7.0 X 106 cells per dish, 500 ,Ag of protein. Gel C: CEF at 14 X 106 cells per dish, 500 ,ug of protein. Gel D: Chicken brain modulator protein, 5 jsg of protein. Arrow, modulator protein.

discontinuous polyacrylamide gel electrophoresis showed a of 7.13 mg of modulator protein. When a homogenate supernatant from normal CEF was fractionated in an identical manner, no modulator protein was detected in the pH 4.0 pellet. Based on these determinations, modulator protein comprises approximately 1.71% of the soluble protein in the homogenate supernatant prepared from CEF infected and transformed with Pr-RSV-C(nd). This compares well with the data in Table 1 and indicates that the recovery of modulator protein from transformed CEF homogenates is quantitative, as has been shown for vertebrate brain (13). The area corresponding to the modulator protein band was cut from an unstained polyacrylamide gel containing a homogenate supernatant from transformed CEF and eluted with 2 volumes of buffer (40 mM Tris-HCl, pH 8.0) at 370 for 8 hr. The volume of the eluted fraction was reduced by lyophilization. This eluted modulator protein fraction stimulated activator-depleted phosphodiesterase to the same extent as did chicken brain modulator protein. Modulator protein in subconfluent normal CEF We have studied the relationship between modulator protein concentration and cell density of subconfluent normal CEF. The cell numbers and conditions were chosen to give a range of cell densities from relatively sparse (3.5 X 106 cells per dish) to nearly confluent (14 X 106 cells per dish). Samples containing 500 ,ug of protein were taken from homogenate supernatants of cells at each cell density and the amount of modulator protein in each supernatant was quantified using discontinuous polyacrylamide gel electrophoresis. As seen in Fig. 4, homogenate supernatants of subconfluent normal CEF appear to contain approximately the same concentration of modulator protein at the three cell densities indicated. The amount of modulator protein detected per 500 ,ug of protein from cultures containing 3.5 X 106, 7.0 X 106, and 14 X 106 cells per dish was 2.6 ,g, 2.1 ,g, and 3.8 ug, respectively. As summarized in Table 1, modulator protein comprises from 0.42 to 0.76% of the soluble protein in homogenates of subconfluent normal CEF, when calculated as percent of protein recovery

DISCUSSION Modulator protein has been shown to be increased in homogenate supernatants of CEF after infection and transformation by an avian RNA tumor virus. Quantification of the amount of modulator protein in homogenate supernatants prepared from transformed CEF shows that modulator protein comprises 1-2% of the soluble protein. It is interesting to note that this is approximately the same concentration of modulator protein found in neurosecretory tissue which has previously been reported to contain relatively high levels of modulator protein (13, 14) compared to many other nonsecretory tissues. The increased levels of Coomassie blue staining material that comigrates in discontinuous polyacrylamide gel electrophoresis with purified chicken brain modulator protein represents a true increase in modulator protein. We have previously demonstrated (13, 14) the utility of this method for detecting and quantifying modulator protein, and have previously discussed (13) the limitations and inaccuracies inherent in procedures for quantifying modulator protein that do not use chelating agents, and in procedures that use a modulator protein activity standard that is quantified spectrophotometrically. We have shown in these studies that the material from normal and transformed CEF has the same electrophoretic mobility as chicken brain modulator protein during discontinuous polyacrylamide gel electrophoresis. The material from transformed CEF has roughly the same molecular size, charge, and functional properties as chicken brain modulator protein. In addition, modulator protein is quantitatively recovered from transformed CEF homogenate supernatants when the supernatant is fractionated according to the initial steps for purification of brain modulator protein. We have isolated brain modulator protein from several vertebrate species and have found it to have identical physicochemical properties among the species studied (13, 14). Bovine brain and heart modulator protein have also been found to possess identical physicochemical properties (13, 21). In addition, modulator protein from all species and tissues studied contains approximately 1 mole of an unidentified ninhydrinpositive compound (compound x) per mole of protein (13). Modulator protein isolated from transformed CEF also contains this unidentified compound (data not shown). The data reported here show that the increased levels of modulator protein in CEF are associated with transformation, since infection of CEF with a transformation-defective mutant of Rous sarcoma virus does not result in the large increase in the amount of modulator protein observed with the nondefective virus. Similarly, high modulator protein levels have been observed in normal rat kidney cells transformed by avian oncornaviruses and in myeloblasts from avian myeloblastosis virusinfected leukemic chickens (D. M. Watterson and T. C. Vanaman, unpublished observations). The correlation of high modulator protein levels with transformation of CEF is intriguing in view of the studies by Pastan and coworkers (22) on the association between decreased cyclic AMP levels in fibroblasts and some of the abnormal properties of transformed cells. Cyclic AMP levels in normal fibroblasts are also decreased during periods of rapid growth and proliferation, but these levels increase as the cells become confluent (24). Our results indicate that the modulator protein concentration in normal CEF at low cell densities is intermediate between that found in confluent normal CEF and in transformed CEF. Thus, modulator protein levels are elevated under conditions in which

Biochemistry:

Watterson et al.

CEF are undergoing rapid growth and have decreased cyclic AMP levels. Cyclic AMP levels in CEF might be decreased through stimulation of cyclic nucleotide phosphodiesterase activity as a result of increased modulator protein levels. However, recent reports indicate that modulator protein also stimulates the activity of solubilized adenylate cyclase in the presence of calcium (7, 8). The physiological significance of the activation of both synthetic and degradative enzymes for cyclic AMP by this regulatory protein is unclear at present. In addition to stimulation of phosphodiesterase and adenylate cyclase, modulator protein may have other functions in CEF. Calcium has been implicated in the control of fibroblast proliferation (23-25), in the modulation of the effects of cyclic nucleotides on fibroblasts (26), and in the regulation of fibroblast motility (24). The isolation of myosin and actin from fibroblasts (27, 28) and the morphological evidence for actin filaments in fibroblasts (29) suggest that these cells might have a calciumcontrolled contractile protein system resembling that in skeletal muscle. These proteins could provide the cell with a contractile and structural system for intracellular and cellular movements. Such a mechanism would appear extremely appropriate for conditions of rapid growth. We have shown that modulator protein isolated from brain is a calcium-binding protein that is structurally similar, but not identical, to muscle troponin C, the regulatory subunit of the troponin complex. The modulator protein from brain also has functional similarities to troponin C, i.e., it will function as a calcium-dependent stimulator of the ATPase activity of reconstituted skeletal muscle actomyosin.t The concurrent involvement of modulator protein in the regulation of a calcium-dependent contractile system in fibroblasts and in the regulation of cyclic nucleotide metabolism would provide a direct link between the mechanochemical systems and the metabolic processes of the cell. This work was supported by N.I.H. Grants CA 12323 and NS 10123. D.M.W. is a recipient of N.I.H. Postdoctoral Fellowship NS 05132, and L.J.V.E. is a recipient of N.S.F. Predoctoral Fellowship no. 338-0019. We thank Dr. J. Gavin and Dr. R. Bums for their critical reading of the manuscript. 1. Pastan, I. & Johnson, G. S. (1974) in Advances in Cancer Research, eds. Klein, G., Weinhouse, S. & Haddow, A. (Academic Press, New York), Vol. 19, pp. 303-329. 2. Anderson, W. B. & Pastan, I. (1975) in Advances in Cyclic Nucleotide Research, eds. Drummond, G. I., Robison, G. A. & Greengard, P. (Raven Press, New York), Vol. 5, pp. 681-698. 3. Otten, J., Johnson, G. S. & Pastan, I. (1972) J. Biol. Chem. 247, 7082-7087. 4. Dulbecco, R. (1974) Cold Spring Harbor Symp. Quant. Biol. 39, 1-7.

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5. Otten, J., Bader, J., Johnson, G. S. & Pastan, I. (1972) J. Biol. Chem. 247, 1632-1633. 6. Chlapowski, F. J., Kelley, L. A. & Butcher, R. A. (1975) in Advances in Cyclic Nucleotide Research, eds. Greengard, P. & Robison, G. A. (Raven Press, New York), Vol. 6, pp. 245-338. 7. Cheung, W. Y., Bradham, L. S., Lynch, R. J., Lin, Y. M. & Tallant, E. A. (1975) Biochem. Biophys. Res. Commun. 66, 10551062. 8. Brostrom, C. O., Huang, Y. C., Breckenridge, B. M. & Wolff, D. J. (1975) Proc. Natl. Acad. Sci. USA 72,64-68. 9. Lin, Y. M. & Cheung, W. Y. (1975) Int. J. Biochem. 6, 271280. 10. Teo, T. S. & Wang, J. H. (1973) J. Biol. Chem. 248, 59505955. 11. Cheung, W. Y. & Lin, Y. M. (1974) in Methods in Enzymology, eds. Hardman, J. G. & O'Malley, B. W. (Academic Press, New York), Vol. 38, pp. 223-239. 12. Lin, Y. M., Liu, Y. P. & Cheung, W. Y. (1974) J. Biol. Chem. 249, 4934-4954. 13. Watterson, D. M., Harrelson, W. G., Keller, P. M., Sharief, F. & Vanaman, T. C. (1976) J. Biol. Chem., in press. 14. Vanaman, T. C., Harrelson, W. G. & Watterson, D. M. (1975) Fed. Proc. 34,307. 15. Smith, R. E. & Bernstein, E. H. (1973) Appl. Microbiol. 25, 346-353. 16. Wang, L.-H., Duesberg, P., Beemon, K. & Vogt, P. K. (1975) J. Virol. 16, 1051-1070. 17. Garapin, A. C., McDonnell, J. P., Levinson, W. & Bishop, J. M.

(1970) J. Virol. 6,589-598. 18. Cheung, K.-S., Smith, R. E., Stone, M. P. & Joklik, W. K. (1972) Virology 50, 851-864. 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 20. Bolognesi, D. P. & Bauer, H. (1970) Virology 42,1097-1112. 21. Stevens, F. C., Walsh, M., Ho, H. C., Teo, T. S. & Wang, J. H. (1976) J. Biol. Chem., in press. 22. Anderson, W. B., Russell, T. R., Carchman, R. A. & Pastan, I. (1973) Proc. Natl. Acad. Sci. USA 70,3802-3805. 23. Balk, S. D., Whitfield, J. F., Youdale, T. & Braun, A. C. (1973) Proc. Natl. Acad. Sci. USA 70,675-679. 24. Gail, M. H., Boone, C. W. & Thompson, C. S. (1973) Exp. Cell Res. 79, 386-390. 25. Dulbecco, R. & Elkington, J. (1975) Proc. Natl. Acad. Sci. USA 72, 1584-1588. 26. Berridge, M. J. (1975) in Advances in Cyclic Nucleotide Research, eds. Greengard, P. & Robison, G. A. (Raven Press, New York), Vol. 6, pp. 1-98. 27. Yang, Y.-Z. & Perdue, J. F. (1972) J. Biol. Chem. 247, 45034509. 28. Ostlund, R. E., Pastan, I. & Adelstein, R. S. (1974) J. Biol. Chem. 249,3903-3907. 29. Goldman, R. D., Lazarides, E., Pollack, R. & Weber, K. (1975) Exp. Cell Res. 90, 333-334.