ARCHIVES
Vol.
OF BIOCHEMISTRY
187, No. 2, April
Cyclic
AND
BIOPHYSICS
30, pp. 406-413,
Nucleotide
Phosphodiesterase
A. L. SINGER,’ Cellular
and Molecular
1978
Biology
Rabbit
Tissues’
A. DUNN,
AND
Program, California,
Received
and Protein
Department Los Angeles,
June 29,1977;
Activator
M. M. APPLEMAN
of Biological Sciences, California 9ooo7
revised
in Fetal
January
University
of Southern
5,1978
Cyclic nucleotide phosphodiesterase activity (EC 3.1.4.17) was studied in fetal and newborn rabbit brain, heart, liver, kidney, and lung. Kinetic analysis of phosphodiesterase activity from homogenates of organs from the 25-day embryo suggested the presence of a high Km and a low K,,, activity for both cyclic AMP and cyclic GMP hydrolysis. The addition of 1 PM cyclic GMP to the assay stimulated the hydrolysis of cyclic AMP by whole homogenates of liver, brain, lung, and kidney, but not heart, at all of the ages studied. The addition of micromolar levels of calcium ion stimulated cyclic GMP hydrolysis by homogenates of fetal brain, heart, and kidney, with or without added protein activator. Cyclic GMP phosphodiesterase activity was not stimulated by the addition of calcium ion in homogenates of early fetal rabbit liver and lung, but stimulation was detected in the late embryo and newborn. The presence of the heat-stable protein activator was demonstrated in brain, heart, kidney, liver, and lung tissue at all of the fetal ages studied, and in the newborn rabbit. DEAE-cellulose chromatography demonstrated the presence of three separable enzymes in brain and liver at 15 days, heart at 19 days, and lung and kidney at 25 days of gestation, with no changes in the kinetic properties of the isolated enzymes during development. These experiments suggest that all of the organs studied have the mature array of phosphodiesterases early in development, but an enzyme from liver and lung becomes sensitive to regulatory control by calcium only late in gestation.
Many developmental events are believed to be triggered by changes in the circulating levels of appropriate hormones, possibly mediated by cyclic nucleotides and the enzymes of cyclic nucleotide metabolism. The appearance of hormone sensitivity of adenylate cyclase has been reported to occur at specific times during the development of fetal mammalian organs ( 1,2). In the rodent heart, the capacity for norepinephrine stimulation is an early property of adenylate ’ Supported by grants from the American Diabetes Association, Southern California Affiliate, and the American Heart Association, Los Angeles County Affiliate; National Institutes of Health Grants AM 16367 and AM 07215; and Biomedical Sciences Support Grant RR 07012-07. M. Michael Appleman is a NIH Research Career Development awardee. ’ Present address: Department of Pathology, University of Southern California Medical School, Los Angeles, Caliioruia 90033. 3 To whom correspondence should be addressed.
cyclase (3), while glucagon sensitivity appears postnatally at the time of anatomical maturation (4). In the fetal liver and lung, specific hormone sensitivities appear prior to increased circulating levels of these hormones, in apparent readiness to trigger new developmental phenomena (5,6). In order for a balanced response to increased cyclic nucleotide levels to exist, the means for degrading these messengers must also be present. The cyclic nucleotide phosphodiesterases (EC 3.1.4.17) of mammalian tissues have been shown to consist of at least three enzymes, differing in substrate specificity, kinetic characteristics, regulatory properties, and subcellular localization (7). A low molecular weight, heat-stable protein activator, specific for one of the phosphodiesterases, has been described (8). Within themselves, these enzymes provide considerable variation in the means of regulating cyclic nucleotide levels. 406
0003-9861/78/1872-0406$02.00/O Copyright All rights
D 1978 by Academic Press, of reproduction in any form
Inc. reserved.
FETAL
RABBIT
TISSUE
PHOSPHODIESTERASE
The presence of phosphodiesterase in late fetal and newborn rat brain (9) and liver (5) and changes in the enzyme levels in the late fetal guinea pig tissues (10) have been reported. The protein activator for phosphodiesterase has been described in the brain and liver of the late fetal and newborn rat (11, 12), and the Ca2+ and activator-sensitive enzyme has been measured in chick embryo liver (13). This study was undertaken in order to determine which of the phosphodiesterases are present in early fetal mammalian organs, and whether changes in their regulation by cyclic GMP or by Ca2+ and the protein activator occur during development. MATERIALS
AND
METHODS
Chemicals and reagents. [3H]Adenosine
3’:5’-cyclic phosphate (38 Ci/mol) and [3H]guanosine 3’:5’-cyclic phosphate (3.5 Ci/mol) were obtained from New England Nuclear Corp., Boston, Massachusetts. Cyclic AMP and crude 5’-nucleotidase (Ophiophagus hunnah or Crotalus atrox snake venom) were obtained from Sigma Chemical Co., St. Louis, Missouri. Cyclic GMP, Tris, Mops,“ and bovine serum albumin (Fraction V) were purchased from Calbiochem. La Jolla, California. EGTA was from J. T. Baker Chemical Co., Phillipsburg, New Jersey. DEAE-cellulose and the anion-exchange resin (AGl-X2, 200- to 400-mesh) were from Bio-Rad Laboratories, Richmond, California. Exchangers were routinely prepared by alternate washes of 1 N NaOH and HCl, followed by distilled water, prior to use. Tissue samples. Newborn and timed pregnant rabbits were obtained from ABC Caviary and Rabbitry, Pomona, California. Newborn rabbits were sacrificed by decapitation. Pregnant rabbits were sacrificed by air embolism and fetal tissues were removed immediately. Organs were homogenized in cold 0.05 M Tris-HCl, pH 7.4, containing 2.5 mM 2-mercaptoethanol in a Potter-Elvehjem homogenizer. Samples for chromatographic analysis were sonicated for 30 s/ml in a Biosonic 1 at low energy and clarified by centrifugation at 30,000g for 30 min. Phosphodiesteruse assay. Cyclic nucleotide phosphodiesterase activity was measured using the twostep assay system of Russell et al. (14). Assay tubes contained the indicated concentrations of unlabeled cyclic nucleotide plus 75,000 dpm of 3H-labeled cyclic nucleotide, 5 mM MgClz, 40 mM Mops-NaOH, pH 7.4, and enzyme, in a total volume of 400 pl. The enzyme 4 Abbreviations used: Mops, 4-morpholinepropanesulfonic acid; EGTA, ethylene glycol bis(P-aminoethyl ether); DEAE, diethylaminoethyl.
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hydrolyzes 3’:5’-cyclic AMP or GMP to the 5’-nucleotides, which are converted to the nucleosides by snake venom 5’-nucleotidase. Unreacted substrate (cyclic AMP or cyclic GMP) is removed by the direct addition of an anion-exchange resin, and the 3H-labeled nucleoside remaining in the supernatant fluid is counted by liquid scintillation. The relatively low pH of the resin separation step (7.4) gives quite reasonable and reproducible recoveries of the nucleoside (70-75% for adenosine and 60-65% for guanosine as measured on labeled standards). All values are corrected for the loss that does occur (15, 16). Inosine is not a major by-product in these tissues, but again, the low pH allows reasonable recoveries (17). Care was taken in using this assay, as major variations in the behavior of different batches of resin have been observed. The unit of phosphodiesterase activity is picomoles of cyclic AMP or cyclic GMP hydrolyzed per minute under the assay conditions. Assay blank tubes, containing heat-denatured enzyme but otherwise identical to assay tubes, gave values of less than 1% of the radioactivity added. Enzyme dilutions were adjusted so that less than 30% of the substrate was hydrolyzed during the lo- to 20-min incubation period. Reaction rates were kept linear with respect to protein and time. Protein was measured by the method of Lowry et al. (18) using bovine serum albumin as the standard. DEAE-cellulose chromatography. DEAE-cellulose was used to separate the different phosphodiesterases. Chromatography was performed in 1 x &cm columns, employing a linear gradient from 0.05 M (the equihbration buffer) to 1.0 M sodium acetate (14). On very small samples, a stepwise gradient was used, with sequential additions of 0.2, 0.35, 0.45, and 0.65 M sodium acetate to elute the various activities (19).
Preparation and assay of the protein activator of phosphodiesterase. The activator has the capacity, in the presence of calcium ion, to stimulate the phosphodiesterase activity which preferentially hydrolyzes cyclic GMP at low substrate concentrations (7). Determinations of the level of protein activator were carried out as previously described (19), using aliquota of tissue homogenates heated for 3 min at 100°C and centrifuged at 109Og for 10 min to remove coagulated proteins. This crude preparation was diluted to normalize the protein concentration at 2.0 pg per assay. A standard calcium-sensitive phosphodiesterase for the assay of activator levels was prepared from rat brain using a fraction eluted from DEAE-cellulose at 0.2 M sodium acetate. This enzyme was not stimulated by the addition of calcium ion alone, but was activated more than 300% when rat brain protein activator was also added. Reaction tubes for the determination of activator levels contained the phosphodiesterase, 40 mM Mops-NaOH, pH 7.4,5 mM MgClx, 1.0 mM EGTA, 1.25 mM calcium chloride, 1 pM cyclic GMP, and r3HJ cyclic GMP, in a total volume of 400 al. The addition of 2 pg of activator preparation (described above) increased the basal enzyme activity from 10 to 150%.
408
SINGER,
DUNN,
AND
The assay for the calcium-sensitive enzyme contained the tissue homogenate diluted to provide about 75 pg of protein, 40 mM Mops-NaOH, pH 7.4, 5 mM MgClx, 1 pM cyclic GMP, [3H]cyclic GMP, and 1.0 rnM EGTA, in a final volume of 400 pl. The ability of the enzyme to be activated was reported as the increment in activity upon the addition of 20 cg of a standard rat brain protein activator, prepared by the method of Chueng (20), plus 1.25 mM calcium chloride to this basal assay. RESULTS
Hydrolytic Activities of Homogenates Cyclic nucleotide phosphodiesterase activities of organ homogenates of the 15-, 19-, and 25-day fetus and the newborn (31day) rabbit were measured at 1 PM substrate to approach physiological concentrations (Fig. 1). Hydrolysis of cyclic AMP and cyclic GMP by liver homogenates did not appear to change significantly during this period of development. Brain and heart activities increased phosphodiesterase steadily with the age of the animal. Lung
140
LIVER .
100 60 i
.
Kinetic Analysis of Homogenates Although kinetic analyses of crude tissue preparations cannot provide definitive enzyme characterization, they can indicate the hydrolytic capabilities of the organ as well as the possible occurrence of multiple activities. Hydrolysis of cyclic AMP and cyclic GMP was measured over a substrate concentration range of 1 to 100 PM in 25day fetal liver, brain, heart, lung, and kidney homogenates, and the results were analyzed using the double-reciprocal method. Plots of cyclic AMP hydrolysis were biphasic, indicating for all tissues the possibility of two enzymes with different affinities for cyclic AMP. Extrapolation yielded
I
.
: .
enzyme levels increased to peak values at Day 25, but decreased in the newborn animal. Cyclic AMP hydrolytic activity was from one-third to one-half of cyclic GMP hydrolytic activity in all tissues except heart, where the two nucleotides were hydrolyzed at the same rate at all ages.
BRAIN .
120
APPLEMAN
500
8
400
I
:
300
:
200 z 3
.
.
:A
l
% A
44
I5
i
100
L
IS
20
z
HEART
25
30
I.-
NE
IS
20
25
LUNG
30
NB
. .
500
400 300
200 :; 100
l
I-+
IS
GESTATIONAL
AGE
I A
I
?%
20
25
30
NB
(days)
FIG. 1. Specific activity of phosphodiesterase in fetal rabbit liver, brain, heart, and lung. Fetal rabbit organs of the indicated ages were rapidly excised and homogenized, and phosphodiesterase activity was measured at 1 pi cyclic AMP (A) or cyclic GMP (0). Units are defined as picomoles of substrate hydrolyxed per minute per 0.4~ml reaction volume. Each point represents the average of duplicate determinations on pooled tissues from a separate litter of fetal rabbits. Values for newborn (NB) rabbit organs are expressed as the means plus or minus the standard errors of the means from three separate determinations.
FETAL
RABBIT
TISSUE
PHOSPHODIESTERASE
apparent Km values of approximately lo-” and 10m4M. Kinetic analysis of cyclic GMP hydrolysis by 25-day fetal rabbit heart, brain, and kidney also revealed biphasic plots, suggesting the involvement of at least two enzymes in cyclic GMP hydrolysis with apparent Km values close to 10m6 and lob5 M. Fetal lung and liver cyclic GMP phosphodiesterase exhibited linear kinetics, with the higher apparent K,,, values. Earlier fetal rabbit brain (17 day) also produced biphasic double-reciprocal plots for cyclic AMP and cyclic GMP hydrolysis. All fetal liver homogenates were biphasic for cyclic AMP, but linear kinetic plots were obtained for cyclic GMP hydrolysis by livers from a fetal age of 14 days through the newborn animal. It should again be recalled that linear kinetic plots of enzyme activity in homogenates might indicate a single enzyme or “kinetic dominance” by a high-K,,, enzyme (14), while nonlinear plots may be produced by two distinct enzymes, by a single enzyme exhibiting negative cooperativity in substrate binding (21), or by a variety of more complex situations. Regulatory
AND
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ACTIVATOR
not activated at any stage of development. A soluble phosphodiesterase activity which preferentially hydrolyzes cyclic GMP at low substrate concentrations has been demonstrated to be susceptible to activation by a low molecular weight protein in the presence of calcium ion (7). Homogenates of each of the fetal tissues were studied both for the presence of this protein (Fig. 3A) and the ability to respond to it (Fig. 3B). The concentration of the activator relative to total tissue proteins appeared to increase with development in brain, heart, and lung, but not in the fetal liver. Rabbit brain appeared to have the highest levels of activator, and heart the lowest. Phosphodiesterase activities of brain and
Properties
The homogenate activities were investigated for their responses to factors reported to influence the different enzymatic activities of mature tissues. At high substrate concentrations, each cyclic nucleotide can act as a competitive inhibitor of hydrolysis of the other by the soluble enzyme activities. In fetal rabbit tissue homogenates, at each of the ages studied, 1 PM cyclic GMP degradation was reduced by the addition of 50 PM cyclic AMP. The inhibition for the 25-day fetal organs was: liver, 64%; brain, 47%; heart, 58%; lung, 38%; and kidney, 44%.
Cyclic AMP hydrolysis by a soluble phosphodiesterase is known to be stimulated by low concentrations of cyclic GMP. Fetal rabbit liver was the tissue most sensitive to cyclic GMP stimulation (Fig. 2); at ail ages studied, cyclic AMP phosphodiesterase was activated greater than 100%. Brain and lung homogenate cyclic AMP phosphodiesterase activities were also significantly elevated in the presence of 1 PM cyclic GMP, but the enzyme in rabbit heart homogenates was
GESTATIONAL
AGE
(in
days)
FIG. 2. Effect of cyclic GMP on cyclic AMP hydrolysis by fetal tissue homogenates. Fetal rabbit organs were rapidly excised, homogenized, and assayed for cyclic AMP phosphodiesterase activity. The bars represent the increase in activity upon the addition of 1 pM unlabeled cyclic GMP to the standard assay. Each bar represents the average of two separate experiments involving pooled tissues from two different litters of rabbits at the indicated ages. All assays were done in duplicate. NB, newborn.
410
SINGER,
DUNN,
AND
APPLEMAN G
GESTATIONAL
AGE
Mays)
FIG. 3. (A) Phosphodiesterase activator levels of fetal rabbit tissues. Heat-treated extracts of the fetal organs containing 2 pg of protein were tested at each of the indicated ages with added calcium, as described under Materials and Methods. Preparations from pooled tissues of two separate litters were tested in duplicate. (B) Development of activator potential in fetal rabbit tissues. The activation of cyclic GMP hydrolysis by calcium and the protein activator was measured in homogenates of the fetal organs at the indicated ages. Basal activity tubes contained 1.0 mM EGTA. The bars indicate the percentage change in cyclic GMP hydrolysis observed on the addition of 1.25 mM calcium chloride plus 20 pg of rat brain protein activator. NB, newborn.
heart homogenates at all ages were consistently stimulated by calcium ion plus the protein activator, but homogenates of liver and lung were not activatable until the 25th day of gestation (Fig. 3B). The presence of protein activator reported in Fig. 3A was confirmed by the finding that the equivalent activitation was obtained with calcium in the absence of exogenous activator (data not shown). Interestingly, liver and lung appeared to contain significant levels of the activator even at ages when the enzyme does not seem to respond to it. Separation and Characterization of Phosphodiesterases DEAE-cellulose chromatography was used to resolve the phosphodiesterase activities of fetal rabbit heart, lung, brain, liver, and kidney. Each of these tissues exhibited three major peaks of activity as early as the organ could be identified and dissected out in quantities sufficient for chromatographic analysis (19 days of gestation for heart, 15 days of gestation for
brain and liver, 25 days of gestation for kidney and lung). These activities correspond to the enzyme identified in mature rat liver (14) and rabbit heart (7). DEAE-cellulose chromatographic profiles of 19- and 25-day fetal rabbit liver are illustrated in Fig. 4. The chromatographic profile from 25day fetal rabbit liver exhibits a striking decrease in the amount of the Fraction I cyclic GMP phosphodiesterase when compared with column profiles of livers from animals 19 days of gestational age. The profile illustrated in Fig. 4B with a smaller amount of activity in the Fraction I enzyme was observed in all chromatographic profiles from rabbit livers 24 days of gestational age and older; the larger peak was observed in liver preparations from all animals of less than 19 days. All lung column profiles, from Day 24 of gestation through the newborn rabbit, exhibited large Fraction I activities. The kinetic characteristics of the isolated enzymes from fetal liver (Fig. 5) are similar to those of the enzymes identified by Rus-
FETAL
RABBIT
TISSUE
PHOSPHODIESTERASE
AND
ACTIVATOR
FIG. 4. DEAE-cellulose chromatography of cyclic nucleotide phosphodiesterase. A 30,OOOg sonicated supernatant of the tissue extract was applied to a 1 x a-cm DEAE-cellulose column. Enzymes were eluted by a linear gradient from 0.05 to 1.0 M sodium acetate. Aliquots (ZOO ~1) of each 2-ml fraction were assayed directly for enzymatic hydrolysis of 1 j&M cyclic AMP (A-A) or cyclic GMP (M). The major peaks have been labeled Fractions I, II, and III in order of their elution from the column. (A) 19-Day fetal rabbit liver; (B) 25day fetal rabbit liver.
FIG. 5. Kinetic analysis of chromatographically separated phosphodiesterases from 19-day fetal rabbit livers. Velocity is expressed as picomoles of cyclic nucleotide hydrolyzed per minute per 0.4ml assay volume. (A) Cyclic GMP hydrolysis by the Fraction I enzyme (O---O). (B) Cyclic AMP hydrolysis by the Fraction II enzyme, in the presence (M) and absence (A-A) of 1 pM cyclic GMP. (C) Cyclic AMP hydrolysis by the Fraction III enzyme (A-A).
411
412
SINGER,
DUNN,
sell et al. (14). No change in the kinetic properties of the isolated enzymes from liver, lung, or heart was observed at any of the ages studied. Cyclic AMP hydrolysis by isolated Fraction II enzymes from 25day liver, lung, and brain were stimulated by the addition of 1 PM cyclic GMP. Fraction I enzymes isolated from 25-day fetal rabbit heart, brain, and kidney were found to be activated by the addition of protein activator plus calcium ion, but not by either alone. Fraction I enzymes from rabbit liver and lung at early ages were insensitive to the addition of calcium plus activator, whether the activator preparation used was from mature rabbit brain or from the same organ of the same age. Isolated Fraction I enzymes from adult rabbit liver were stimulated by the addition of calcium plus the protein activator. DISCUSSION
The present study demonstrates that, as early as dissection and chromatographic separation are feasible, each of the major organs of the fetal rabbit possesses all of the cyclic nucleotide phosphodiesterase forms found in mature animals, in addition to the protein activator. The presence of these activities early in fetal development suggests that they are all involved in critical aspects of cellular function. Studies of tissue homogenates provide some indication of which forms of cyclic nucleotide phosphodiesterase are present in fetal organs, but the results obtained may be influenced by the relative proportion of each activity. Cyclic AMP hydrolysis by liver homogenates is greatly stimulated by micromolar amounts of cyclic GMP because the liver contains a high level of the enzyme sensitive to this nucleotide (Fraction II). This activity in heart homogenates does not appear to be stimulated because the heart contains high levels of the other forms of cyclic AMP phosphodiesterase which are not responsive to cyclic GMP. Similarly, the assessment of “calcium sensitivity” in a homogenate is i&hienced by the relative level of the Fraction I enzyme as well as the protein activator. Separation and characterization of the phosphodiesterases provide additional im-
AND
APPLEMAN
portant information concerning the regulation of cyclic nucleotide degradation. Hormonal regulation of cyclic AMP formation can be demonstrated at an early stage in the development of the heart (3, 22); the presence of the cyclic nucleotide phosphodiesterases early in heart development suggests that they may be involved in the modulation of responses to cyclic AMP. In the rabbit and rodent brain, the appearance of the catecholamine sensitivity of adenylate cyclase is a postnatal event (9); the presence of the phosphodiesterases at a much earlier stage suggests an involvement of cyclic nucleotides in other aspects of early brain development. The early fetal liver is essentially a hematopoietic organ (1); glycogen metabolizing enzymes and the cyclic AMP second messenger system become functional in the latter third of the gestation period (23,24). This investigation reports that a decrease in the relative proportion of the cyclic GMP phosphodiesterase and the appearance of sensitivity of homogenate enzymes to calcium activation both occur at approximately this transition time. The fetal rabbit lung also undergoes maturation, possibly cyclic AMP mediated, several days prior to birth (25-27), and the appearance of calcium sensitivity of lung homogenate phosphodiesterase coincides with these events. In summary, the different forms of cyclic nucleotide phosphodiesterase are present very early in fetal development, and there are subtle changes in their relative proportions and regulatory potential as the organs develop sensitivity to hormone action. REFERENCES 1. GREENGARD, 0. (1970) in Biochemical Actions of Hormones (G. Litwack, ed.), Vol. 1, pp. 53-87, Academic Press, New York. 2. PALMER, G. C., AND DAIL, W. G. (1975) Pediat.
Res. 9,98-103. 3. MARTIN,
S., LEVEY,
B. A., ANDLEVEY,
G. S. (1973)
Biochem. Biophys. Res. Commun. 54,949-954. 4. CLARK,
C. M.,
BEATTY,
B., AND ALLEN,
D. 0.
(1973) J. Clin. Znuest. 52, 10X3-1025. 5. CHRISTOFFERSEN, T., MORLAND, J., OSNES, J. B., AND OYE, I. (1973) Biochem. Biophys. Acta 313, 338-349. 6. BARRETT, C. T., SEVANIAN, A., AND KAPLAN, S. A. (1974) Pediat. Res. 8,244-247. 7. APPLEMAN, M. M., AND TERASAKI, W. L. (1975)
FETAL
RABBIT
TISSUE
PHOSPHODIESTERASE
Advan. Cyclic Nucleotide Res. 5, 153-162. 8. TEO, T. S., WANG, T. H., AND WANG, J. H. (1973) J. Biol. Chem. 248.568-595. 9. WEISS, B., AND STRADA, S. J. (1973) in Fetal Pharmacology (L. Boreus, ed.), pp. 205-232, Raven Press, New York. 10. DAVIS, C. W., AND Kuo, J. F. (1976) Biochem. Biophys. Acta 444,554-562. 11. SMOAKE, J. A., SONG, S-Y, AND CHEUNG, W. Y. (1974) Bzbchem. Biophys. Acta 341.402-411. 12. STRADA, S. J., UZUNOV, P., AND WEISS, B. (1974) J. Neurochem. 23,1097-1103. 13. TANICAWA, Y., SHIMOYAMA, M., TAI, J., Fum, K., AND UEDA, I. (1976) Biochem. Biophys. Res. Commun. 73, 19-24. 14. RUSSELL, T. R., TERASAKI, W. L., AND APPLE. MAN, M. M. (1973) J. Biol. Chem. 258, 1334-1340. 15. LYNCH, T. J., AND CHEUNG, W. Y. (1975) Anal. Biochem. 67,130-138. 16. BOIJDREAU, R. J., AND DRUMMOND, G. I. (1975) Anal. Biochem. 63,388-399. 17. RUTTEN, W. J., SCWOOT, B. M., AND DE PONT, J. J. H. H. M. (1973) Biochem. Biophys. Acta 315,
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ACTIVATOR
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378-383. 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 19. SINGER, A. L., SHERWIN, R. P., DUNN, A. S., AND APPLEMAN, M. M. (1976) Cancer Res. 36.60-66. 20. CHEUNG, W. Y. (1971) J. Biol. Chem. 246, 2859-2869. 21. RUSSELL, T. R., THOMPSON, W. J., SCHNEIDER, F. W., AND APPLEMAN, M. M. (1972) Proc. Nat. Acad. Sci. USA 69, 1791-1795. 22. WILDENTHAL, K. (1973) J. Clin. Invest. 52, 2250-2258. 23. WATTS, C., AND GAIN, K. R. (1976) Biochem. J. 160,263-271. 24. SCHWARTZ, A. L., AND RALL, T. W. (1973) Biothem. J. 134,985-993. 25. KIKKAWA, Y., KAIBARA, M., MOTOYAMA, E. K., ORZALESI, M. M., AND COOK, C. D. (1971) Amer. J. Pathol. 64.423-442. 26. BARRETT, C. T., SEVANIAN, A., LAVIN, N., AND KAPLAN, S. A. (1976) Pediat. Res. 10,621-625. 27. BALLARD, P. L., ANDBALLARD, R. A. (1972) Proc. Nat. Acad. Sci. USA 69,2666-2672.
Vol.
OF BIOCHEMISTRY
187, No. 2, April
Cyclic
AND
BIOPHYSICS
30, pp. 406-413,
Nucleotide
Phosphodiesterase
A. L. SINGER,’ Cellular
and Molecular
1978
Biology
Rabbit
Tissues’
A. DUNN,
AND
Program, California,
Received
and Protein
Department Los Angeles,
June 29,1977;
Activator
M. M. APPLEMAN
of Biological Sciences, California 9ooo7
revised
in Fetal
January
University
of Southern
5,1978
Cyclic nucleotide phosphodiesterase activity (EC 3.1.4.17) was studied in fetal and newborn rabbit brain, heart, liver, kidney, and lung. Kinetic analysis of phosphodiesterase activity from homogenates of organs from the 25-day embryo suggested the presence of a high Km and a low K,,, activity for both cyclic AMP and cyclic GMP hydrolysis. The addition of 1 PM cyclic GMP to the assay stimulated the hydrolysis of cyclic AMP by whole homogenates of liver, brain, lung, and kidney, but not heart, at all of the ages studied. The addition of micromolar levels of calcium ion stimulated cyclic GMP hydrolysis by homogenates of fetal brain, heart, and kidney, with or without added protein activator. Cyclic GMP phosphodiesterase activity was not stimulated by the addition of calcium ion in homogenates of early fetal rabbit liver and lung, but stimulation was detected in the late embryo and newborn. The presence of the heat-stable protein activator was demonstrated in brain, heart, kidney, liver, and lung tissue at all of the fetal ages studied, and in the newborn rabbit. DEAE-cellulose chromatography demonstrated the presence of three separable enzymes in brain and liver at 15 days, heart at 19 days, and lung and kidney at 25 days of gestation, with no changes in the kinetic properties of the isolated enzymes during development. These experiments suggest that all of the organs studied have the mature array of phosphodiesterases early in development, but an enzyme from liver and lung becomes sensitive to regulatory control by calcium only late in gestation.
Many developmental events are believed to be triggered by changes in the circulating levels of appropriate hormones, possibly mediated by cyclic nucleotides and the enzymes of cyclic nucleotide metabolism. The appearance of hormone sensitivity of adenylate cyclase has been reported to occur at specific times during the development of fetal mammalian organs ( 1,2). In the rodent heart, the capacity for norepinephrine stimulation is an early property of adenylate ’ Supported by grants from the American Diabetes Association, Southern California Affiliate, and the American Heart Association, Los Angeles County Affiliate; National Institutes of Health Grants AM 16367 and AM 07215; and Biomedical Sciences Support Grant RR 07012-07. M. Michael Appleman is a NIH Research Career Development awardee. ’ Present address: Department of Pathology, University of Southern California Medical School, Los Angeles, Caliioruia 90033. 3 To whom correspondence should be addressed.
cyclase (3), while glucagon sensitivity appears postnatally at the time of anatomical maturation (4). In the fetal liver and lung, specific hormone sensitivities appear prior to increased circulating levels of these hormones, in apparent readiness to trigger new developmental phenomena (5,6). In order for a balanced response to increased cyclic nucleotide levels to exist, the means for degrading these messengers must also be present. The cyclic nucleotide phosphodiesterases (EC 3.1.4.17) of mammalian tissues have been shown to consist of at least three enzymes, differing in substrate specificity, kinetic characteristics, regulatory properties, and subcellular localization (7). A low molecular weight, heat-stable protein activator, specific for one of the phosphodiesterases, has been described (8). Within themselves, these enzymes provide considerable variation in the means of regulating cyclic nucleotide levels. 406
0003-9861/78/1872-0406$02.00/O Copyright All rights
D 1978 by Academic Press, of reproduction in any form
Inc. reserved.
FETAL
RABBIT
TISSUE
PHOSPHODIESTERASE
The presence of phosphodiesterase in late fetal and newborn rat brain (9) and liver (5) and changes in the enzyme levels in the late fetal guinea pig tissues (10) have been reported. The protein activator for phosphodiesterase has been described in the brain and liver of the late fetal and newborn rat (11, 12), and the Ca2+ and activator-sensitive enzyme has been measured in chick embryo liver (13). This study was undertaken in order to determine which of the phosphodiesterases are present in early fetal mammalian organs, and whether changes in their regulation by cyclic GMP or by Ca2+ and the protein activator occur during development. MATERIALS
AND
METHODS
Chemicals and reagents. [3H]Adenosine
3’:5’-cyclic phosphate (38 Ci/mol) and [3H]guanosine 3’:5’-cyclic phosphate (3.5 Ci/mol) were obtained from New England Nuclear Corp., Boston, Massachusetts. Cyclic AMP and crude 5’-nucleotidase (Ophiophagus hunnah or Crotalus atrox snake venom) were obtained from Sigma Chemical Co., St. Louis, Missouri. Cyclic GMP, Tris, Mops,“ and bovine serum albumin (Fraction V) were purchased from Calbiochem. La Jolla, California. EGTA was from J. T. Baker Chemical Co., Phillipsburg, New Jersey. DEAE-cellulose and the anion-exchange resin (AGl-X2, 200- to 400-mesh) were from Bio-Rad Laboratories, Richmond, California. Exchangers were routinely prepared by alternate washes of 1 N NaOH and HCl, followed by distilled water, prior to use. Tissue samples. Newborn and timed pregnant rabbits were obtained from ABC Caviary and Rabbitry, Pomona, California. Newborn rabbits were sacrificed by decapitation. Pregnant rabbits were sacrificed by air embolism and fetal tissues were removed immediately. Organs were homogenized in cold 0.05 M Tris-HCl, pH 7.4, containing 2.5 mM 2-mercaptoethanol in a Potter-Elvehjem homogenizer. Samples for chromatographic analysis were sonicated for 30 s/ml in a Biosonic 1 at low energy and clarified by centrifugation at 30,000g for 30 min. Phosphodiesteruse assay. Cyclic nucleotide phosphodiesterase activity was measured using the twostep assay system of Russell et al. (14). Assay tubes contained the indicated concentrations of unlabeled cyclic nucleotide plus 75,000 dpm of 3H-labeled cyclic nucleotide, 5 mM MgClz, 40 mM Mops-NaOH, pH 7.4, and enzyme, in a total volume of 400 pl. The enzyme 4 Abbreviations used: Mops, 4-morpholinepropanesulfonic acid; EGTA, ethylene glycol bis(P-aminoethyl ether); DEAE, diethylaminoethyl.
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ACTIVATOR
407
hydrolyzes 3’:5’-cyclic AMP or GMP to the 5’-nucleotides, which are converted to the nucleosides by snake venom 5’-nucleotidase. Unreacted substrate (cyclic AMP or cyclic GMP) is removed by the direct addition of an anion-exchange resin, and the 3H-labeled nucleoside remaining in the supernatant fluid is counted by liquid scintillation. The relatively low pH of the resin separation step (7.4) gives quite reasonable and reproducible recoveries of the nucleoside (70-75% for adenosine and 60-65% for guanosine as measured on labeled standards). All values are corrected for the loss that does occur (15, 16). Inosine is not a major by-product in these tissues, but again, the low pH allows reasonable recoveries (17). Care was taken in using this assay, as major variations in the behavior of different batches of resin have been observed. The unit of phosphodiesterase activity is picomoles of cyclic AMP or cyclic GMP hydrolyzed per minute under the assay conditions. Assay blank tubes, containing heat-denatured enzyme but otherwise identical to assay tubes, gave values of less than 1% of the radioactivity added. Enzyme dilutions were adjusted so that less than 30% of the substrate was hydrolyzed during the lo- to 20-min incubation period. Reaction rates were kept linear with respect to protein and time. Protein was measured by the method of Lowry et al. (18) using bovine serum albumin as the standard. DEAE-cellulose chromatography. DEAE-cellulose was used to separate the different phosphodiesterases. Chromatography was performed in 1 x &cm columns, employing a linear gradient from 0.05 M (the equihbration buffer) to 1.0 M sodium acetate (14). On very small samples, a stepwise gradient was used, with sequential additions of 0.2, 0.35, 0.45, and 0.65 M sodium acetate to elute the various activities (19).
Preparation and assay of the protein activator of phosphodiesterase. The activator has the capacity, in the presence of calcium ion, to stimulate the phosphodiesterase activity which preferentially hydrolyzes cyclic GMP at low substrate concentrations (7). Determinations of the level of protein activator were carried out as previously described (19), using aliquota of tissue homogenates heated for 3 min at 100°C and centrifuged at 109Og for 10 min to remove coagulated proteins. This crude preparation was diluted to normalize the protein concentration at 2.0 pg per assay. A standard calcium-sensitive phosphodiesterase for the assay of activator levels was prepared from rat brain using a fraction eluted from DEAE-cellulose at 0.2 M sodium acetate. This enzyme was not stimulated by the addition of calcium ion alone, but was activated more than 300% when rat brain protein activator was also added. Reaction tubes for the determination of activator levels contained the phosphodiesterase, 40 mM Mops-NaOH, pH 7.4,5 mM MgClx, 1.0 mM EGTA, 1.25 mM calcium chloride, 1 pM cyclic GMP, and r3HJ cyclic GMP, in a total volume of 400 al. The addition of 2 pg of activator preparation (described above) increased the basal enzyme activity from 10 to 150%.
408
SINGER,
DUNN,
AND
The assay for the calcium-sensitive enzyme contained the tissue homogenate diluted to provide about 75 pg of protein, 40 mM Mops-NaOH, pH 7.4, 5 mM MgClx, 1 pM cyclic GMP, [3H]cyclic GMP, and 1.0 rnM EGTA, in a final volume of 400 pl. The ability of the enzyme to be activated was reported as the increment in activity upon the addition of 20 cg of a standard rat brain protein activator, prepared by the method of Chueng (20), plus 1.25 mM calcium chloride to this basal assay. RESULTS
Hydrolytic Activities of Homogenates Cyclic nucleotide phosphodiesterase activities of organ homogenates of the 15-, 19-, and 25-day fetus and the newborn (31day) rabbit were measured at 1 PM substrate to approach physiological concentrations (Fig. 1). Hydrolysis of cyclic AMP and cyclic GMP by liver homogenates did not appear to change significantly during this period of development. Brain and heart activities increased phosphodiesterase steadily with the age of the animal. Lung
140
LIVER .
100 60 i
.
Kinetic Analysis of Homogenates Although kinetic analyses of crude tissue preparations cannot provide definitive enzyme characterization, they can indicate the hydrolytic capabilities of the organ as well as the possible occurrence of multiple activities. Hydrolysis of cyclic AMP and cyclic GMP was measured over a substrate concentration range of 1 to 100 PM in 25day fetal liver, brain, heart, lung, and kidney homogenates, and the results were analyzed using the double-reciprocal method. Plots of cyclic AMP hydrolysis were biphasic, indicating for all tissues the possibility of two enzymes with different affinities for cyclic AMP. Extrapolation yielded
I
.
: .
enzyme levels increased to peak values at Day 25, but decreased in the newborn animal. Cyclic AMP hydrolytic activity was from one-third to one-half of cyclic GMP hydrolytic activity in all tissues except heart, where the two nucleotides were hydrolyzed at the same rate at all ages.
BRAIN .
120
APPLEMAN
500
8
400
I
:
300
:
200 z 3
.
.
:A
l
% A
44
I5
i
100
L
IS
20
z
HEART
25
30
I.-
NE
IS
20
25
LUNG
30
NB
. .
500
400 300
200 :; 100
l
I-+
IS
GESTATIONAL
AGE
I A
I
?%
20
25
30
NB
(days)
FIG. 1. Specific activity of phosphodiesterase in fetal rabbit liver, brain, heart, and lung. Fetal rabbit organs of the indicated ages were rapidly excised and homogenized, and phosphodiesterase activity was measured at 1 pi cyclic AMP (A) or cyclic GMP (0). Units are defined as picomoles of substrate hydrolyxed per minute per 0.4~ml reaction volume. Each point represents the average of duplicate determinations on pooled tissues from a separate litter of fetal rabbits. Values for newborn (NB) rabbit organs are expressed as the means plus or minus the standard errors of the means from three separate determinations.
FETAL
RABBIT
TISSUE
PHOSPHODIESTERASE
apparent Km values of approximately lo-” and 10m4M. Kinetic analysis of cyclic GMP hydrolysis by 25-day fetal rabbit heart, brain, and kidney also revealed biphasic plots, suggesting the involvement of at least two enzymes in cyclic GMP hydrolysis with apparent Km values close to 10m6 and lob5 M. Fetal lung and liver cyclic GMP phosphodiesterase exhibited linear kinetics, with the higher apparent K,,, values. Earlier fetal rabbit brain (17 day) also produced biphasic double-reciprocal plots for cyclic AMP and cyclic GMP hydrolysis. All fetal liver homogenates were biphasic for cyclic AMP, but linear kinetic plots were obtained for cyclic GMP hydrolysis by livers from a fetal age of 14 days through the newborn animal. It should again be recalled that linear kinetic plots of enzyme activity in homogenates might indicate a single enzyme or “kinetic dominance” by a high-K,,, enzyme (14), while nonlinear plots may be produced by two distinct enzymes, by a single enzyme exhibiting negative cooperativity in substrate binding (21), or by a variety of more complex situations. Regulatory
AND
409
ACTIVATOR
not activated at any stage of development. A soluble phosphodiesterase activity which preferentially hydrolyzes cyclic GMP at low substrate concentrations has been demonstrated to be susceptible to activation by a low molecular weight protein in the presence of calcium ion (7). Homogenates of each of the fetal tissues were studied both for the presence of this protein (Fig. 3A) and the ability to respond to it (Fig. 3B). The concentration of the activator relative to total tissue proteins appeared to increase with development in brain, heart, and lung, but not in the fetal liver. Rabbit brain appeared to have the highest levels of activator, and heart the lowest. Phosphodiesterase activities of brain and
Properties
The homogenate activities were investigated for their responses to factors reported to influence the different enzymatic activities of mature tissues. At high substrate concentrations, each cyclic nucleotide can act as a competitive inhibitor of hydrolysis of the other by the soluble enzyme activities. In fetal rabbit tissue homogenates, at each of the ages studied, 1 PM cyclic GMP degradation was reduced by the addition of 50 PM cyclic AMP. The inhibition for the 25-day fetal organs was: liver, 64%; brain, 47%; heart, 58%; lung, 38%; and kidney, 44%.
Cyclic AMP hydrolysis by a soluble phosphodiesterase is known to be stimulated by low concentrations of cyclic GMP. Fetal rabbit liver was the tissue most sensitive to cyclic GMP stimulation (Fig. 2); at ail ages studied, cyclic AMP phosphodiesterase was activated greater than 100%. Brain and lung homogenate cyclic AMP phosphodiesterase activities were also significantly elevated in the presence of 1 PM cyclic GMP, but the enzyme in rabbit heart homogenates was
GESTATIONAL
AGE
(in
days)
FIG. 2. Effect of cyclic GMP on cyclic AMP hydrolysis by fetal tissue homogenates. Fetal rabbit organs were rapidly excised, homogenized, and assayed for cyclic AMP phosphodiesterase activity. The bars represent the increase in activity upon the addition of 1 pM unlabeled cyclic GMP to the standard assay. Each bar represents the average of two separate experiments involving pooled tissues from two different litters of rabbits at the indicated ages. All assays were done in duplicate. NB, newborn.
410
SINGER,
DUNN,
AND
APPLEMAN G
GESTATIONAL
AGE
Mays)
FIG. 3. (A) Phosphodiesterase activator levels of fetal rabbit tissues. Heat-treated extracts of the fetal organs containing 2 pg of protein were tested at each of the indicated ages with added calcium, as described under Materials and Methods. Preparations from pooled tissues of two separate litters were tested in duplicate. (B) Development of activator potential in fetal rabbit tissues. The activation of cyclic GMP hydrolysis by calcium and the protein activator was measured in homogenates of the fetal organs at the indicated ages. Basal activity tubes contained 1.0 mM EGTA. The bars indicate the percentage change in cyclic GMP hydrolysis observed on the addition of 1.25 mM calcium chloride plus 20 pg of rat brain protein activator. NB, newborn.
heart homogenates at all ages were consistently stimulated by calcium ion plus the protein activator, but homogenates of liver and lung were not activatable until the 25th day of gestation (Fig. 3B). The presence of protein activator reported in Fig. 3A was confirmed by the finding that the equivalent activitation was obtained with calcium in the absence of exogenous activator (data not shown). Interestingly, liver and lung appeared to contain significant levels of the activator even at ages when the enzyme does not seem to respond to it. Separation and Characterization of Phosphodiesterases DEAE-cellulose chromatography was used to resolve the phosphodiesterase activities of fetal rabbit heart, lung, brain, liver, and kidney. Each of these tissues exhibited three major peaks of activity as early as the organ could be identified and dissected out in quantities sufficient for chromatographic analysis (19 days of gestation for heart, 15 days of gestation for
brain and liver, 25 days of gestation for kidney and lung). These activities correspond to the enzyme identified in mature rat liver (14) and rabbit heart (7). DEAE-cellulose chromatographic profiles of 19- and 25-day fetal rabbit liver are illustrated in Fig. 4. The chromatographic profile from 25day fetal rabbit liver exhibits a striking decrease in the amount of the Fraction I cyclic GMP phosphodiesterase when compared with column profiles of livers from animals 19 days of gestational age. The profile illustrated in Fig. 4B with a smaller amount of activity in the Fraction I enzyme was observed in all chromatographic profiles from rabbit livers 24 days of gestational age and older; the larger peak was observed in liver preparations from all animals of less than 19 days. All lung column profiles, from Day 24 of gestation through the newborn rabbit, exhibited large Fraction I activities. The kinetic characteristics of the isolated enzymes from fetal liver (Fig. 5) are similar to those of the enzymes identified by Rus-
FETAL
RABBIT
TISSUE
PHOSPHODIESTERASE
AND
ACTIVATOR
FIG. 4. DEAE-cellulose chromatography of cyclic nucleotide phosphodiesterase. A 30,OOOg sonicated supernatant of the tissue extract was applied to a 1 x a-cm DEAE-cellulose column. Enzymes were eluted by a linear gradient from 0.05 to 1.0 M sodium acetate. Aliquots (ZOO ~1) of each 2-ml fraction were assayed directly for enzymatic hydrolysis of 1 j&M cyclic AMP (A-A) or cyclic GMP (M). The major peaks have been labeled Fractions I, II, and III in order of their elution from the column. (A) 19-Day fetal rabbit liver; (B) 25day fetal rabbit liver.
FIG. 5. Kinetic analysis of chromatographically separated phosphodiesterases from 19-day fetal rabbit livers. Velocity is expressed as picomoles of cyclic nucleotide hydrolyzed per minute per 0.4ml assay volume. (A) Cyclic GMP hydrolysis by the Fraction I enzyme (O---O). (B) Cyclic AMP hydrolysis by the Fraction II enzyme, in the presence (M) and absence (A-A) of 1 pM cyclic GMP. (C) Cyclic AMP hydrolysis by the Fraction III enzyme (A-A).
411
412
SINGER,
DUNN,
sell et al. (14). No change in the kinetic properties of the isolated enzymes from liver, lung, or heart was observed at any of the ages studied. Cyclic AMP hydrolysis by isolated Fraction II enzymes from 25day liver, lung, and brain were stimulated by the addition of 1 PM cyclic GMP. Fraction I enzymes isolated from 25-day fetal rabbit heart, brain, and kidney were found to be activated by the addition of protein activator plus calcium ion, but not by either alone. Fraction I enzymes from rabbit liver and lung at early ages were insensitive to the addition of calcium plus activator, whether the activator preparation used was from mature rabbit brain or from the same organ of the same age. Isolated Fraction I enzymes from adult rabbit liver were stimulated by the addition of calcium plus the protein activator. DISCUSSION
The present study demonstrates that, as early as dissection and chromatographic separation are feasible, each of the major organs of the fetal rabbit possesses all of the cyclic nucleotide phosphodiesterase forms found in mature animals, in addition to the protein activator. The presence of these activities early in fetal development suggests that they are all involved in critical aspects of cellular function. Studies of tissue homogenates provide some indication of which forms of cyclic nucleotide phosphodiesterase are present in fetal organs, but the results obtained may be influenced by the relative proportion of each activity. Cyclic AMP hydrolysis by liver homogenates is greatly stimulated by micromolar amounts of cyclic GMP because the liver contains a high level of the enzyme sensitive to this nucleotide (Fraction II). This activity in heart homogenates does not appear to be stimulated because the heart contains high levels of the other forms of cyclic AMP phosphodiesterase which are not responsive to cyclic GMP. Similarly, the assessment of “calcium sensitivity” in a homogenate is i&hienced by the relative level of the Fraction I enzyme as well as the protein activator. Separation and characterization of the phosphodiesterases provide additional im-
AND
APPLEMAN
portant information concerning the regulation of cyclic nucleotide degradation. Hormonal regulation of cyclic AMP formation can be demonstrated at an early stage in the development of the heart (3, 22); the presence of the cyclic nucleotide phosphodiesterases early in heart development suggests that they may be involved in the modulation of responses to cyclic AMP. In the rabbit and rodent brain, the appearance of the catecholamine sensitivity of adenylate cyclase is a postnatal event (9); the presence of the phosphodiesterases at a much earlier stage suggests an involvement of cyclic nucleotides in other aspects of early brain development. The early fetal liver is essentially a hematopoietic organ (1); glycogen metabolizing enzymes and the cyclic AMP second messenger system become functional in the latter third of the gestation period (23,24). This investigation reports that a decrease in the relative proportion of the cyclic GMP phosphodiesterase and the appearance of sensitivity of homogenate enzymes to calcium activation both occur at approximately this transition time. The fetal rabbit lung also undergoes maturation, possibly cyclic AMP mediated, several days prior to birth (25-27), and the appearance of calcium sensitivity of lung homogenate phosphodiesterase coincides with these events. In summary, the different forms of cyclic nucleotide phosphodiesterase are present very early in fetal development, and there are subtle changes in their relative proportions and regulatory potential as the organs develop sensitivity to hormone action. REFERENCES 1. GREENGARD, 0. (1970) in Biochemical Actions of Hormones (G. Litwack, ed.), Vol. 1, pp. 53-87, Academic Press, New York. 2. PALMER, G. C., AND DAIL, W. G. (1975) Pediat.
Res. 9,98-103. 3. MARTIN,
S., LEVEY,
B. A., ANDLEVEY,
G. S. (1973)
Biochem. Biophys. Res. Commun. 54,949-954. 4. CLARK,
C. M.,
BEATTY,
B., AND ALLEN,
D. 0.
(1973) J. Clin. Znuest. 52, 10X3-1025. 5. CHRISTOFFERSEN, T., MORLAND, J., OSNES, J. B., AND OYE, I. (1973) Biochem. Biophys. Acta 313, 338-349. 6. BARRETT, C. T., SEVANIAN, A., AND KAPLAN, S. A. (1974) Pediat. Res. 8,244-247. 7. APPLEMAN, M. M., AND TERASAKI, W. L. (1975)
FETAL
RABBIT
TISSUE
PHOSPHODIESTERASE
Advan. Cyclic Nucleotide Res. 5, 153-162. 8. TEO, T. S., WANG, T. H., AND WANG, J. H. (1973) J. Biol. Chem. 248.568-595. 9. WEISS, B., AND STRADA, S. J. (1973) in Fetal Pharmacology (L. Boreus, ed.), pp. 205-232, Raven Press, New York. 10. DAVIS, C. W., AND Kuo, J. F. (1976) Biochem. Biophys. Acta 444,554-562. 11. SMOAKE, J. A., SONG, S-Y, AND CHEUNG, W. Y. (1974) Bzbchem. Biophys. Acta 341.402-411. 12. STRADA, S. J., UZUNOV, P., AND WEISS, B. (1974) J. Neurochem. 23,1097-1103. 13. TANICAWA, Y., SHIMOYAMA, M., TAI, J., Fum, K., AND UEDA, I. (1976) Biochem. Biophys. Res. Commun. 73, 19-24. 14. RUSSELL, T. R., TERASAKI, W. L., AND APPLE. MAN, M. M. (1973) J. Biol. Chem. 258, 1334-1340. 15. LYNCH, T. J., AND CHEUNG, W. Y. (1975) Anal. Biochem. 67,130-138. 16. BOIJDREAU, R. J., AND DRUMMOND, G. I. (1975) Anal. Biochem. 63,388-399. 17. RUTTEN, W. J., SCWOOT, B. M., AND DE PONT, J. J. H. H. M. (1973) Biochem. Biophys. Acta 315,
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ACTIVATOR
413
378-383. 18. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 19. SINGER, A. L., SHERWIN, R. P., DUNN, A. S., AND APPLEMAN, M. M. (1976) Cancer Res. 36.60-66. 20. CHEUNG, W. Y. (1971) J. Biol. Chem. 246, 2859-2869. 21. RUSSELL, T. R., THOMPSON, W. J., SCHNEIDER, F. W., AND APPLEMAN, M. M. (1972) Proc. Nat. Acad. Sci. USA 69, 1791-1795. 22. WILDENTHAL, K. (1973) J. Clin. Invest. 52, 2250-2258. 23. WATTS, C., AND GAIN, K. R. (1976) Biochem. J. 160,263-271. 24. SCHWARTZ, A. L., AND RALL, T. W. (1973) Biothem. J. 134,985-993. 25. KIKKAWA, Y., KAIBARA, M., MOTOYAMA, E. K., ORZALESI, M. M., AND COOK, C. D. (1971) Amer. J. Pathol. 64.423-442. 26. BARRETT, C. T., SEVANIAN, A., LAVIN, N., AND KAPLAN, S. A. (1976) Pediat. Res. 10,621-625. 27. BALLARD, P. L., ANDBALLARD, R. A. (1972) Proc. Nat. Acad. Sci. USA 69,2666-2672.
