Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea

ARCHIVES

OF BIOCHEMISTRY

Vol. 185, No. 1, January

Receptors

AND BIOPHYSICS

15, pp. 126-133, 1978

for Gonadotropins and Prostaglandins Bovine Corpora Lutea S. MITRA

Departments

of

Obstetrics-Gynecology

AND

CH.

and Biochemistry, Louisville, Kentucky

v.

in Lysosomes

of

RAO’

University 40202

Received June 24, 1977; revised August

of Louisville

School

of Medicine,

25, 1977

The total mitochondrial fraction of bovine corpus luteum specifically bound 13Hlprostaglandin (PG) E,, 13HlPGF,a, and ‘251-labeled human lutropin (hLH) despite very little 5’-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also resulted in 13HlPGE,, 13HlPGF,ol, and lz51-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results in addition to plasma membranes, suggest that lysosomes, but not mitochondria, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.

Recent work from our laboratory demonstrated that subcellular fractions, in addition to plasma membranes, exhibited 13Hlprostaglandin (PGYE,, L3HlPGFZa, and Y-labeled human lutropin (hLH) specific binding (1). Although not as high as plasma membranes, enrichment of binding was also observed in the mitochondrial fraction (1). Since the mitochondrial fractions isolated by conventional centrifugation techniques contain lysosomes as well, it was difficult to ascertain whether the observed binding was due to ’ To whom enquiries should be addressed. 2 Abbreviations used: PG, prostaglandin; hLH, human lutropin; hCG, human choriogonadotropin; [‘2511-, lZSI-labeled. 126 0003-9861/78/1851-0126$02.00/O Copyright 0 197s by Academic Press, Inc. All rights of reproduction in any form reserved.

mitochondria and/or lysosomes. Therefore, we attempted to subfractionate the mitochondrial fraction into lysosomes and mitochondria, assess their relative purity and cross-contamination with appropriate marker enzymes, and examine the [3H]PGs and [‘9]hLH specific binding. The results indicate that lysosomes in the mitochondrial fraction contributed to the observed PG and gonadotropin binding. MATERIALS AND METHODS Unlabeled PGE, and PGF,or were generously supplied by Dr. John Pike of the Upjohn Co. The following items were purchased from the indicated commercial sources: [3HlPGE, (sp act, 89.5 Ci/ mmol), 13HlPGF,ol (sp act, 179 Ci/mmol), and 13HlUDP-galactose (sp act, 14.6 Cilmmol) from New England Nuclear Corp.; carrier-free lz51-labeled Na

GONADOTROPIN

AND

PROSTAGLANDIN

from Amersham/Searle Corp.; sodium salt of AMP, cytochrome c, disodium phenyl phosphate, 4-aminoantipyrine, N-acetylglucosamine, sodium succinate, NADH, phenolphthalein mono-P-glucuronic acid, and phenolphthalein from Sigma Chemical Co.; Gelman Metricel filters (0.45pm pore size) from Scientific Products. Procedures for checking the purity of [3HlPGs and their purification, if needed, were the same as those described earlier for 13HlPGF,a except the solvent system, i.e., toluene:dioxane:acetic acid (2O:lO:l) (2). Unlabeled human lutropin (hLH) (generously donated by Dr. L. Reichert, Jr.; LER-960, 4620 IU/mg) and unlabeled human choriogonadotropin (hCG) (a gift from the Center for Population Research, NICHD; CR119; 11,600 IU/mg) were iodinated by the lactoperoxidase technique essentially as described by Miyachi et al. (3). The specific activities of [lZ51]hLH and [iZSI]hCG were 80.4 and 56.3 &iIwg, respectively. In the presence of excess plasma membranes, 25.1% of the added [12”I]hLH and 48.3% of the added [iZ511hCG were specifically bound. The rest of the details on iodination were the same as described before for hCG (4). Bovine corpora lutea have been shown to be functionally active (5, 6) and contain receptors for PGE,, PGF,a, and hCG (7, 8) throughout pregnancy; therefore, they have been selected for fractionation studies. Corpora lutea of pregnancy were collected in a local slaughterhouse and placed in ice-cold homogenizing buffer which consisted of 10 mM Tris-HCl, pH 7.3, 250 mM sucrose, and 1 mM Ca’+. The luteal tissue was scraped off from the rest of the ovarian tissue and homogenized with a Polytron homogenizer (PCU-2-110) at a setting of 6 using three 10-s bursts. The homogenates were filtered through four layers of cheesecloth. After saving an aliquot of homogenate, the rest was subjected to differential and discontinuous sucrose gradient centrifugations to obtain mitochondrial and plasma membrane fractions (9). The so-called F, and F,, plasma membrane fractions (9) were pooled. The mitochondria at this stage were designated as the total mitochondrial fraction. After saving an aliquot of this fraction, the rest was subjected to flotation discontinuous sucrose density gradient centrifugation to separate lysosomes from mitochondria (10, 11). The mitochondrial and lysosomal fractions were separately collected. The protein in an aliquot of the subcellular fractions was determined, following digestion (l), according to Lowry et al. (12) using bovine serum albumin as the standard. The inorganic phosphate (in the 5’nucleotidase assay) was measured according to Fiske and Subbarow (13) using KH,PO, as the standard. Cytochrome c oxidase was assayed according to Cooperstein and Lazarow (14), 5’-nucleotidase according to Emmelot and Bos (15), acid phosphatase

LYSOSOMAL

RECEPTORS

127

according to Kind and King (16), NADH-cytochrome c reductase according to Mahler (171, galactosyl transferase using [3HlUDP-galactose according to Treloar et al. (la), succinic dehydrogenase according to Green et al. (19), p-glucuronidase according to Ignarro (20), and catalase according to Beers and Sizer (21). The conditions for the above marker enzyme assays were, however, standardized with respect to assay buffer (0.1% Triton X-100 was present in the case of acid phosphatase and galactosyl transferase), time, and amount of subcellular fraction protein to use so that linear reaction rates were obtained. The enzyme specific activities were calculated from linear rate data. Lysosomes and plasma membranes were frozen by immersing in a dry ice-acetone bath and were thawed by placing in water at room temperature. The above fractions were also preincubated for 1 h at 4°C with 0.05% Triton X-100. Following the above procedures, the fractions were centrifuged at 131,000 g for 1 h, washed (only Triton X-loo-treated lysosomes and membranes), and resuspended in homogenizing buffer. The control lysosomes and plasma membranes were handled in a similar manner in every respect except freezing and thawing and addition of Triton X-100. Freezing and thawing resulted in 21.8 (5x) and 36.8% (20x) losses, whereas Triton X-100 exposure resulted in a 24.1% loss of lysosomal protein. The corresponding values for plasma membranes were: 3.4 (5x), 8.4 (20x), and 13.1% (Triton X-100 exposure). The binding studies were conducted with 100 pg of protein at 38°C for 1 h for 13HlPGE,, 22°C for 2 h for 13H]PGF,a, and 38°C for 2 h for 1’2511gonadotropins. About 0.1 &i of 13HlPGs was used per tube in the binding studies. Because of the differences in specific activities, the concentrations were different for each prostaglandin, i.e., -5 nM [“HlPGF*a and -10 nM [3H]PGE,. The concentration of [““Ilgonadotropins used in the binding studies was 1.0 x 10-i’ M. The separation of bound and free 13HlPGs and [12jI]gonadotropins was accomplished by filtration through Metricel filters, the details of which are the same as those described earlier (2, 22, 23). Nonspecific binding was determined in each experiment using the same amount of protein, [3H]PGs, and [iz51]gonadotropins as were used in the total binding tubes but in the presence of excess corresponding unlabeled ligands (PGs, 2.8 x 10e5 M; hCG, 1.3 x lo-’ M). We could not use unlabeled hLH to assess [‘251]hLH nonspecific binding because of a lack of sufficient hormone. Furthermore, use of unlabeled hCG for this purpose appears to be valid primarily because l’251]hLH at the concentrations used appears to bind to the same sites as [1251]hCG but with lower affinity (24). The magnitude of nonspecific binding was essentially the same in all of the subcellular fractions. The specific binding

128

MITRA

was the difference between total and nonspecific binding. The femtomoles bound was calculated using molecular weights of 354 for [“HIPGs and 30,000 for [Y]hLH. The enzyme activities were determined using two different protein concentrations, and binding experiments were run in quadruplicates. Each experimental value represents the means and its standard error of 4 to 16 independent observations in two to four experiments. RESULTS

AND

AND

RAO

H TM M LY PM

-

HOMOGENATE TOTAL MITOCHONORIA MITOCHONORIA LYSOSOMES PLASMA MEMBRANES

DISCUSSION

Figure 1 shows that 5’-nucleotidase, a marker for plasma membranes, was enriched, with respect to homogenate, in plasma membranes, cytochrome c oxidase, a marker for mitochondria, was enriched in total mitochondria and mitochondria, and acid phosphatase, a marker for lysosomes, was enriched in lysosomes. Succinic dehydrogenase and /?-glucuronidase, marker enzymes for mitochondria and lysosomes, respectively, were also measured in some of the above experiments (data not shown). Succinic dehydrogenase activity paralleled cytochrome c oxidase, and p-glucuronidase activity paralleled acid phosphatase. Although the above marker enzymes were markedly enriched in the appropriate fractions, they were either undetectable or detectable only to a minor extent in other fractions. These results suggest that the above isolated subcellular fractions were relatively pure with little or no detectable cross-contamination among them. When [3HlPGE,, 13HlPGF,a, and [ lz51]hLH specific bindings were examined in these subcellular fractions, the binding of all three ligands was greatly enriched in plasma membranes (Fig. 2). Although not as high as in plasma membranes, the total mitochondrial fraction also exhibited enrichment of binding despite a lack of 5’nucleotidase activity enrichment (see Fig. 1). The subfractionation of the total mitochondria resulted in decreased binding in mitochondria and increased binding in lysosomes. The lysosomal binding was enriched 2.7- to 3.0-fold depending on the ligand despite the lack of 5’-nucleotidase activity enrichment. Although enrichment data in Figs. 1 and 2 were very impressive, the recovery

FIG. 1. Marker enzyme distribution in subcellular fractions of bovine corpus luteum. Each bar in Figs. 1 to 3 represents the mean and its standard error. ND denotes nondetectable.

data were less impressive (Table I). Generally there is better recovery of marker enzymes compared to 13HlPGs and [lz5]hLH binding. Despite the overall poor recoveries, marker enzyme recoveries were the highest in appropriate fractions and binding recovery was the highest in plasma membranes. Similar to the trend observed in Fig. 2, the subfractionation of total mitochondria resulted in a drastic decrease in recovery of receptors in mitochondria and relatively high recovery in lysosomes. The lysosomal recovery was,

GONADOTROPIN

H TM M LY PM

H

-

AND

PROSTAGLANDIN

HOMOGENATE TOTAL MITOCHONORIA MITOCHONDRIA LYSOSOMES PLASMA MEMBRANES

TM

M

LY

PM

FIG. 2. The specific binding of ?HlPGE,, [3HlPGF,~, and [‘Z511hLH to subcellular fractions of bovine corpus luteum.

however, considerably lower than that of total mitochondria. This discrepancy appears primarily due to losses because there was also a loss of about 41% total mitochondrial protein during subfractionation. Although the data on overall low recoveries are disturbing, it must be realized, however, that fractionation procedures employed in these studies were designed to give maximal purity with severe compromises on the yield. Therefore, severe losses could partly explain the poor recoveries. Problems such as this are commonly encountered in studies of this type re-

LYSOSOMAL

RECEPTORS

129

ported in the literature (9, 25). Therefore, the recovery data, at this time, should be viewed with caution. However, it should be noted that, using either the specific activity or the recovery, the point emerges very clearly that binding observed in total mitochondria was primarily due to lysosomes. The residual binding in mitochondria could not be due to plasma membranes because there was no detectable 5’-nucleotidase activity in this fraction. Instead, this binding could be attributable to lysosomal contamination because there was a detectable acid phosphatase activity. It has been reported earlier that detergent administration to animals was a prerequisite for complete separation of lysosomes from mitochondria (11). This was not done in our experiments for obvious reasons, which might explain residual lysosomal contamination in mitochondria. Since lysosomes were not totally free from 5’-nucleotidase activity, the possibility that lysosomal binding was due to minor contamination with plasma membranes should be considered. If this were the case, the ratios of 5’-nucleotidase activity to binding should be the same for plasma membranes and lysosomes for each ligand. On the contrary, as can be seen in Table II, the ratios were widely different for plasma membranes and lysosomes. Therefore, the binding observed in lysosomes should be intrinsic to this organelle. Since lysosomes intrinsically contain PGs and gonadotropin receptors, the question of whether plasma membrane binding was due to lysosomal contamination should be raised. The use of the above reasoning, i.e., using ratios between acid phosphatase activity to binding in lysosomes and plasma membranes, makes it clear that plasma membrane binding could not be explained by lysosomal contamination. Since the binding enrichment and recovery were higher in plasma membranes compared to lysosomes (see Fig. 2) and they do not correlate with acid phosphatase or 5’-nucleotidase activities (see Fig. 1 and Table II), respectively, these findings should only be taken to mean that plasma membranes contain a greater number of PGs and gonadotropin receptors

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MITRA

AND TABLE

RECOVERY

OF PROTEIN,

MARKER

ENZYMES,

FRACTIONS

Parameter

OF BOVINE

Total mitochondria

TABLE

5.6 2.7 64.3 22.7 11.3 7.5 9.1

ENZYMES AND AND LYSOSOMES

Ratio” 5’-NE in PM binding in PM

2.8

2.9

13.0

5’-NE in LY binding in LY

0.4

0.3

0.8

AP in PM binding in PM

0.08

0.08

0.4

AP in LY binding in LY

4.3

3.0

8.6

u The specific activities were used in calculating the ratios. NE, 5’-nucleotidase; PM, plasma membranes; LY, lysosomes; AP, acid phosphatase.

as compared to intact lysosomes (however, to be described shortly, lysosomes overall contain a greater number of PGs and gonadotropin receptors than plasma membranes), and both were intrinsic to these organelles. In this communication, the assumption was made that 5’-nucleotidase and receptors were uniformly distributed on the luteal cell surface. Although this assumption is by no means unequivocal at this time, it should be considered valid until such time that plasma membrane regions rich in either receptors or 5’-nucleotidase are shown to exist. Furthermore, if the above situation should prevail, it is logical to expect that these specialized plasma membrane regions contaminate subcellular organelles at random. This means that one should also be able to see 5’-nucleotid-

BINDING

IN SUBCELLULAR

CORPUS LUTEUM

recovery

Mitochondria 1.3 0 18.9 0.6 0.2 0.5 0.8

II

THE RATIOS BETWEEN MARKER BINDING IN PLASMA MEMBRANES

I

AND [3HIPGs AND [““I]hLH

Percentage

Protein 5’-Nucleotidase Cytochrome c oxidase Acid phosphatase [“HIPGE, binding 13HIPGF,o binding [‘*“I]hLH binding

RAO

with respect to homogenate Lvsosomes

Plasma membranes

1.4 1.4 0.6 42.0 4.3 4.1 3.7

1.2 36.0 0 2.6 14.8 9.4 5.6

ase activity without any detectable binding in some other organelles. However, we have not seen significant 5’-nucleotidase activity enrichment in any subcellular organelle other than plasma membranes. But, similar to lysosomes, we found that Golgi vesicles and rough endoplasmic reticulum exhibited binding enrichment despite little or no 5’-nucleotidase activity. We recently discovered that Golgi vesicles and rough endoplasmic reticulum prepared from the total endoplasmic reticulum fraction exhibited [3H]PGs and [12511hCGbinding enrichment with respect to homogenate, despite very little or no detectable 5’-nucleotidase activity (Ref. 26; manuscript in preparation). These fractions were also enriched with appropriate marker enzymes, i.e., galactosyl transferase in Golgi vesicles and NADH-cytochrome c reductase in rough endoplasmic reticulum (Ref. 26; manuscript in preparation). The question arises, therefore, of whether lysosomal binding can be explained by contaminating Golgi vesicles and rough endoplasmic reticulum in lysosomes. To examine the above possibility, galactosyl transferase and NADH-cytochrome c reductase activities were measured in lysosomes. Catalase, a marker enzyme for peroxisomes, was also measured. Table III shows that galactosyl transferase was detectable but was not enriched. The other two enzymes (NADHcytochrome c reductase and catalase) were undetectable in lysosomes. Therefore, these data suggest that lysosomal binding could not be explained by possible contamination with Golgi vesicles, rough endo-

GONADOTROPIN TABLE

AND

PROSTAGLANDIN

131

RECEPTORS

III

GOLGI, ROUGH ENDOPLASMIC RETICULUM, PEROXISOME MARKER ENZYME ACTIVITIES PURIFIED LYSOSOMES

Marker

LYSOSOMAL

enzyme

Galactosyl transferase NADH-cytochrome c reductase Catalase

AND IN

51” t

[?il

300 t

[‘-I]

PGE,

Enrichment with respect to homogenate (n-fold) 0.79 0 0

plasmic reticulum, or peroxisomes. Further convincing evidence that lysosomal and plasma membrane binding was truly intrinsic to these organelles comes from the data presented in Fig. 3. Freezing and thawing five times had greatly increased 13HlPGs and slightly increased [‘2sIlhCG binding to lysosomes. The increase in binding to lysosomes was even more dramatic when the freezing and thawing cycles were increased to 20 times. Contrary to lysosomes, freezing and thawing cycles had little or no effect on plasma membrane binding. The increase in lysosomal binding following freezing and thawing was most likely due to a greater number of receptors in the same amount of protein in treated fractions as a result of protein losses. The finding of a marked increase in lysosomal binding with concomitant losses of protein (see Materials and Methods) due to freezing and thawing infers that PG and gonadotropin receptors are associated with the membranes but not the content of lysosomes. Since the increased bindings do not strictly parallel protein losses, the increased bindings indicate the presence of latent PG and gonadotropin receptors. These latent receptors may be present on the inside of the lysosomal membranes which become exposed due to freezing and thawing. In view of the presence of latent PG and gonadotropin receptors in lysosomes but not in plasma membranes, lysosomes overall should be considered to contain a greater number of receptors compared to plasma membranes. Pretreatment of lysosomes and plasma membranes with 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. Decreases in binding by Triton

1 hCG

-I CONTROL

5X Frozen LL T”aved

20X Frozen 8 Thawed

Exporurc to O.O5%Triton

X-100

FIG. 3. Effect of freezing and thawing and exposure to Triton X-100 on specific binding of 13H]PGE,, [3H]PGF,a, and [iz51]hCG to lysosomes and plasma membranes. The specific binding in control lysosomes and plasma membranes was taken as 100%. Although only one bar is shown for the control, there were separate controls for plasma membranes and lysosomes for freezing and thawing and for exposure to Triton X-100 (see Materials and Methods for further details). ND denotes nondetectable.

X-100 were due to receptor solubilization (27). The lower lysosomal binding losses compared to plasma membranes were due to a relatively greater number of receptors in a given amount of lysosomal protein and/or to differential solubilization of lysosomal and plasma membranes sites. Although the above data convincingly demonstrate that lysosomes, in addition to plasma membranes, contain receptors for PGE,, PGF2q and gonadotropins, at the present time we have neither the evidence for hormone entry (relevant in the

132

MITRA

case of PGF,o and gonadotropins) into bovine luteal cells to bind to lysosomes under physiological conditions nor the data to support that these sites were indeed physiological receptors and not just binding sites. The answers to the points raised above are being actively sought at the present time in our laboratory. We previously reported on the detailed characterization (with respect to affmity, specificity, and other factors) of PG and gonadotropin receptors in plasma membranes (2, 22, 28). Such detailed characterization studies were not completed on lysosomal receptors. Such studies, which are in progress in our laboratory, are anticipated to shed some light on whether or not lysosoma1 and plasma membrane receptors are similar. The possible physiological significance of the presence of gonadotropin and PGs receptors in bovine corpus luteum lysosomes can be only speculative at this time. There is a growing belief that membrane receptors are internalized and catabolized in lysosomes. If this were true, the presence of receptors in lysosomes should not be a totally unexpected finding. Besides the catabolic mechanism, a lysosomal receptor representing a in the biosynthetic cycle sequence 2 I L (Golgi + plasma membranes + lysosomes) and/or possibly contributing to tissue responsiveness to gonadotropins and PGs should also be considered possible unless proven otherwise. There is already some evidence for lysosomal involvement in steroid and protein hormone action (29). Finally, it should be pointed out that there are three recent reports in the literature which presented evidence for lysosomal uptake (30,311 and lysosomal binding sites (25) for proteins that were once believed to bind exclusively to the cell surface. ACKNOWLEDGMENTS We thank Mr. Fred Carman, Jr., for his excellent help in some of the binding experiments. This work was supported by Grant HD09557 from the National Institutes of Child Health and Human Development. This work was presented at the 28th Annual Fall Meeting of the American Physiological Society (32).

AND

RAO REFERENCES

1. RAO, CH. V., AND MITRA, S. (1977) Biochem. Biophys. Res. Commun. 76, 636-643. 2. RAO, CH. V. (1976) Mol. Cell Endocrinol. 6, l16. 3. MIYACHI, Y., VAITUKAITIS, J. L., NEISCHLAG, E., AND LIPSETT, M. B. (1972) J. Clin. Endocrinol. Metab. 34, 23-28. 4. RAO. CH. V.. GRIFFIN, L. P., AND CARMAN, F. R.‘, JR. (19;7) Amer. J. Obstet. Gynecol. 128, 146-153. 5. STORMSHAK, F., AND ERB, R. E. (1961) J. Dairy Sci. 44, 310-320. MILLS, R. C., AND MORRISSETTE, M. C. (1970) J. Reprod. Fertil. 22, 435-440. RAO, CH. V. (1975) Fertil. Steril. 26, 1185-1189. RAO, CH. V. (1976) Biol. Reprod. 15, 134-139. GOSPODAROWICZ, D. (1973) J. Biol. Chem. 248, 5050-5056. LO. TROUET, A. (1964) Arch. Intern. Physiol. Biothem. 72, 698-699. 11. LEIGHTON, F., POOLE, B., BEUFAY, H., BAUDHIN, P., CAFFEY, J. W., FOWLER, S., AND DE DUVE, C. (1968) J. Cell Biol. 37, 482-513. 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275.

13. FISKE, C. H., AND SUBBAROW, Y. (1925) J. Biol. Chem. 66, 375-400. 14. COOPERSTEIN, S. J., AND LAZAROW, A. (1951) J. Biol. Chem. 189, 665-670. 15. EMMELOT, P., AND Bos, C. J. (1966) Biochim. Biophys. Acta 120, 369-382. 16. KIND, P. R. N., AND KING, E. J. (1954) J. Clin. Pathol. 7, 322-326. 17. MAHLER, H. R. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.) Vol. 2, 688-693, Academic Press, New York. 18. TRELOAR, M., STURGESS, J. M., AND MOSCARELLO, M. A. (1974) J. Biol. Chem. 249, 66286632. 19. GREEN, D. E., MII, S., AND KOHOUT, R. M. (1955) J. Biol. Chem. 217, 551-567. 20, 20. IGNARRO, L. J. (1971) Biochem. Pharmacol. 2847-2860. 31 BEERS, R. F., JR., AND SIZER, I. W. (1952) J. --. Biol. Chem. 195, 133-140. 22. RAO, CH. V. (1974) J. Biol. Chem. 249, 72037209. 23. RAO, CH. V. (1975) Mol. Cell Endocrinol. 3, 255-271. 24. RAO, CH. V. (1977) Endocrinology (suppl.) 100, 171 (abstr.). 25. PRICER, W. E., JR., AND ASHWELL, G. (1976) J. Biol. Chem. 251, 7539-7544. 26. MITRA, S., AND RAO, CH. V. (1977) Physiologist 20, 64 (abst).

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PROSTAGLANDIN

27. RAO, CH. V. (1977) Life Sci. 20, 2013-2022. 28. RAO, CH. V. (1974) J. Biol. Chem. 249, 28642872. 29. &EGO, C. M. (1974) Recent Progr. Hormone Res. 30, 171-222. 30. CHEN, T. T., ABEL, J. H., JR., MCCLELLAN, M. C., DIEKMAN, M. A., AND NISWENDER, G. D.

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(1976) in The Program of the Ninth Annual Meeting of the Society for the Study of Reproduction, Abstr. No. 41. 31. ASCOLI, M., AND PUETT, D. (1977) FEBS Lett. 75, 77-82. 32. RAO, CH. V., AND MITRA, S. (1977) Physiologist 20, 78 (abst).