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Characteristics of prolactin binding to rat liver plasma membranes Silverstein, Alan Michael 1978

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CHARACTERISTICS OF PROLACTIN BINDING TO RAT LIVER PLASMA MEMBRANES by ALAN MICHAEL SILVERSTEIN Sc., York University, Toronto, Ontario, THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 19 77 fT) Alan Michael S i l v e r s t e i n , 1977 i n In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I further agree that permission for extensive copying of th i s thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thesis fo r f inanc ia l gain sha l l not be allowed without my written permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date srflj^r- Ho/7 7 i i ABSTRACT Binding s i t e s for p r o l a c t i n have been i d e n t i f i e d and characterized in a plasma membrane enriched f r a c t i o n i s o -lated from l i v e r s of mature female rats. By chemical and enzymatic analysis the membrane preparation was shown to have s l i g h t contamination with nuclei and endoplasmic reticulum, while mitochondria were not detected. Sided-ness analysis indicated that the membrane preparation was 125 largely composed of inside-out v e s i c l e s . I-oPRL pre-pared by the lactoperoxidase method had a s p e c i f i c a c t i v i t y of 40-60 ^(Ci/^g. Competition studies using iodoprolactin indicated that iodination of the hormone did not a f f e c t i t s a f f i n i t y for the receptor as compared to the native hormone. 125 Binding of I-oPRL was inhi b i t e d by p r o l a c t i n from various species including ovine, bovine and rat p r o l a c t i n while bGH, pACTH and AVP had no e f f e c t on binding. The binding of 125 I-oPRL was activated by both bivalent and monovalent cations - bivalent cations exerting a greater e f f e c t than monovalent cations. In the presence of 10 mM CaCl 2 , binding 125 of I-oPRL was equal to the binding i n the presence of the physiological concentration of NaCI. The association of 125 I-oPRL with the membrane was a time and temperature dependent process, being maximal at 37°. The d i s s o c i a t i o n 125 of I-oPRL was time and temperature dependent only with 150 mM NaCl at 3 7-° while at a l l other temperatures and in 1 i i i the presence of 10 mM CaCl 2 d i s s o c i a t i o n was not.observed. 125 The binding of I-oPRL was strongly influenced by pH with an optimum observed at pH 6.5. Receptor a c t i v i t y was des-troyed by pronase and phospholipase C, while neuraminidase increased binding. Treatment of the membranes by RNase and 125 DNase did not e f f e c t the binding. Binding of I-oPRL was in h i b i t e d by p-chloromercuribenzoic acid, d i t h i o t h r e i t o l , and by b r i e f exposure to high temperatures. Scatchard anal-125 y s i s of the binding of I-oPRL to receptors indicates that p r o l a c t i n has a high a f f i n i t y for i t s receptor. iv ACKNOWLEDGEMENTS The work i n this thesis was performed under the super-v i s i o n of Dr. J. F. Richards; his assistance and advice was greatly appreciated. I would also l i k e to thank Mr. A r i s t o t l e Azad for his technical assistance and for the many enlighten-ing discussions on biomembranes. V TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS i v LIST OF TABLES v i i LIST OF FIGURES y i i i LIST OF ABBREVIATIONS ix INTRODUCTION 1 General 1 In t r a c e l l u l a r s i t e of p r o l a c t i n receptors.... 2 Mechanism of action 5 Pro l a c t i n receptors i n normal tissue 9 A. Mammary gland 10 B. Ovary 12 C. Liver 13 The present investigation 16 MATERIALS 18 METHODS • 19 1. Isolation of rat l i v e r plasma membranes 19 2. Marker assays for subcellular fractions 21 a. 5 '-nucleotidase 21 b. Glucose-6-phosphatase 21 c. Succinate dehydrogenase 22 d. Diphenylamine test for DNA 23 v i Page 3. Sidedness Assays 24 a. Acetylcholinesterase 24 b. S i a l i c acid 25 125 4. Preparation of I-oPRL 26 5. Preparation of iodoprolactin 27 6. Radioreceptor assay . 28 7. Radioimunoassay 29 8. Polyacrylamide gel electrophoresis 30 RESULTS 31 A. Fractionation and characterization of LPO-iodinated oPRL 31 B. Characterization of rat l i v e r plasma membranes 37 C. Activation of binding by cations 40 D. Effects of time and temperature on the binding and d i s s o c i a t i o n of 1 2 5I-oPRL 44 125 E. S p e c i f i c i t y of binding of I-oPRL to isolated rat l i v e r plasma membranes.... 50 125 F. E f f e c t of pH on the binding of I-oPRL.. 52 G. E f f e c t of membrane protein concentration.. 54 125 H. E f f e c t of I-oPRL concentration 57 I. E f f e c t of enzymes and i n h i b i t o r s 57 DISCUSSION 62 CONCLUSION 69 BIBLIOGRAPHY 70 v i i LIST OF TABLES TABLE PAGE I D i s t r i b u t i o n of subcellular markers in fractions i s o l a t e d from rat l i v e r homogenate 3 8 II D i s t r i b u t i o n of sidedness markers in p u r i f i e d rat l i v e r plasma membranes 41 III E f f e c t of cations on rate and quantity of binding of 1 2 5I-oPRL 47 IV E f f e c t of enzyme treatment on binding 60 V E f f e c t of i n h i b i t o r s on binding 61 v i i i LIST OF FIGURES FIGURE PAGE 1. Time course of the e f f e c t of pr o l a c t i n on the a c t i v i t i e s of protein kinase i n mammary explants 6 2. Time course of the e f f e c t of p r o l a c t i n on various molecular synthetic events in mammary explants 6 125 3. Fractionation of I-oPRL by gel f i l t r a t i o n 32 4. Displacement of antibody-bound 1 2 5I-oPRL by unlabelled oPRL 34 125 5. D i s t r i b u t i o n of I-oPRL and unlabelled oPRL on a polyacrylamide gel 35 6. S t a b i l i t y of 1 2 5I-oPRL v.s. time 36 7. Activation of binding by cations 43 8. E f f e c t of time and temperature on binding 125 of I-oPRL to rat l i v e r membranes 45-9. E f f e c t of time and temperature on di s s o c i a -••J 125 tion of I-oPRL from rat l i v e r membranes.... 49 125 10. S p e c i f i c i t y of binding of I-oPRL to rat l i v e r membranes 51 125 11. E f f e c t of pH on the binding of I-oPRL to rat l i v e r membranes -. 53 12. E f f e c t of pH on the displacement of 125 membrane bound I-oPRL by iodoprolactin and native p r o l a c t i n 55 13. E f f e c t of membrane protein concentration on binding 56 125 14. E f f e c t of I-oPRL concentration on binding 58/ LIST OF ABBREVIATIONS oPRL - ovine p r o l a c t i n 12 5 125 I-oPRL - I-la b e l l e d ovine p r o l a c t i n bPRL - bovine p r o l a c t i n rPRL - rat p r o l a c t i n bGH - bovine growth hormone pACTH - porcine adrenocorticotropic hormone AVP - arginine vasopressin RRA - radioreceptor assay RIA - radioimunoassay LPO - lactoperoxidase 51-AMP - 5' adenosine monophosphate DTNB - 5,5 1-dithiobis-(2-nitrobenzoic acid) PAGE - polyacrylamide gel electrophoresis pCMBA - p-chloromercuribenzoic acid DTT - d i t h i o t h r e i t o l DNA - deoxyribonucleic acid RNA - ribonucleic acid Na/K ATPase - sodium and potassium activated adenosine triphosphatase BSA - bovine serum albumin mM - millimolar /,( moles - micro moles f moles - femto moles nm - nanometer ng - nanogram partition coefficient specific counts bound total counts in incubation specific counts bound in absence of unlabelled hormone inorganic phosphate standard deviation standard error of the mean INTRODUCTION It i s generally accepted that i n mammals the p r i n c i p a l target tissue for p r o l a c t i n i s the mammary gland. P r o l a c t i n receptors from mammary gland have been isol a t e d and charac-t e r i z e d (1) and th e i r role i n the regulation of l a c t a t i o n i s becoming c l a r i f i e d (2). I t has recently been shown that p r o l a c t i n binds to tissues other than mammary gland (3). Work of a more preliminary nature has been carried out on these lactogenic binding s i t e s , however, the significance of this binding i s d i f f i c u l t to assess since; (i) these receptors bind a family of hormones and; ( i i ) the s i t e and mechanism of action and metabolic effects have not been i d e n t i f i e d . Also,binding studies i n other tissues made use of the receptor assay o r i g i n a l l y derived for binding studies i n the mammary gland. I t may be possible that t h i s methodology i s not applicable to other tissues. The p r i n c i p l e s involved i n evaluating hormone receptors have been derived from the study of the int e r a c t i o n of peptide hormones with s p e c i f i c antibodies. Labelled hormone binds to a receptor and can be progressively displaced by increasing concentrations of unlabelled hormone which competes for the limited number of binding s i t e s . S t r u c t u r a l l y d i s s i m i l a r hormones cannot e f f e c t t h i s displacement even when present i n huge excess. Thus the s p e c i f i c i t y of the receptor can be evaluated. 2 The procedure used i n most p r o l a c t i n binding studies has been described by Shiu et a l (4). Membranes, are'incub-125 ated with l a b e l l e d hormone, usually I l a b e l l e d ovine p r o l a c t i n , i n the presence of 10 mM MgCl2- or C a C l 2 . The reaction i s terminated by cooling and membrane-bound and free hormone are separated by centrifugation. Using t h i s method s p e c i f i c p r o l a c t i n binding has been detected i n a number of tissues. These hormone-receptor interactions have provided presumptive evidence for a role of the hor-mone i n the target organ function. Although a role for p r o l a c t i n i n lactogenesis has been reasonably well estab-lished the physiological function of p r o l a c t i n receptors in other tissues i s not well understood. C e l l u l a r Site of Action As with many other peptide hormones p r o l a c t i n i s thought to bind to a s p e c i f i c surface membrane receptor of a target c e l l . This binding supposedly triggers an i n t r a c e l l u l a r series of events culminating i n the tissue response without entry of the hormone into the c e l l . Although t h i s theory has been tested using several d i f f e r e n t experimental tech-niques i t has not yet been confirmed. The retention of b i o l o g i c a l a c t i v i t y i n an i n v i t r o system by p r o l a c t i n coupled to an insoluble matrix suggested that receptor s i t e s were located on the external surface of the c e l l membrane (5). However, recent evidence has shown that the hormonal response could be attributed to a small 3 amount of pr o l a c t i n that became s o l u b i l i z e d during the i n -cubation period (6). This does not mean that p r o l a c t i n enters the c e l l but neither does i t prove that p r o l a c t i n does not do so. Even more s t r i k i n g was the demonstration that prolac-t i n , at physiological concentrations, could stimulate RNA synthesis i n nuclei isolated from rat and mouse mammary glands ( 7 ) . This e f f e c t was not detected i n nuclei i s o l -ated from other tissues which bind p r o l a c t i n suggesting that p r o l a c t i n may have a d i f f e r e n t mechanism of action i n these tissues. However, i t i s not known to what extent these effects observed with isolated nuclei r e f l e c t events which occur i n the inta c t c e l l . Other investigations were carried out using h i s t o l o g i c a l techniques. Autoradiographic studies of mammary glands i s o l -125 ated from rabbits injected with I - lab e l l e d p r o l a c t i n or of mammary explants incubated with the lab e l l e d hormone, showed a selective l o c a l i z a t i o n of r a d i o a c t i v i t y on or near the serosal side of the alveolar secretory c e l l membrane (8). Irrespective of method of administration or time i n t e r -v a l no r a d i o a c t i v i t y was ever found l o c a l i z e d i n the cytoplasm nor i n and around the nucleus. In contrast, immunohisto-chemical staining for endogenous p r o l a c t i n in rat mammary glands detected the presence of pr o l a c t i n both on the c e l l membrane and in the cytoplasm of the alveolar c e l l s (9). 4 Since no b i o l o g i c a l end point was measured i n these i n v e s t i -gations i t i s impossible to prove whether p r o l a c t i n was acting i n t r a c e l l u l a r l y , or was there passively, or was there as a r e s u l t of being metabolized. It i s worth noting that immunoreactive p r o l a c t i n i s present i n milk (10.y '11) and the p o s s i b i l i t y exists that p r o l a c t i n , l i k e immunoglobulins, enters the alveolar c e l l s on i t s way into the milk. The importance of prolactin-membrane interactions was investigated i n studies using antiserum against a p u r i f i e d receptor (2). P r o l a c t i n receptors, from mammary gland, pur-i f i e d by a f f i n i t y chromatography (12) were used to produce guinea pig antiserum against them. This antiserum s p e c i f i -c a l l y i n h i b i t e d the binding of pr o l a c t i n to i t s membrane receptors, and also s e l e c t i v e l y blocked prolactin-stimulated casein synthesis and amino acid transport, but was without e f f e c t on the binding of i n s u l i n and insulin-mediated events in cultured mammary explants. These results support the hypothesis that the membrane structures which bind p r o l a c t i n are essential for mediating several actions of the hormone. However, the problem of whether p r o l a c t i n enters the c e l l or not i s s t i l l not resolved by these r e s u l t s . I t i s possible that the receptor i s a s p e c i f i c protein which transports p r o l a c t i n into the c e l l where i t carr i e s out i t s b i o l o g i c a l function. I t i s worth noting that for p r o l a c t i n , i n contrast to other polypeptide hormones, no receptor-second messenger system has yet been i d e n t i f i e d . 5 Mechanism of Action The metabolic effects of many polypeptide hormones are mediated by adenyl cyclase. Limited evidence has suggested that p r o l a c t i n exerts i t s e f f e c t i n mammary gland by a di f f e r e n t mechanism (13). There i s limited evidence that in the prostate, p r o l a c t i n may act by elevating c y c l i c AMP concentrations (14) however no increase i n c y c l i c AMP i n response to p r o l a c t i n could be found i n the mammary gland (13). In mammary explants the e a r l i e s t response to prolac-t i n was the induction of the protein kinase to which c y c l i c nucleotides bind (Fig. 1) (15). This induction was rapid, with half maximal leve l s being reached within 30 min., and maximum levels i n 1 - 2 hrs. Subsequently, marked increases in the rates of phosphorylation of proteins was observed, with plasma membrane proteins reaching a maximum l e v e l by 8 hrs., ribosomal proteins showing a maximum value at 16 hrs. and nuclear proteins by 24 hrs. P a r a l l e l i n g these events a c h a r a c t e r i s t i c sequence of molecular biosynthetic processes take place (Fig. 2) (15). During the f i r s t 20 min. there was an increase i n the rate of synthesis of rapidly l a b e l l e d nuclear RNA, followed closely by an increased rate of ribosomal RNA synthesis. The appearance of an increased population of polysomes i n the cytoplasm i s i n close temporal association with the synthesis of casein, which i s undetectable u n t i l 4 hours after addition of p r o l a c t i n . F i n a l l y , a f t e r 12 hours HOURS AFTER ADDITION OF PROLACTIN Figure 1. Time course of the e f f e c t of p r o l a c t i n on the a c t i v i t i e s of.protein kinase assayed with c y c l i c AMP and c y c l i c AMP-binding protein i n r e l a t i o n to the phosphorylation of proteins i n ribosomes • • , plasma membranesO O , and nucleus #)—• i n mammary explants (15) . HOURS AFTER ADDITION OF PROLACTIN Figure 2. Time course of the eff e c t s of p r o l a c t i n on various t r a n s c r i p t i o n a l events i n the induction of casein and the galactosyl transferase and oC - l a c -talbumin •? components of lactose synthetase i n mouse mammary explants. O——O / RNA polymerase;* • , nRNA-H; Q-T--• , P-labelled c a s e i n ; , ! — • / polysomes; x-x, rRNA- H; A A , galactosyl" transferase; A A , ot -lactalbumin (15) . 7 of incubation, increases in the enzymatic a c t i v i t i e s of the two components of lactose synthetase, the galactosyl trans-ferase and (x. -lactalbumin were observed. Thus the response of mammary explants to p r o l a c t i n results i n a co-ordinated, time-dependent set of i n t r a c e l l u l a r events, beginning with the stimulation of the c y c l i c AMP dependent protein kinase. However evidence has shown that p r o l a c t i n cannot stimulate adenyl cyclase i n isola t e d mammary e p i t h e l -i a l c e l l membranes, nor can c y c l i c AMP substitute for the action of p r o l a c t i n (13). Therefore i t seems un l i k e l y that the generation of c y c l i c AMP represents a rate l i m i t i n g step in the action of p r o l a c t i n . The mechanism by which receptor a c t i v a t i o n by p r o l a c t i n serves to stimulate t r a n s c r i p t i o n i s s t i l l unknown. It has been suggested that the response to p r o l a c t i n i n the mammary gland may be mediated by both polyamines and prostaglandins (16). Prolactin-stimulation of RNA synthesis i n mammary explants could be abolished by i n h i b i t o r s of prostaglandin biosynthesis while addition of prostaglandins restored the a c t i v i t y (17). Prostaglandins by themselves, however, had no e f f e c t on casein synthesis. In a similar manner i t was found that p r o l a c t i n stimulation of casein synthesis could be blocked by i n h i b i t o r s of spermidine synthesis while addition of spermidine restored the a c t i v i t y (16). Spermi-dine by i t s e l f could stimulate phosphorylation of casein (18) 8 but not the formation of nascent chains. Spermidine i n combination with prostaglandins, however, resulted in a p r o l a c t i n l i k e stimulation of casein synthesis. The mechanism by which receptor a c t i v a t i o n serves to stimulate these c e l l u l a r processes i s s t i l l uncertain. One theory i s that the action of p r o l a c t i n on RNA synthesis i n the mammary gland may be triggered by an a c t i v a t i o n of mem-brane-associated phospholipase A, with the consequential release of unsaturated free fatty acids and the synthesis of protaglandins (19). However, the data i n favour of t h i s theory i s questionable, since phospholipase A a c t i v i t y could only be stimulated by extremely high concentrations of pro-l a c t i n . These conditions may present a completely distorted picture of the biochemical effects of the hormone, because i t has previously been shown that effects which are c l e a r l y apparent at low p r o l a c t i n concentrations disappear, or even become reversed at high ones (7, 14). An alternate mechanism suggested by recent evidence, could be through the mediation of ornithine decarboxylase (ODC) a c t i v i t y brought about by a p r o l a c t i n induced a l t e r a -t i o n of i n t r a c e l l u l a r cation concentrations (20, 21, 22). ODC i s the rate l i m i t i n g enzyme in the pathway which leads to the formation of the polyamines putrescine, spermidine and spermine. There i s considerable evidence that these polyamines have a d i r e c t e f f e c t on t r a n s c r i p t i o n and trans-l a t i o n i n a number of eukaryote systems (23). P r o l a c t i n , at 9 physiological concentrations has been shown to stimulate ODC a c t i v i t y in mammary explants (21, 24) causing an increase i n i n t r a c e l l u l a r spermidine concentrations (18, 21). In two independent studies i t was shown that ODC a c t i v i t y was influenced by i n t r a c e l l u l a r cation concentrations (22, 25) and i n another that these concentrations could be altered by a d i r e c t e f f e c t of p r o l a c t i n on ouabain sensitive Na/K ATPase (20). It may then be possible that p r o l a c t i n acts by stimula-ti n g the Na/K ATPase which causes a change i n the concentration of i n t r a c e l l u l a r cations, ac t i v a t i n g ODC a c t i v i t y which i n turn results i n elevated polyamine concentrations. Since there i s only a single homogenous population of p r o l a c t i n receptors i n the mammary gland (1, 26) i t would appear unlike l y that pro-l a c t i n would stimulate Na/K ATPase and phospholipase A through separate receptor interactions. It may be possible that one i s stimulated as a consequence of the other. I t may also be possible that d i f f e r e n t actions of p r o l a c t i n are mediated through d i f f e r e n t second messengers. Pr o l a c t i n Receptors in Normal Tissues Pr o l a c t i n binding has been found i n many tissues, includ-ing mammary gland, ovary, uterus, l i v e r , kidney, seminal vesi c l e s and adrenal cortex. In general, most p r o l a c t i n bind-ing s i t e s have shown a s p e c i f i c i t y for lactogenic hormones, including human growth hormone and placental lactogen. Pro-l a c t i n seems to induce i t s own receptor in some tissues, 10 while i n others steroids appear to be the c o n t r o l l i n g factor. Of the tissues i n which s p e c i f i c p r o l a c t i n receptors have been found, more detailed studies of mammary, ovary and l i v e r receptors have been made. A. Mammary Gland Sp e c i f i c receptors for p r o l a c t i n have been demonstrated in rabbit (1), rat (26) and mouse (27) mammary gland. The receptors were s p e c i f i c for lactogenic hormones only. They w i l l bind p r o l a c t i n i s o l a t e d from other species as well as placental lactogen and human growth hormone. This finding i s consistent with reports by Forsyth (28) and Frantz et a l (29) that both human growth hormone and placental lactogen produce lactogenic responses i n i n v i t r o mouse mammary gland assays while growth hormones from other species had no e f f e c t s . Several other peptide hormones, f o l l i c l e stimulating hormone, l u t e i n i s i n g hormone, chorionic gonadotrophin, thyroid stimula-ting hormone, adrenocorticotrophin, i n s u l i n and glucagon had no e f f e c t on the binding of p r o l a c t i n to either i s o l a t e d plasma membranes (4) or in t a c t cultured c e l l s (27). Time course studies showed that at 23° binding' reached a maximum after 2 hrs. with whole cultured c e l l s (26) and 6 hrs. with iso l a t e d membranes (1). Using a p u r i f i e d soluble receptor p r o l a c t i n binding had not reached a plateau after 20 hours (12). Dissociation rates were influenced i n a simi-l a r manner. The shortest h a l f - l i f e was obtained with whole 11 c e l l s (27) while other subcellular preparations had much slower d i s s o c i a t i o n rates. These differences may be a t t r i -buted to many factors including synthesis and degradation of receptors i n whole c e l l s and ioni c conditions of the buffers used. The preparation of the receptors may have an influence also since i t has been shown that the a f f i n i t y constant for a soluble mammary receptor was fi v e times higher than the part i c u l a t e receptor (12). Receptor a c t i v i t y could be destroyed by treatment with trypsin or phospholipase C (1) indicating that protein and phospholipid constituents are stru c t u r a l components that are necessary for receptor a c t i v i t y . Steroid hormones, nucleotides and other low molecular weight compounds had no e f f e c t on pro-l a c t i n binding to isola t e d mammary e p i t h e l i a l c e l l membranes (1) suggesting that these substances do not play any s i g n i f i c a n t role i n the formation of the hormone receptor complex. The binding capacity of the mammary gland p a r a l l e l s the serum p r o l a c t i n concentrations during pregnancy and l a c t a t i o n (26, 30). Both serum p r o l a c t i n concentrations and mammary receptor levels remain low during pregnancy but increase dramatically near term. Immediately after b i r t h and during the following 20 days receptor levels remain f i v e to seven times higher than i n non pregnant rats. P r o l a c t i n and progesterone both appear to influence the pro l a c t i n binding capacity in mammary gland (30). Injection of pseudopregnant rabbits with p r o l a c t i n resulted i n a f i v e 12 fol d increase i n receptor s i t e s but administration of proges-terone abolished t h i s e f f e c t . This observation was consistent with the fact that progesterone i n h i b i t s lactogenesis (31). However, i n ovariectomized and hysterectomized rats, proges-terone had no e f f e c t on p r o l a c t i n binding, suggesting that other factors which may be influenced by progesterone may play a role i n the regulation of mammary receptors. B. Ovary Sp e c i f i c binding s i t e s for p r o l a c t i n have been demonstra-ted i n rat, cow, human (32) and porcine (33) ovaries. The s p e c i f i c i t y of these receptors i s similar to that of mammary gland p r o l a c t i n receptors. The ovary bound a l l lactogenic hormones, placental lactogen and human growth hormone included but f o l l i c l e stimulating hormone and l u t e i n i s i n g hormone had no ef f e c t on binding. Binding of pr o l a c t i n to ovary receptors was also time and temperature dependent. Equilibrium was reached after 4 to 5 hrs. in the porcine ovary while i n the rat ovary equilibrium was reached within 2 hrs. at 37°. The.binding capacity of rat ovaries varied during the oestrus cycle (32) with binding capacity at i t s lowest during metoestrus but i n -creasing rapidly during dioestrus. A maximum was reached at pro-oestrus followed by a rapid decrease at oestrus. These values p a r a l l e l e d serum concentrations of p r o l a c t i n and l u t e i n -i s i n g hormone which are low throughout the cycle, except during pro-oestrus, where the serum concentrations of these two hormones increase ten fo l d (34). During pregnancy i n the 13 rat, ovarian binding capacity for pr o l a c t i n increases only s l i g h t l y as do the serum concentrations of p r o l a c t i n (34). These c y c l i c a l fluctuations of receptor s i t e s suggests a role for p r o l a c t i n in the control of ovarian function. Recent evidence has shown that p r o l a c t i n receptor lev e l s i n the rat ovary are influenced by l u t e i n i s i n g hormone but not pr o l a c t i n (35, 36). Depletion of serum p r o l a c t i n or adminis-t r a t i o n of large amounts of the hormone had no e f f e c t on ovar-ian p r o l a c t i n receptors in rats (36). However, administration of l u t e i n i s i n g hormone i n the absence of p r o l a c t i n resulted i n a f i v e f o l d increase i n p r o l a c t i n receptors. Subsequent administration of p r o l a c t i n resulted i n an increased capacity to produce progesterone (36). These results would explain the requirement for p r o l a c t i n during embryo implantation and the i n i t i a l growth phases of the fetus (37), since progesterone i s necessary for these developments. C. Liver A role for p r o l a c t i n i n l i v e r was f i r s t recognized when dwarf mice, d e f i c i e n t in p r o l a c t i n , showed a stimulation of hepatic RNA synthesis following i n j e c t i o n with the hormone (38). It was subsequently shown that l i v e r s of female rats, rabbits, sheep, pigeons and frogs contained s p e c i f i c l a c t o -genic binding s i t e s (3). Males of these same species did not have these s p e c i f i c receptors. Hepatic p r o l a c t i n receptors demonstrated the same s p e c i f i c i t y as shown by mammary and ovary receptors. They bound a l l lactogenic hormones including plac-ental lactogen and human growth hormone, while a l l other polypeptide hormones showed no a c t i v i t y (3, 39). Age and pregnancy had a dramatic e f f e c t on l i v e r binding capacity (40). In f e t a l rat l i v e r the binding cap-aci t y was very low compared to adult female rats but binding increased three f o l d 40 days afte r b i r t h . The maternal l i v e r showed no s i g n i f i c a n t increase in binding capacity from early to mid-pregnancy but by day 20 of pregnancy p r o l a c t i n binding had more than doubled. The p o s s i b i l i t y , as suggested by these observations, that sex steroids might play a role i n the regu-l a t i o n of hepatic binding capacity was demonstrated by Friesen and co-workers (39). Administration of estrogen induced a marked increase i n p r o l a c t i n binding to l i v e r s of i n t a c t but not hypophysectomized rats. Further investigation revealed that estrogen was stimulating p r o l a c t i n secretion and that the p r o l a c t i n was inducing i t s own hepatic receptors (41). The influence of other hormones was also investigated. , Gelato and co-workers (42) found that thyroidectomy decreased p r o l a c t i n binding i n the rat l i v e r . Administration of thy-roxine returned p r o l a c t i n binding a c t i v i t y i n l i v e r s of thyroidectomized rats to the l e v e l observed in in t a c t controls. The mechanism by which the thyroid influences p r o l a c t i n binding s i t e s i n the l i v e r i s unknown. However, i t i s known that thy-roidectomy can decrease synthesis of l i v e r proteins i n general (43) and i t i s possible that proteins from p r o l a c t i n binding si t e s also may have been decreased. Since only the binding of p r o l a c t i n was investigated i t i s unknown whether the e f f e c t of thyroidectomy i s s p e c i f i c for lactogenic hormones or i f i t 15 a f f e c t s s p e c i f i c b i n d i n g s i t e s f o r a l l other hormones at the same time. S t e r o i d hormones, other than estrogens, appear to have a d i r e c t i n f l u e n c e on the l e v e l of h e p a t i c l a c t o g e n i c r e c e p t o r s (44). A d m i n i s t r a t i o n of C o r t i s o l , t e s t o s t e r o n e or medroxy-progesterone s i g n i f i c a n t l y reduced the number of h e p a t i c b i n d -ing s i t e s f o r p r o l a c t i n i n normal and o v a r i e c t o m i z e d female r a t s . These hormones had no e f f e c t on serum p r o l a c t i n concen-t r a t i o n s and were as e f f e c t i v e i n estrogen t r e a t e d hyperpro-l a c t i n e m i c r a t s as i n animals with normal p r o l a c t i n c oncentra-t i o n s . The o b s e r v a t i o n t h a t C o r t i s o l decreased b i n d i n g c o r r e l a t e s with another independent study which showed t h a t adrenalectomy caused i n c r e a s e d b i n d i n g of p r o l a c t i n to r a t l i v e r membranes (45). These e f f e c t s appear to be s p e c i f i c f o r l a c t o g e n i c hormones s i n c e the same r e s u l t s were obtained with both p r o l a c t i n and human growth hormone wh i l e i n s u l i n b i n d i n g was not a f f e c t e d . The e f f e c t s of i n h i b i t o r s of p r o t e i n and RNA s y n t h e s i s on b i n d i n g have a l s o been i n v e s t i g a t e d (45). Actinomycin D, an i n h i b i t o r of RNA s y n t h e s i s , had no e f f e c t on r e c e p t o r formation i n estrogen primed r a t s . T h i s suggests t h a t the messenger RNA i n v o l v e d i n the s y n t h e s i s of the b i n d i n g s i t e s f o r l a c t o g e n i c hormones has a r e l a t i v e l y long h a l f - l i f e . In c o n t r a s t cycloheximide, an i n h i b i t o r of t r a n s l a t i o n , s i g n i f i -c a n t l y reduced b i n d i n g i n estrogen primed r a t s , r e a c h i n g the lowest a f t e r 3 h r s . and r e t u r n i n g to c o n t r o l l e v e l s w i t h i n 16 24 - 48 h r s . T h i s suggests t h a t the r e c e p t o r p r o t e i n s have an extremely s h o r t h a l f - l i f e and must be c o n t i n u o u s l y renewed. I t i s s t i l l u n c e r t a i n as to what f u n c t i o n p r o l a c t i n p l a y s i n h e p a t i c metabolism. Recent work has shown t h a t p r o l a c t i n may s t i m u l a t e o r n i t h i n e decarboxylase a c t i v i t y (46) and the pr o d u c t i o n o f somatomedin (47). I t may a l s o r e g u l a t e estrogen r e c e p t o r l e v e l s (48). These o b s e r v a t i o n s suggest a b i o l o g i -c a l l y important r o l e f o r h e p a t i c l a c t o g e n i c b i n d i n g s i t e s . The Present I n v e s t i g a t i o n The p r o l a c t i n r e c e p t o r from mammary gland has been i s o l a -ted and i t s b i n d i n g c h a r a c t e r i s t i c s have been analyzed (1). However, the c h a r a c t e r i s t i c s of p r o l a c t i n b i n d i n g i n other t i s s u e s has not been s y s t e m a t i c a l l y s t u d i e d . I t i s now be-coming e v i d e n t t h a t p r o l a c t i n may p l a y an important r o l e i n r e g u l a t i n g l i v e r metabolism and modulating the l i v e r ' s r e -sponse to other hormones. T h e o r e t i c a l l y a s i n g l e primary event, the b i n d i n g of p r o l a c t i n to a s p e c i f i c r e c o g n i t i o n s i t e i n i t i a t e s the sequence o f steps which culminate i n the hormonal response. The r e f o r e f a c t o r s which i n f l u e n c e the p r o l a c t i n - r e c e p t o r i n t e r a c t i o n would a l t e r the hormonal r e s -ponse of the t i s s u e . In t h i s i n v e s t i g a t i o n the c h a r a c t e r i s t i c s of p r o l a c t i n b i n d i n g to i s o l a t e d r a t l i v e r plasma membranes was s t u d i e d . The i n f l u e n c e of v a r i o u s f a c t o r s which have been shown to e f f e c t b i n d i n g i n the mammary gland have a l s o been examined. Previous s t u d i e s on p r o l a c t i n b i n d i n g to other t i s s u e s 17 were carried out using a receptor assay which was derived for mammary gland (4). This assay made use of a crude un-characterized membrane preparation and was performed i n the presence of 10 mM CaC^- Since the normal serum concentration of calcium i s approximately 2.5 mM, there i s no certainty that the r e s u l t s of these investigations are applicable to binding under physiological conditions. Therefore, the present inves-t i g a t i o n was carr i e d out using a r e l a t i v e l y pure, well charac-terized rat l i v e r plasma membrane preparation. Binding assays were carr i e d out both i n physiological ionic conditions and i n the presence of 10 mM CaC^. The findings are compared with published data on p r o l a c t i n binding by preparations from mammary gland. 18 MATERIALS AND METHODS M a t e r i a l s Ovine, bovine and r a t p r o l a c t i n s were obtained from the N a t i o n a l I n s t i t u t e of A r t h r i t i s and M e t a b o l i c Diseases, N a t i o n a l I n s t i t u t e s of Hea l t h , Bethesda, Md., U.S.A. Bovine growth hormone was purchased from M i l e s L a b o r a t o r i e s and a r g i n i n e v a s o p r e s s i n was purchased from Spectr ;um Chemical I n d u s t r i e s . P o r c i n e a d r e n o c o r t i c o t r o p h i c hormone, g l u c o s e -6-phosphate, d i t h i o t h r e i t o l , a c e t y l t h i o c h o l i n e c h l o r i d e , 5 , 5 1 - d i t h i o b i s - 2 - n i t r o b e n < z o i c a c i d , sodium metaperiodate, t h i o b a r b i t u r i c a c i d , neuraminidase and phospholipase C were obtained from Sigma Chemical Company. Lactoperoxidase, bov-vin e serum albumin ( f r a c t i o n V ) , normal r a b b i t serum ( l y o p h i l -ized) , deoxyribonuclease, r i b o n u c l e a s e and pronase were ob-t a i n e d from Calbiochem. Rabbit anti-oPRL, 5'-AMP and human gamma g l o b u l i n ( f r a c t i o n II) were obtained from Schwarz-Mann. Cyclohexanone, sodium a r s e n i t e and 30% hydrogen peroxide were obtained from M a l l i n k r o d t Laboratory Chemicals. H i g h l y p o l y -merized c a l f thymus DNA was s u p p l i e d by Worthington B i o c h e m i c a l C o r p o r a t i o n . P o l y e t h y l e n e g l y c o l 6000 was purchased from J.T. Baker Chemical Company. T r i t o n X-100 was purchased from K. & K. L a b o r a t o r i e s . P a r a c h l o r o m e r c u r i benzoic a c i d was purchased from Kent L a b o r a t o r i e s . Acetaldehyde was purchased from E a s t -125 man Kodak. Na I ( c a r r i e r free) was purchased from New England Nuclear. Sephadex G-100 was purchased from Pharmacia 19 Fine Chemicals and the ingredients for polyacrylamide gel electrophoresis were purchased from Bio-Rad Laboratories. A l l other chemicals and reagents used were purchased from Fisher S c i e n t i f i c Company. Female Wistar rats were obtained from the University of B r i t i s h Columbia animal unit. Methods 1. Isolation of Rat Liver Plasma Membranes Rat l i v e r plasma membranes were prepared by the method of N e v i l l e (49) as described by Emmelot et a l (50). Three mature female Wistar rats (200-300g each) were k i l l e d by a blow on the head and t h e i r l i v e r s , corresponding to about 30g wet weight of tissue, were removed immediately and c h i l l e d i n ice cold ImM sodium bicarbonate pH^ 7. 5 . V L A l l : further;., operations were carried out at 0-4°C with p r e c h i l l e d materials. Portions corresponding to about 5g of l i v e r were minced with scissors and homogenized in 20 ml of bicarbonate using a Potter-Elvehjem homogenizer. The pestle was driven at about 600 r.p.m. during the 3 - 4 up-and-down movements. The homogenized l i v e r prepera-tions were coll e c t e d i n a beaker and dil u t e d to a f i n a l volume of 250 ml with bicarbonate medium and vigorously s t i r r e d for 2 minutes. The homogenate was then centrifuged at 1500g for 10 minutes i n a Sorvall RC-2B refrigerated centrifuge, after which the fat t y layer f l o a t i n g at the surface and the super-natant were removed-with-,suction.. Ten ml bicarbonate medium was added to each tube and the p e l l e t was suspended in 20 t o t o by s t i r r i n g with a g l a s s rod. F u r t h e r suspension was c a r r i e d out u s i n g a Potter-Elvehjem; ihomogenizer with 3 up-and-down movements with the p e s t l e .remaining s t a t i o n a r y . The suspensions were again c e n t r i f u g e d f i r s t a t lOOg f o r 5 minutes and then the c e n t r i f u g e was a c c e l e r a t e d to lOOOg f o r 10 minutes. The r e s u l t i n g p e l l e t c o n s i s t e d of two e a s i l y d i s t i n g u i s h a b l e l a y e r s : an upper l a y e r of l o o s e l y packed membranes, pa l e tan i n appearance and a lower much l a r g e r l a y e r c o n s i s t i n g o f dark red n u c l e a r m a t e r i a l and other d e b r i s . The supernatant was drawn o f f by a p i p e t t e with water-pump a s p i r a t i o n . Bicarbonate medium was l a y e r e d over the p e l l e t and the f l u f f y upper l a y e r was suspended w i t h a g l a s s rod l e a v i n g the bottom l a y e r i n t a c t . The suspended membranes were t r a n s f e r r e d with a Pasteur p i p e t t e to the homogenizer and made up to 20 mis wit h b i c a r b o n a t e med-ium and f u r t h e r suspended w i t h three s t r o k e s . The lower n u c l e a r p e l l e t was then resuspended a l s o u s i n g 3 up-and-down s t r o k e s and the procedure was repeated i n order t o e x t r a c t more membranes. The r e s u l t i n g membrane suspension was then r e c e n t r i f u g e d at lOOg f o r 5 min. then lOOOg f o r 10 min. The supernatants were removed l e a v i n g behind a p e l l e t which c o n s i s t e d of a f l u f f y l a y e r of plasma membranes above a small amount of n u c l e a r m a t e r i a l which stuck to the bottom of the tube. The f l u f f y l a y e r was suspended, homogenized as before and c e n t r i f u g e d f o r 5 min. a t lOOg then 10 min. a t 1000. The washing procedure was repeated. A f t e r - t h e l a s t c e n t r i f u g a t i o n the supernatant was no longer t u r b i d . The r e s u l t i n g creamy white p e l l e t was suspended i n b i c a r b o n a t e medium using the homogenizer. An a l i q u o t of the membrane sus-21 pension was s o l u b i l i z e d by heating i n IN NaOH for 60 min on a 60° water bath. Protein was determined by the Lowry procedure (51). The remainder was stored frozen at -20° at a f i n a l protein concentration of about 3.0 mg/ml. When re-quired for assay the suspensions were thawed and the p a r t i c l e s were dispersed using an a l l glass hand driven homogenizer immediately before use. 2. Marker Assays for Subcellular Fractions a. 5' - Nucleotidase 5' - Nucleotidase a c t i v i t y was assayed as described by Widnell (52). Incubations were carried out at 37° for 10 minutes i n a buffer containing 0.IM Tris-HCl pH 8.5, 10 mM 5'AMP, 10 mM MgCl 2 and 30-100 ^ g protein i n a f i n a l volume of 1.0 ml. Control incubations were carried out under the same conditions with enzyme i n the absence of 5'AMP, and 5'AMP i n the absence of enzyme. The re-action was terminated by the addition of 2 ml ice cold 8% TCA. Assay tubes were centrifuged at 3000 g for 5 minutes to remove protein. Aliquots of the supernatants were removed and assayed for inorganic phosphate by the method of Ames (53). b. Glucose-6-Phosphatase Glucose-6-phosphatase a c t i v i t y was assayed by the method of Aronson and Touster (54). Incubations were carried out at 37° for 10 min. i n a buffer containing 18 mM h i s t i d i n e pH 6.5, 20 mM glucose-6-phosphate, 4mM 22 EDTA, 60-200 yfg protein and 2 mM NaF, to i n h i b i t acid phosphatase (55), i n a f i n a l volume of 1.0 ml. Control incubations were carried out under the same conditions with enzyme i n the absence of glucose-6-phosphate and glucose-6-phosphate i n the absence of enzyme. The re-action was terminated by the addition of 2 mis ice cold 8% TCA. Assay tubes were centrifuged and inorganic phos-phate was assayed for as described for 5' nucleotidase, c. Succinate Dehydrogenase Succinate dehydrogenase a c t i v i t y was assayed as des-cribed by Veeger et a l (56). The reaction took place i n a 3 ml spectrophotometer cuvette thermostated at 25° in a buffer containing 0.06M K 2HP0 4 pH 7.6, 0.6 mM EDTA, 25 mM succinate, 0.06% (wt/vol) BSA, 3 mM K-,Fe (CN) r and 0.6 mM KCN i n a f i n a l volume of 2.5 ml. The reaction was i n i t i a t e d by the addition of enzyme and the decrease i n absorbance was measured at 455 nm using a G i l f o r d 240 recording spectrophotometer. A blank rate, a l l reagents except succinate, was determined separately. In t h i s reaction 1 mole of succinate reduces 2 moles of K^Fe(CN)g. Knowing that the extinction c o e f f i c i e n t for K_Fe(CN) c at 455 nm i s 150 M cm ^ the rate of oxidation of succinate was determined by the following equation; ^^(+) succinate succinate 455 455 X 2.5 X 1 = moles succinate oxid. 150 1000 2 min. 23 d. Diphenylamine T e s t f o r DNA The presence of DNA was t e s t e d i n the membrane p r e p a r a t i o n s as an i n d i c a t i o n of n u c l e a r contamina-t i o n . The procedure used was s i m i l a r to t h a t o f S e i b e r t (57). i . E x t r a c t i o n of DNA To an a l i q u o t o f t i s s u e p r e p a r a t i o n c o o l e d to 0° .on i c e was • added -.an equal volume.'..of i c e c o l d 10% TCA. The mixture was c e n t r i f u g e d a t 2000g f o r 10 minutes and the supernatant was d i s -carded. The p e l l e t was suspended i n 10% NaCl and p l a c e d i n a b o i l i n g water bath f o r 15 minutes. The suspension was c e n t r i f u g e d a t 2,000g f o r 10 minutes and the p e l l e t was d i s c a r d e d . To the supernatant was added 2.5 volumes of i c e c o l d 95% e t h a n o l and the mixtures were s t o r e d a t -20° o v e r n i g h t . The s o l u t i o n s were then c e n t r i f u g e d at 2,000g f o r 15 minutes and the supernatants were d i s c a r d e d . i i . Diphenylamine T e s t To the DNA e x t r a c t s was added 0.5 ml 0.1 N NH^OH and 1 ml 1.5 N HCIO^. The tubes were stoppered and heated a t 70° f o r 15 minutes. Two volumes of diphenylamine reagent, c o n s i s t i n g of 1.5 g dipheny-lamine, 8 mg acetaldehyde and 1.5 ml c o n c e n t r a t e d H 5SO d i n 100 ml g l a c i a l a c e t i c a c i d , was then added. 24 The tubes were sealed, mixed thoroughly and l e f t to s i t overnight (16 hrs.). The o p t i c a l density was then read at 600 nm. Highly polymerized c a l f thymus DNA, used as the stan-dard, was also put through the same procedure. 3. Sidedness Assays a. Acetylcholinesterase Acetylcholinesterase a c t i v i t y was assayed as des-cribed by Steck (58). Aliquots of 45-150xg membrane protein i n 15-50 nil were pipetted into a?5'-nil cuvette. An equal volume of 5'>mM sodium phosphate pH~8.0 or 0.05%-0.4% ( ,v/v) TritonX-,100 i n 5.mM sodium phosphate pH 8.0 was mixed well with the membranes. The detergent condi-tions were optimized so that a l l latent enzymatic a c t i v i t y would be released with l i t t l e or no i n a c t i v a -t i o n . The volume was made up to 0.7ml with 100;- mM sodium phosphate pH 7 . 5 then 50M1 of DTNB [5,5 1-dithiobis- < (2-nitrobenzoic acid)] stock solution was added. This solution contained 1'0,'mM DTNB, lOOmM sodium phosphate pH' .7'.0and 3mg NaHC03 per 8mg of DTNB. The reaction was i n i t i a t e d by addition of 50/(l of acetylthiocholine chloride (12.5 mM i n water). The cuvette contents were mixed by inversion and the cuvette was placed i n a recording spectrophotometer. The increase i n absor-bance at 412nm was measured. A blank rate, a l l reagents except membranes, was determined separately i n the presence and absence of Tri t o n X-100. b. S i a l i c Acid S i a l i c acid was assayed for by the method of Steck (59). A 1 mg/ml solution of neuraminidase in d i s t i l l e d water containing 0.1 mg/ml BSA was pre-pared. Aliquots of 300/«Yg membrane protein i n 100^1 were mixed with 100^1 of the neuraminidase solution previously d i l u t e d 10 f o l d (0.1 mg/ml), i n 0.1 M T r i s -acetate pH 5.6 either containing or lacking 0.5% (v/v) Triton X-100. Digestions were allowed to take place at room temperature for 30 min. In order to check the potency of the enzyme and to measure t o t a l s i a l i c acid 300^ aliquots of membrane protein i n 100>yl were mixed with 100^1 0.1 N H 2S0 4 and incubated at 80° for 1 hr. Released s i a l i c acid was then determined by the method of Warren (60). To both neuraminidase digests and acid hydrolyzed solutions was added 100^1 of Na metaperiodate (0.2 M in 9 M phosphoric acid). The solutions were thoroughly mixed and allow-ed to react for 20 min. at room temperature. One and a half ml of Na arsenite solution (10% (w/v) i n 0.5 M Na 2S0 4) was mixed i n vigorously and the tubes were incubated at room temperature for 2 min. Then 3.0 mis of t h i o b a r b i t u r i c acid solution (0.6% (w/v) i n 0.5 M Na 2S0 4) was mixed i n . The tubes were capped and placed i n a b o i l i n g water bath for 15 min. afte r 26 which they were cooled i n tap water to room tempera-ture. Two ml of t h i s solution were removed and vigorously mixed with 2ml." cyclohexanone. The phases were separated by centrifugation at 2000g for 10 min. at room temperature. Each upper phase was removed and transferred to a 3ml cuvette and the absorbance was determined at 549nm. A blank consisting of a l l reagents except neuraminidase was put through the same procedure i n order to correct for interference from other organic molecules (e.g. 2 deoxyribose) (60). The quantity of s i a l i c acid was determined using a molar extinction c o e f f i c i e n t of 57,000. 125 4. Preparation of I-labelled oPRL Ovine p r o l a c t i n was iodinated by the method of Thorell and Johansson (61) as modified by Rogol and Chramback (62). A 2.7 mg/ml. solution of lactoperoxidase i n 0.IM sodium acetate pH 5.6 was prepared, aliquoted and stored at -20°C. A lOO'^g/ ml solution of oPRL i n 0.05M Na2HP04,pH7.4 was prepared and stored i n 2^g aliquots i n 6 x 50mm Pyrex culture tubes. The iodination was performed at room temperature. To 2/rg oPRL 1 2 5 was added 10/fl of 0. 4M sodium acetate, pH5.6, 200 ACI Na I (2pil) , 1^1 of lactoperoxidase solution and ljul of d i l u t e H 20 2 (30%, freshly diluted 1:15,000 in H 20). After 30 seconds incubation the reaction was stopped by adding 100^1 of a solution of 16% (wt/vol) sucrose, 1% (wt/vol) KI, 0.02% (wt/ vol) NaN_. The solution was immediately applied under a 27 layer of buffer to a 1.5x87cm column of Sephadex G-100 equilibrated with 25mM Tris-HCl, pH 7.5 and 0.1% (wt/vol) bovine serum albumin maintained at 4°C. Elution was c a r r i e d out using the same buffer. One ml fractions were coll e c t e d at a flow rate of 0.2 ml/min. Fractions were counted for r a d i o a c t i v i t y and analyzed by radioimmunoassay and radio-125 receptor assay. Tubes containing I-oPRL were pooled, aliquotted- and stored at -20°C. 5. Preparation of Iodoprolactin In order to investigate the effects of radioiodinating oPRL, cold l a b e l l e d iodoprolactin was prepared and i t s a b i l i t y 125 to displace I-oPRL from rat l i v e r p r o l a c t i n receptors was compared to native oPRL at d i f f e r e n t pH's. (See section (F) of results.) 125 The reaction was performed as described for I-oPRL except that a 1 mg/ml solution of oPRL in 0.05M Na 2HP0 4 pH 7.4, and a 5 mg/ml solution of LPO i n 0.IM sodium acetate pH, 5; 6 and a ImM solution of Nal i n 0.IN NaOH were prepared. To 20 oPRL (1 nmole in 20^1) was added 10 of 0. 4M sodium acetate pK.5.6, 2^1 Nal (2rnmoles) and 1^1 of LPO solution (5^g). In order to follow the recovery of product and to 4 125 determine the extent of lodmation 5x10 cpm Na I was added as a tracer. The reaction was i n i t i a t e d by addition of L>|1 of d i l u t e H 20 2 (30% freshly diluted 1:15,000 in H20) . After 5 min. a second 1^1 aliquot of H 2 0 2 was added for a second 5 min. a c t i v a t i o n period followed by a t h i r d !#! 28 aliquot of H 20 2 and a t h i r d a c t i v a t i o n period. The reaction 125 was stopped i n the same way as for I-oPRL. Iodoprolactin was desalted at 4° on a 1 x 40 cm Sephadex G-100 column using 25 mM Tris-HCl pH 7.5, 0.1% BSA as eluent. F i f t y per cent of the r a d i o a c t i v i t y was found associated with the p r o l a c t i n there-fore the product contained 1 iodine atom per molecule of p r o l a c t i n . The reaction was repeated i n duplicate as des-125 cribed above but without the Na I tracer. After the reaction was stopped the duplicates were pooled and desalt-ed as described. Tubes containing iodoprolactin were pooled and concentrated. The f i n a l concentration of iodoprolactin was determined by radioimmunoassay. The iodoprolactin obtained from this procedure was used i n the experiment described i n Section E of the r e s u l t s . 6. Radioreceptor Assay The procedure used was similar to that of Shiu and 125 3 Friesen (63). I-labelled oPRL (80 x 10 cpm) was incubated with 150 jnq of membrane protein i n a f i n a l volume of 0.5 ml containing 25 mM Tris-HCl pH 7.5, 10 mM CaCl 2 or 150 mM NaCl (see results) and 0.1% (wt/vol) BSA. For each determination a p a r a l l e l incubation was performed i n the presence of 5 J(g unlabelled oPRL. The incubations were carried out i n 10 x 75 disposable glass culture tubes at room temperature for 6 hrs. The incubations were terminated by cooling the tubes to 0° 125 i n an ice bath. Bound and free I-oPRL were separated by centrifugation at 3000 g for 30 min. i n a refrig e r a t e d c e n t r i -fuge. The supernatants were decanted and the mouths of the 29 tubes were blotted with absorbent papers. The p e l l e t s were then counted i n a gamma s c i n t i l l a t i o n spectrometer. Total binding refers to the counts bound to the p e l l e t i n the absence of unlabelled hormone. Nonspecific binding refers to the counts bound i n the presence of excess unlabelled hormone. S p e c i f i c ; b i n d i n g was obtained by subtracting non-s p e c i f i c from t o t a l binding and expressing i t as a percentage of the t o t a l r a d i o a c t i v i t y added to the incubation mixture. Under these conditions 20-30% of the la b e l l e d hormone was s p e c i f i c a l l y bound. 7. Radioimmunoassay Immunoreactivity of the column fractions were evaluated by measuring the binding by hormone s p e c i f i c antibody of the eluent r a d i o a c t i v i t y . Aliquots of 0.1ml of selected fractions were pipetted into 0.5ml f i n a l volume of a buffer containing 0.01M sodium phosphate pH.7.6, 0.15M NaCl, 0.1% NaN^ r 1.0% (w/v). BSA and rabbit anti-oPRL di l u t e d 1:1000. After allowing the mixture to incubate at room temperature for 24 hrs. the assay tubes were cooled on ice and 0.5 ml of ice cold 0.1% (w/v), human gamma globulin (fraction II) was added followed by 1 ml of ice cold 25% polyethylene g l y c o l . The suspensions were mixed thoroughly and then centrifuged at 3,000g for 60 min. in a refrigerated centrifuge. The supernatants were poured off and the p e l l e t s were counted i n a gamma s c i n t i l l a t i o n spectrometer. In order to construct a radioimmunoassay 30 standard curve incubations were carried out as described 125 above except 15000cpm I-oPRL was incubated with unlabelled oPRL (1-100 ng/ml), in the presence of the antibody. 8. Disc Gel Electrophoresis Discontinuous polyacrylamide gel electrophoresis of oPRL was carried out as described by Davis (64). The stacking gel containing 3.25% acrylamide was run at pH' 8-. 3. The separating gel containing 7.5% acrylamide was run at pH9.5. The reser-vo i r buffer was 25mM Tri s - g l y c i n e pEjJ.8.3. Electrophoresis was carried out at 4° at 230 volts and 4 ma per tube. Approximate 125 50/jg protein or 15,000cpm I-oPRL was applied to each g e l . Bromphenol blue was used to mark the s a l t front. Gels were stained with coomassie blue or s l i c e d into 2mm sections and counted i n a gamma s c i n t i l l a t i o n counter. RESULTS 125 A. Fractionation and Characterization of I-labelled oPRL Lactoperoxidase iodination of oPRL yielded a product l a b e l l e d to high s p e c i f i c a c t i v i t y , with l i t t l e or no damage. The elution p r o f i l e of the iodination mixture fractionated by gel f i l t r a t i o n on Sephadex G-100 yielded four peaks of radio-a c t i v i t y ( f i g . 3A) characterized by Kav 0.02(1), 0.17(11), 0.44(111), 1.0(IV). A large proportion of the r a d i o a c t i v i t y was found divided evenly between peaks III and IV while peaks I and II contained much less r a d i o a c t i v i t y . Figure 3B shows the r e l a t i v e binding to anti-oPRL and to rat l i v e r membranes of the various LPO-iodinated components as a function of Kav in gel f i l t r a t i o n . Peaks I and III contained immunoactive 125 and receptor active I-oPRL while peaks II and IV showed no such a c t i v i t y . It has been shown that LPO elutes from Sephadex G-100 with a Kav of 0.15 (62) therefore peak II i s most l i k e l y s e l f iodinated LPO. The peak at the void volume was considered to be aggre-gated hormone and although low in concentration showed con-siderable a c t i v i t y in both radioligand assays. Aggregation of the hormone was considered not to be a r e s u l t of the iodination procedure because gel f i l t r a t i o n of native un-treated oPRL on Sephadex G-100 yielded a peak of protein at 125 the same position. The immunologic quality of I-oPRL derived from peak III was gauged i n two ways - binding to 32 Figure 3. Fractionation of radioiodinated oPRL by gel f i l -t r a t i o n . A-Isotppe analysis. B-Immunoassay and receptor assay analysis. Gel f i l t r a t i o n on Sephadex G-100 and radio-ligand assays were carr i e d out as described i n the methods section. 33 antibody t f i g . 3B) and i t s a b i l i t y t o be d i s p l a c e d from the antibody by non i o d i n a t e d oPRL ( f i g . 4 ) . F i g u r e 3B shows t h a t a t an antibody d i l u t i o n of 1:1000 approximately 75% of 125 the I-oPRL i n peak 3 was bound. F i g u r e 3B a l s o shows t h a t . 125 I-oPRL bound to r a t l i v e r r e c e p t o r s i n a p a t t e r n s i m i l a r to t h a t observed with a n t i b o d i e s . F i g u r e 4 shows t h a t the 125 I-oPRL d e r i v e d from peak I I I c o u l d be d i s p l a c e d from a n t i -oPRL by i n c r e a s i n g amounts of u n l a b e l l e d oPRL. P o l y a c r y l a -125 mide g e l e l e c t r o p h o r e s i s of both I-oPRL and u n l a b e l l e d oPRL y i e l d e d s i m i l a r p r o f i l e s ( f i g . 5). Both had major peaks 125 c h a r a c t e r i z e d by Rf 0.41 and 0.47 and 0.52. I-oPRL had an a d d i t i o n a l minor peak wi t h Rf 0.68. Po l y a c r y l a m i d e g e l e l e c -t r o p h o r e s i s i n the presence of SDS y i e l d e d o n l y one band (data not shown). These r e s u l t s i n d i c a t e t h a t the mo l e c u l a r 125 i n t e g r i t y of I-oPRL has been pr e s e r v e d . 125 R e f i l t r a t i o n of the I-oPRL from peak I I I on Sephadex G100 y i e l d e d one peak wi t h Kav 0.43 ( f i g . 6). A f t e r one month of storage a t -20° two a d d i t i o n a l peaks were observed a t Kav 0.0 and 1.0 and the c e n t r a l peak was s l i g h t l y d i m i n i s h e d i n s i z e . A f t e r 4 months storage a t -20° the r a d i o a c t i v i t y was spread evenly among the th r e e peaks as i l l u s t r a t e d i n f i g u r e 6. The appearance of the a d d i t i o n a l peaks i s most l i k e l y a 125 r e s u l t of continuous damage of I-oPRL due to r a d i o l y s i s d u r i n g storage. Accurate e s t i m a t i o n of s p e c i f i c a c t i v i t y thus becomes v e r y d i f f i c u l t . While the s p e c i f i c a c t i v i t y 100 o o o 00 CO 80 60 40 20 J I I I I I 1 I ' i i i i i i n 10 oPRL (ng/ml) 100 125 Figure 4. Displacement of antibody bound I-oPRL as function of increasing concentration of unlabelled oPRL Radioimmunoassay was carried out as described i n the methods section. Ordinate: r a t i o of counts bound to counts bound at zero dose. 35 0.0 05 to Figure 5. D i s t r i b u t i o n of I-oPRL and native oPRL on a^jZ.5% polyacrylamide gel. Approximately 15,000 cpm of I-oPRL or 50/(g native oPRL was added to each g e l . Electrophoresis procedure and gel staining are described under methods. Figure 6. S t a b i l i t y of,-- I-oPRL in storage. Approx-imately 150,000 cpm of I-oPRL was layered on a 1 x 40 Sephadex G-100 column equilibrated with 25mM Tris-HCl, pH7.5, 0.1% (w/v) BSA. Gel f i l t r a t i o n was carried out at 4°C with the same buffer. F r a j ^ o n s were coll e c t e d and monitored for r a d i o a c t i v i t y . I-oPRL was stored at •^20°C immediately following iodination and samples were removed aft e r , 3 days , A- - A ; 1 month, o-^  o; and 4 months, —-• . 37 of the '^"'j.-oPRL was 40-60 C i / g as determined by the method of Rogol and Chramback (62) , these values can only be con-sidered as approximations. However, precaution was taken that 125 I-oPRL was used within two weeks of preparation. B. Characterization of Plasma Membranes Plasma membranes obtained from r a t l i v e r as described i n the methods section were analyzed for enzymatic and struc-t u r a l markers i n order to determine purity and morphology. Table I shows that the s p e c i f i c a c t i v i t y of 5 1-nucleotidase, a marker enzyme for plasma membranes, increased approximately 12 f o l d i n the f i n a l membrane preparation while the s p e c i f i c a c t i v i t i e s of glucose-6-phosphatase and succinate dehydrogen-ase, marker enzymes for endoplasmic reticulum and mitochondria respectively, decreased. The plasma membranes were contam-inated by small amounts of endoplasmic reticulum and nu c l e i while mitochondria were not detected. However, the absence of succinate dehydrogenase a c t i v i t y i n the f i n a l membrane preparation could have resulted from the assays i n a b i l i t y to detect low concentrations of t h i s enzyme. The highest bind-ing per mg of protein was observed i n the plasma membrane fr a c t i o n (Table I ) . Since the other marker a c t i v i t i e s were decreased to a minimum i n t h i s f r a c t i o n i t i s reasonable to assume that the p r o l a c t i n receptors are located on the c e l l surface membranes. TABLE I DISTRIBUTION OF MARKER ASSAYS IN SUBCELLULAR FRACTIONS FROM RAT LIVER HOMOGENATE Homogenization conditions, procedures for i s o l a t i n g subcellular fractions and 125. determination of enzyme a c t i v i t i e s , protein and s p e c i f i c binding of are described i n the methods section. N = 4 ± S.E.M. I-oPRL Marker Assay and Subcellular Fraction Specific A c t i v i t y P u r i f i c a t i o n Y i e l d 5 1-Nucleotidase homogenate 1500g p e l l e t membranes nmoles Pi/min/mg,.protein 73.4+11 153.0+16 887.6+131 2.1+0.4 12.1+1.5 100 62.7+9.3 11.5+2.1 Glucose-6-phosphatase homogenate 1500g p e l l e t membranes ,nmoles Pi/min/mg protein 134.0+16. 132.0+22 31.2+9 100 49.6+10 0.6+0.2 Succinate dehydrogenase • nmoles/min/mg protein homogenate 0.68+0.3 - 100 1500g p e l l e t 0.03+0.02 - 9.3+2.0 membranes 0 - 0 TABLE I - (Continued) Marker Assay and Spec i f i c A c t i v i t y P u r i f i c a t i o n Y i e l d % Subcellular Fraction Diphenylamine t e s t f o r DNA /jg DNA/mg p r o t e i n homogenate 5.6+0.5 1 100 1500g p e l l e t 8.0+0.3 1.4+0.4 85.9+5.0 membranes 1.8+0.5 - 1.8+0.4 P r o l a c t i n b i n d i n g c a p a c i t y fmoles I-oPRL/mg p r o t e i n homogenate 65.2+8 . 1 10 0 1500g p e l l e t 100.3+16 1.6+0.4 43 + 7 membranes 352.1+40 5.4+1.2 4.9+0.8 40 The morphology of the plasma membrane p r e p a r a t i o n was determined by ass a y i n g f o r s i a l i c a c i d and a c e t y l c h o l i n e s -t e r a s e a c t i v i t y . Both of these markers are known to be a c c e s s i b l e o n l y from the outer s u r f a c e of plasma membranes (58). The data i n Table I I shows t h a t most of the a c e t y l -c h o l i n e s t e r a s e and s i a l i c a c i d are i n a c c e s s i b l e i n the absence of T r i t o n X-100 however i n the presence of t h i s d e t e r -gent the membrane components become s o l u b i l i z e d and the t o t a l s i a l i c a c i d and a c e t y l c h o l i n e s t e r a s e are d e t e c t e d . T h i s data i n d i c a t e s t h a t approximately 20% of the t o t a l outer s u r f a c e of the membrane i s a c c e s s i b l e to the medium. There f o r e the m a j o r i t y of the membranes appear to e x i s t as t i g h t l y s e a l e d i n s i d e - o u t v e s i c l e s while a small p r o p o r t i o n are e i t h e r r i g h t s i d e - o u t v e s i c l e s or membrane sheets or a mixture of both. Although t h i s suggests t h a t a l a r g e p r o p o r t i o n of the r e c e p t o r s may remain undetected the assay c o n d i t i o n s used i n the RRA and sidedness assays were q u i t e d i f f e r e n t and t h i s may a l t e r the r a t i o s shown i n Table II (58). C. A c t i v a t i o n of B i n d i n g by Cat i o n s Previous s t u d i e s on p r o l a c t i n r e c e p t o r s made use of 10, mM cal c i u m or magnesium to a c t i v a t e b i n d i n g (1-4, 12, 39-42, 45). However no e x p l a n a t i o n was given f o r the use of these high c o n c e n t r a t i o n s c o n s i d e r i n g t h a t i n normal r a t serum the con-c e n t r a t i o n s of c a l c i u m and magnesium are 2.5JmM and 1.0 mM 41 TABLE I I DISTRIBUTION OF "SIDEDNESS" MARKERS IN PURIFIED RAT LIVER PLASMA MEMBRANES I s o l a t i o n of r a t l i v e r plasma membranes and de t e r m i n a t i o n of a c e t y l c h o l i n e s t e r a s e a c t i v i t y and s i a l i c a c i d are de s c r i b e d i n the methods s e c t i o n . n = 2 ± S.D. S i a l i c A c i d A c e t y l c h o l i n e s t e r a s e '.(nmoles/mg p r o t e i n ) (nmoles/min/mg p r o t e i n ) without T r i t o n X-100 0.72+0.08 2.9+0.11 with T r i t o n X-100* 4.5+0.3** 12.9+0.01 r a t i o ( i n s i d e - o u t : r i g h t s i d e - o u t ) 6:1 4:1 The c o n c e n t r a t i o n s o f T r i t o n X-100 used were, 0.25% f o r s i a l i c a c i d and 0.4% f o r a c e t y l c h o l i n e s t e r a s e . T o t a l s i a l i c a c i d r e l e a s e d by a c i d h y d r o l y s i s was 3.7+0.5 nmoles/mg p r o t e i n . 42 r e s p e c t i v e l y . In another independent study i t was shown t h a t i n the absence of c a l c i u m and magnesium, 150.mM sodium c o u l d a c t i v a t e b i n d i n g (64). Since t h i s i s the concentra-t i o n of sodium i n normal r a t serum i t would appear t h a t i n v i t r o b i n d i n g s t u d i e s under these c o n d i t i o n s , r a t h e r than lOmM c a l c i u m of magnesium, would be more a p p l i c a b l e to what i s happening i n v i v o . In a l l of these s t u d i e s the e f f e c t of v a r i o u s c o n c e n t r a t i o n s of these c a t i o n s on b i n d i n g was not examined. Ther e f o r e a c t i v a t i o n of b i n d i n g was i n -v e s t i g a t e d by v a r y i n g the c o n c e n t r a t i o n s of c a l c i u m , magne-sium, sodium and potassium. F i g u r e 7A and 7B show the 125 b i n d i n g of I-oPRL to r a t l i v e r membranes at v a r i o u s con-c e n t r a t i o n s of these c a t i o n s . In the absence of any c a t i o n s 125 approximately 2 to 3 per cent of the I-oPRL was s p e c i f i c a l l y bound whether the membrane p r e p a r a t i o n was d i a l y z e d before use or not. A d d i t i o n of e i t h e r calcium, magnesium, sodium or potassium r e s u l t e d i n a marked i n c r e a s e i n s p e c i f i c b i n d i n g . Calcium was most e f f e c t i v e i n a c t i v a t i n g b i n d i n g w i t h h a l f + 2 maximal l e v e l s reached a t 2.5mM Ca while magnesium was s l i g h t l y l e s s e f f e c t i v e , with h a l f maximal b i n d i n g reached at 4 mM Both sodium and potassium were much l e s s e f f e c t i v e with h a l f maximal b i n d i n g reached at e i t h e r 40 frM sodium or potassium. The unusual shape of the b i n d i n g curve obtained i n the presence of c a l c i u m was a unique c h a r a c t e r i s t i c of t h i s c a t i o n . S u c c e s s i v e r e p e t i t i o n s of the experiment y i e l d e d the same p a t t e r n ; b i n d i n g i n c r e a s e d to a maximum at 18 mM c a l c i u m 0 20 40 60 0 100 200 300 C O N C E N T R A T I O N ( m M ) Figure 7. Activation of binding by cations. Incubations were carried out as described i n the methods section except that either A; CaCl-, • • or MgCl 2, O — O was added or B; NaCl,A A or KCl, A & was added to give the f i n a l concentrations indicated. Each point i s the average of three i n d i v i d u a l determinations. Nonspecific binding, • • . 44 f o l l o w e d by a decrease at 20..mM..calcium a f t e r which a second i n c r e a s e occurred, r e a c h i n g a p l a t e a u between 4 0 and 60.mM calc i u m . T h i s p a t t e r n was not observed with the other c a t i o n s t e s t e d . N o n - s p e c i f i c b i n d i n g i n the presence of sodium or potassium was 1-2% l e s s than with c a l c i u m or magnesium but remained r e l a t i v e l y constant at a l l c a t i o n c o n c e n t r a t i o n s . As shown i n f i g u r e s 7A and 7B with e i t h e r 150 mM sodium 125 or lO'.'mM c a l c i u m approximately 24% of the I-oPRL was s p e c i f i c a l l y bound. T h i s would j u s t i f y the use of 10 mM c a l -cium s i n c e i t a c t i v a t e d b i n d i n g to the same l e v e l as the p h y s i o l o g i c a l c o n c e n t r a t i o n of sodium. However, t h i s does not mean t h a t other b i n d i n g c h a r a c t e r i s t i c s o f the r e c e p t o r would be the same i n the presence of e i t h e r lCrttiM c a l c i u m or 150.;mM; sodium. Ther e f o r e both of these c a t i o n s were used to a c t i v a t e b i n d i n g i n the f o l l o w i n g i n v e s t i g a t i o n s and t h e i r e f f e c t s were compared. D. E f f e c t of Time and Temperature on the Bi n d i n g and D i s s o c i a t i o n of P r o l a c t i n  125 The b i n d i n g o f I-oPRL to r a t l i v e r membranes i n the presence of 10 rnM CaCl-p was time, and temperature dependent ( f i g u r e 8). The r a t e of b i n d i n g was g r e a t e s t a t 37°' f o r the f i r s t hour f o l l o w e d by a much slower r a t e of a s s o c i a t i o n f o r the next 3 hours. At 30°-' b i n d i n g was slower than a t 37°". The r a t e appeared to be constant u n t i l a maximum was reached at 1.5 hours a f t e r which a slow decrease i n b i n d i n g o c c u r r e d . At 23° the r a t e of b i n d i n g v/as l e s s than h a l f the r a t e a t 30 TIME (hr) 125 F i g u r e 8. E f f e c t o f time and temperature on b i n d i n g of I-oPRL to r a t l i v e r membranes. Incu b a t i o n mixtures were prepared as d e s c r i b e d i n the methods sec-t i o n w i t h the e x c e p t i o n t h a t the volumes were s c a l e d up ten times. Assay b u f f e r s and membrane p r e p a r a t i o n s were preincubated s e p a r a t e l y at the temperatures i n d i -c ated then mixed to s t a r t the r e a c t i o n . At the times i n d i c a t e d on the graphic.. 5 ml a l i q u o t s were removed and analyzed f o r s p e c i f i c b i n d i n g . 37- • • ; 30 O O ; 23° ,A A ; 0° & A . 46 37 reaching a plateau at 6 hours. The maximum binding ob-tained at 2 3°' .after 6 hours was less than that obtained at 37° after 4 hours. There was very l i t t l e binding at 0°.,. Non-specific binding (not shown) increased with time and temperature i n a similar manner. The effects of d i f f e r e n t cations on the rate of binding and t o t a l amount bound at equilibrium at 2 3° i s shown i n 125 Table I I I . The t o t a l amount of I-oPRL bound at equilibrium was not s i g n i f i c a n t l y d i f f e r e n t for the d i f f e r e n t combinations of cations. The combination of fO mM CaCl 2 and 150"mM' NaCl did not have an additive e f f e c t as compared to the binding obtained with individual cations. The i n i t i a l rates of binding show only s l i g h t differences. The fastest rate was obtained with 10 mM CaCl 2. The average i n i t i a l rate for 150'mM NaCl was slower but the va r i a t i o n with th i s cation was very large. A combina-tio n of • 10 mMCaCl„ and 150 mM NaCl increased the i n i t i a l rate above that obtained with 150 mM NaCl„- i- alone but i t was s t i l l lower than with 10 mM CaCl 2 alone. Addition of 130 mM NaCl and 20 mM KC1 to give a f i n a l concentration of 150 mM of monovalent cations resulted i n the lowest i n i t i a l rate. 125 Dissociation of I-oPRL from rat l i v e r plasma membranes does not appear to be dependent on time or temperature in the presence of 10 mM CaCl 2 ( f i g . 9A). The di s s o c i a t i o n at the three temperatures 37° , 23° and 0° fluctuated within a narrow range for the f i r s t 8 hours. After 20 hours there was a net decrease i n binding but the lowest value observed at TABLE I I I EFFECT OF CATIONS ON RATE AND QUANTITY OF BINDING OF PROLACTIN Incubations were c a r r i e d out a t 23 f o r 30 minutes to measure i n i t i a l r a t e s and f o r 12 hours to ensure e q u i l i b r i u m had been reached f o r t o t a l b i n d i n g . S p e c i f i c b i n d i n g was determined as d e s c r i b e d i n the methods s e c t i o n . Each value i s the mean (+ S.E.M.) of fou r d e t e r m i n a t i o n s . CATIONS INITIAL RATE TOTAL BINDING 10 mM CaCl 2 150 mM NaCI 10 mM Ca C l 2 + 150 mM NaCI 150 mM NaCI + 2.5 mM KC1 130 mM NaCI + 20 mM KC1 (fmoles/min/mg p r o t e i n ) 1.86+0.02 1.64+0.29 1.79+0.09 1.73+0.40 1.41+0.09 (fmoles/mg p r o t e i n ) 180.0+0.43 185.3+4.3 173.2+6.9 171.5+2.6 185.3+2.6 23 was s t i l l only 12% less than the zero time control. As shown i n figure 9A successive repetitions of the experiment gave r i s e to large deviations at each sample time. In the presence of 150\mM;' NaCl ( f i g . 9B) di s s o c i a t i o n fluctuated widely at 0° and 23°' but at 37° . d i s s o c i a t i o n appears to be time and temperature dependent. However di s s o c i a t i o n i s very slow with approximately 35% of the hormone dissociated after 20 hours. The same results were observed when the preincubated hormone-receptor complex was dil u t e d into buffers containing up to 40#g/mi" : unlabelled oPRL. Also a one hundred f o l d d i l u t i o n into these buffers had no af f e c t on the di s s o c i a t i o n rate. This data demonstrates that the di s s o c i a t i o n i s d i f f e r -ent with 15"0mM•• sodium as compared to 10 mM calcium. However in either case the di s s o c i a t i o n rate i s extremely slow and since the h a l f - l i f e of the hepatic p r o l a c t i n receptor in rat has been shown to be less than 3 hours (42) i t appears that the interaction observed here i s unique. Other peptide hormones dissociate from t h e i r receptors with r e l a t i v e l y 125 short h a l f - l i f e s . For example I-bGH d i s s o c i a t i o n from mouse l i v e r receptors i s half complete aft e r 4 hours (65). 125 Even more s t r i k i n g i s the fact that I-oPRL d i s s o c i a t i o n from rabbit mammary gland receptors i s also half complete after 4 hours at 37° i n the presence of lbrrnM. calcium (1) . 49 A 0 5 10 15 20 TIME (hr) F i g u r e 9. E f f e c t of time and temperature on d i s s o c i a t i o n of I-oPRL from r a t l i v e r membranes 300 /(q membrane p r o t e i n was incubated with 5 x 10 cpm I-oPRL a t 23 i n the presence of e i t h e r 10 mM CaCl„ or 150 mM NaCI as d e s c r i b e d i n the methods s e c t i o n . A f t e r 8 h r s . 3 mis of 25 mM T r i s - H C l pH 7.5 wit h 10 mM C a C l 2 or 150 mM NaCI, 0.1% BSA c o n t a i n i n g 3,4g/ml u n l a b e l l e d p r o l a c t i n p r e i n c u b a t e d a t the i n d i c a t e d temperatures was added and i n c u b a t i o n was c a r r i e d out a t 3 7 ,• • ; 2 3 ,0——O; and 0 A . At the i n d i c a t e d times a 0.5 ml a l i q u o t was removed and s p e c i f i c b i n d i n g was determined as d e s c r i b e d i n the methods s e c t i o n . A- 10 mM C a C l 2 , B- 150 mM NaCI. Bars r e p r e s e n t s t a n -dard d e v i a t i o n of f o u r d e t e r m i n a t i o n s . 50 E. S p e c i f i c i t y of B i n d i n g of P r o l a c t i n to Receptors The s p e c i f i c i t y of p r o l a c t i n b i n d i n g to r a t l i v e r plasma membranes has p r e v i o u s l y been demonstrated i n gre a t d e t a i l u s i n g both lO.mM c a l c i u m (39) and 150 mM sodium (64). T h e r e f o r e o n l y a c u r s o r y i n v e s t i g a t i o n was performed here u s i n g o n l y lO.mM ca l c i u m to a c t i v a t e b i n d i n g . S p e c i f i c i t y of the i n t e r a c t i o n was examined by i n c u b a t i n g 125 r a t l i v e r plasma membranes with I-oPRL i n the presence of v a r i o u s doses of s e v e r a l d i f f e r e n t u n l a b e l l e d hormones. The 125 b i n d i n g of I-oPRL to r a t l i v e r plasma membranes was i n -h i b i t e d by i n c r e a s i n g amounts of u n l a b e l l e d oPRL, bPRL and rPRL while pACTH and AVP had no e f f e c t ( f i g . 10). The s l i g h t 125 displacement of I-oPRL by hig h bGH c o n c e n t r a t i o n s may be due to bPRL contamination of t h i s hormone. The rPRL was l e s s e f f e c t i v e than oPRL and bPRL i n d i s -125 p l a c i n g the I-oPRL because i t had a potency of 11 I.U./mg while the oPRL and bPRL had a potency of 20-25 I.U./mg as measured by c o n v e n t i o n a l b i o a s s a y s . F i g u r e 10 shows t h a t there i s good c o r r e l a t i o n between the estimates of potency obtained by these two assay methods. The l i n e a r range of the oPRL and bPRL displacement curve was between 5 and 100 ng/ml. In r a t s , serum p r o l a c t i n c o n c e n t r a t i o n s may range from 10 to 250 ng/ml. Th e r e f o r e t h i s assay c o u l d be a u s e f u l method of measuring serum pro-l a c t i n c o n c e n t r a t i o n s . 51 1001 I 1 I I J - J 0.1 1 10 100 1000 2000 U N L A B E L L E D H O R M O N E ( n g / m l ) 125 F i g u r e 10. S p e c i f i c i t y of b i n d i n g of I-oPRL to^jjat l i v e r membranes. Procedures f o r determining b i n d i n g of I-oPRL are d e s c r i b e d i n the methods s e c t i o n . U n l a b e l l e d hormones were added to g i v e the f i n a l c o n c e n t r a t i o n s i n d i c a t e d on the a b s c i s s a . oPRL, • • ; bPRL, A ; rPRL,o O; bGH , • • ; pACTH and AVP, • • . Each p o i n t i s the average of two de t e r m i n a t i o n s . 52 F. E f f e c t of pH on the B i n d i n g of P r o l a c t i n 125 The s p e c i f i c b i n d i n g of I-oPRL to r e c e p t o r s i n the presence of 10 mM C a C ^ o c c u r r e d over a r e l a t i v e l y wide pH range. Maximal b i n d i n g o c c u r r e d at pH "'6/5 ( F i g . 11) and d e c l i n e d to h a l f maximal l e v e l s at rpH 5.5 and pH 8.75. At 125 pH values l e s s than 6.5 I-oPRL p r e c i p i t a t e s from s o l u -t i o n under the i n c u b a t i o n c o n d i t i o n s used, thus causing the high n o n - s p e c i f i c values observed at the lower pH 1s. In the presence of 150 mM NaCl the pH optimum and g e n e r a l shape of the curve was i d e n t i c a l to t h a t obtained with 10 mM C a C ^ • The p o s s i b i l i t y was c o n s i d e r e d t h a t the pH optimum ob-125 served f o r I-oPRL may not be the same as f o r n a t i v e pro-l a c t i n . I o d i n a t i o n of t y r o s y l r e s i d u e s produces two opposing e f f e c t s : (1) an i n c r e a s e i n the h y d r o p h o b i c i t y of the phenoxy r i n g and ( i i ) a r e d u c t i o n i n the pKa f o r the hydroxyl group of t y r o s i n e from 10.2 i n the n a t i v e t y r o s i n e to 8.0 i n monoiodinated t y r o s i n e . I f the i o d i n a t e d t y r o s y l r e s i d u e s are c r i t i c a l l y i n v o l v e d i n the i n t e r a c t i o n between p r o l a c t i n and r e c e p t o r , then a major e f f e c t due to i o d i n a t i o n can be expected. In order to determine whether i o d i n a t i o n of pro-l a c t i n a f f e c t s b i n d i n g the a b i l i t y of n a t i v e p r o l a c t i n and • - i . 125 i o d o p r o l a c t i n ; i to compete with the b i n d i n g of I-oPRL was s t u d i e d a t pH 6.5 and pH 7.5. ; As shown i n f i g u r e 12A n a t i v e p r o l a c t i n was s l i g h t l y more e f f e c t i v e than i o d o p r o l a c t i n i n 125 competing with I-oPRL atpH 6.5.' ; Half-maximal oc c u p a t i o n of the r e c e p t o r s o c c u r r e d with 12 ng/ml n a t i v e p r o l a c t i n and 53 Q Z D O CD 125 Figure 11. E f f e c t of pH on the binding of I-oPRL to rat l i v e r membranes. A combination of acetate, T r i s , and bicar-bonate buffers were used to obtain the observed pH's. Measurements of pH were taken before and afte r the incubation period. S p e c i f i c binding was determined as described i n the methods section. S p e c i f i c binding, • • ; nonspecific, O O, with 18.2 ng/ml i o d o p r o l a c t i n . At pH 7.5 ( f i g . 12B) n a t i v e p r o l a c t i n and i o d o p r o l a c t i n were e q u a l l y e f f e c t i v e i n com-125 p e t i n g with I-oPRL. The i o n i z a t i o n of the t y r o s i n e hydroxyl group i n both i o d o p r o l a c t i n and n a t i v e p r o l a c t i n i s n e g l i g i b l e a t pH. 6.5, while a t pH 7.5:. approximately 30% of the i o d o p r o l a c t i n would be i o n i z e d but l e s s than 1% of the n a t i v e p r o l a c t i n would be i o n i z e d . Therefore i o d i n a t i o n of oPRL does not a f f e c t i t s a f f i n i t y f o r the r e c e p t o r at^pH 7.5* and thus i n f o r m a t i o n obtained from b i n d i n g s t u d i e s a t t h i s 125 pH u s i n g I-oPRL would r e f l e c t what would be o c c u r r i n g with the n a t i v e hormone. Since at the pH optimum of the r e c e p t o r , i o n i z a t i o n of the i o d o p r o l a c t i n i s n e g l i g i b l e , the decrease i n a f f i n i t y may be a t t r i b u t e d to i n c r e a s e d h y d r o p h o b i c i t y claimed to be c o n t r i b u t e d by the i o d i d e atom (66). •G-* E f f e c t of Membrane P r o t e i n C o n c e n t r a t i o n As shown i n f i g u r e 13 there was a l i n e a r r e l a t i o n s h i p 125 between I-oPRL s p e c i f i c b i n d i n g and membrane p r o t e i n con-c e n t r a t i o n up to a p r o t e i n c o n c e n t r a t i o n of lOO^fg per tube. T h e r e a f t e r , the i n c r e a s e i n s p e c i f i c b i n d i n g with i n c r e a s i n g p r o t e i n c o n c e n t r a t i o n was s l i g h t l y reduced. N o n s p e c i f i c b i n d i n g i n c r e a s e d o n l y s l i g h t l y from 5.5% to 7.5% of the t o t a l l a b e l l e d hormone added. I t appears t h a t n o n s p e c i f i c b i n d i n g i s a consequence of a d s o r p t i o n of the l a b e l l e d hormone to the i n n e r s u r f a c e of the assay tubes s i n c e i t was., s t i l l , 'apparent i n the absence of any membrane p r o t e i n . 55 o M 50 Z UJ LU O < _ l CL cn 100 50 100 W/^  J ' I I I 111 ' ' I I / I 111 I I I I I 111 B p H 7.5 I Mi l l J I I I I I I I I ' I I M I I 10 10' 10^ H O R M O N E A D D E D ( n g / m l ) 125. F i g u r e 12. E f f e c t o f p H o n c o m p e t i t i o n o f b i n d i n g o f " " I - o P R L w i t h n a t i v e p r o l a c t i n a n d i o d o p r o l a c t i n . B i n d i n g a s s a y s w e r e c a r r i e d o u t a s d e s c r i b e d i n t h e m e t h o d s s e c t i o n w i t h t h e a d d i t i o n o f h o r m o n e t o g i v e t h e f i n a l c o n c e n t r a t i o n s i n d i c a t e d . T h e b i n d i n g o f n a t i v e p r o l a c t i n O — O a n d i o d o p r o l a c t i n • • w a s s t u d i e d a t pH6.5 (A) a n d pH7.5 ( B ) . B a r s r e p r e s e n t s t a n d a r d d e v i a t i o n o f f o u r d e t e r m i n a t i o n s . MEMBRANE PROTEIN (jjg/tube) Figure 13. E f f e c t of membrane protein concentration on binding. Determination of s p e c i f i c binding and protein i s described i n the methods section. S p e c i f i c b i n d i n g , * — — • non s p e c i f i c binding, O- O . H. E f f e c t of I-oPRL C o n c e n t r a t i o n 125 The e f f e c t of i n c r e a s i n g the c o n c e n t r a t i o n of I-oPRL on s p e c i f i c and n o n s p e c i f i c b i n d i n g i s shown i n f i g u r e 14A. The s p e c i f i c b i n d i n g i n c r e a s e d l i n e a r l y up to 2x10^ cpm/tube and t h e r e a f t e r began to f a l l o f f . N o n s p e c i f i c b i n d i n g i n -creased i n a l i n e a r f a s h i o n over the whole c o n c e n t r a t i o n 125 range of I-oPRL used. A Scatchard (67) p l o t ( f i g . 14B) of the data from f i g u r e 14A y i e l d e d an apparent a s s o c i a t i o n 9- --1 constant, Ka, of 6.9x10 "M . . The d i s s o c i a t i o n constant Kd -10*'' i s t h e r e f o r e 1.45x10 ;,'M or approximately 3 ng/ml... The same r e s u l t s were obtained whether the b i n d i n g was a c t i v a t e d by 10'mM' C a C l 2 or 150 mM. ,NaCl. A n a l y s i s of the displacement curve of oPRL i n fugure 10 a l s o y i e l d e d the same r e s u l t s . These values should be regarded as approximations f o r , though the analyses were done at a steady s t a t e of b i n d i n g i t i s not c l e a r i f t h i s r e f l e c t s t r u e e q u i l i b r i u m . As was shown i n 125 f i g u r e 6 continuous damage of the I-oPRL i s always o c c u r r -i n g and i t i s not known to what extent t h i s may e f f e c t the e q u i l i b r i u m . I. E f f e c t o f Enzyme Treatment and I n h i b i t o r s on Receptor A c t i v i t y  The s t r u c t u r a l c h a r a c t e r i s t i c s of the p r o l a c t i n r e c e p t o r s i t e .-were, i n v e s t i g a t e d by t r e a t i n g r a t l i v e r plasma membranes with v a r i o u s enzymes (Table IV) and i n h i b i t o r s (Table V ) . Exposure of the membranes to pronase r e s u l t e d i n an 8 0% de-125 crease of the b i n d i n g of I-oPRL suggesting t h a t p r o t e i n LEAF 58 OMITTED IN PAGE NUMBERING. 58a F i g u r e 14A. E f f e c t of I-oPRL c o n c e n t r a t i o n on b i n d i n g . Incubation c o n d i t i o n s and procedures f o r determining s p e c i f i c b i n d i n g were i d e n t i c a l to those d e s c r i b e d i n the methods s e c t i o n . S p e c i f i c b i n d i n g , * • ; n o n s p e c i f i c b i n d i n g , O 0 . 58b BOUND (f m o l e s ) F i g u r e 14B. Sc_atchard a n a l y s i s of the data o b t a i n e d from the s p e c i f i c b i n d i n g curve i n f i g u r e 14A. M o l e c u l a r weight of oPRL was taken to be 20,000. 59 i s a f u n c t i o n a l l y important p a r t of the r e c e p t o r . Phos-p h o l i p a s e C treatment a l s o leads to a s i g n i f i c a n t decrease of r e c e p t o r a c t i v i t y suggesting t h a t p h o s p h o l i p i d s may a l s o p l a y a s i g n i f i c a n t r o l e i n the b i n d i n g of p r o l a c t i n . Neuraminidase treatment r e s u l t e d i n i n c r e a s e d b i n d i n g suggesting t h a t s i a l i c a c i d may i n f l u e n c e b i n d i n g but t h a t i t i s not e s s e n t i a l f o r the i n t e r a c t i o n . The r o l e t h a t s i a l i c a c i d has i n b i n d i n g i s d i f f i c u l t to assess s i n c e the nature of the i n c r e a s e was not examined. The absence of any e f f e c t of r i b o n u c l e a s e and deoxyribonuclease on r e c e p t o r a c t i v i t y suggests t h a t n u c l e i c a c i d s are not i n v o l v e d i n the b i n d i n g of p r o l a c t i n to i t s r e c e p t o r . I n h i b i t i o n of b i n d i n g by low c o n c e n t r a t i o n s of d i t h i o -t h r e i t o l and p-'~"cnlbromercur^ i n d i c a t e the presence of very r e a c t i v e s u l f h y d r y l groups which are e s s e n t i a l f o r b i n d i n g a c t i v i t y . S ince a molecule of p r o l a c t i n has s i x h a l f -c y s t e i n e s i t was c o n s i d e r e d t h a t the s u l f h y d r y l reagents may be r e a c t i n g w i t h them and thus causing the i n h i b i t i o n . As 125 shown i n Table V, pretreatment of I-oPRL with p-chloromer-c u r i b e n z o i c a c i d had no e f f e c t on subsequent b i n d i n g of the hormone to i t s r e c e p t o r . Loss of b i n d i n g a c t i v i t y upon b r i e f exposure of the membrane suspension to high temperature i n -d i c a t e s t h a t the r e c e p t o r i s extremely l a b i l e . TABLE IV EFFECT OF ENZYME TREATMENT ON RECEPTOR ACTIVITY P o r t i o n s o f membrane suspension were incubated a t 3 7° .< i n the presence of d i f f e r e n t enzymes f o r 30 min. i n the same b u f f e r as was used f o r b i n d i n g s t u d i e s . For neuraminidase treatment, membranes were suspended i n 0.IM T r i s - a c e t a t e • -< V. o ' pH -5.6 and incubated at 23 f o r 30 minutes (59) . At the end of the i n c u b a t i o n p e r i o d the tubes were c e n t r i f u g e d o • f o r 20 minutes a t 3000g at 4 ' The p e l l e t s were washed three times with c o l d b u f f e r before resuspension i n the i n c u b a t i o n b u f f e r . Minus enzyme c o n t r o l s were c a r r i e d 125 through the same procedure. S p e c i f i c b i n d i n g of I-oPRL v/as determined e x a c t l y as d e s c r i b e d i n the methods s e c t i o n . ENZYME CONCENTRATION SPECIFIC BINDING (^g/ml) (% c o n t r o l ) C o n t r o l - 100 DNase.. • 20 108 .RNase • 20 9 8 Pronase 20 18 Phospholipase C 20 31 Neuraminidase 20 132 TABLE V EFFECT OF INHIBITORS ON BINDING ACTIVITY Incubations were c a r r i e d out as d e s c r i b e d i n the methods s e c t i o n with i n h i b i t o r s present at the c o n c e n t r a t i o n s 125 noted. In a d d i t i o n p o r t i o n s of I-oPRL were incubated with I'mM'pCMBA at 23°,. f o r 30 minutes. The s o l u t i o n was then p l a c e d on a 1x40cm Sephadex G100 column and e l u t e d with•..25\mM Trf's-HCl., pH.'7.5, 0.1% BSA. The 1 2 5 I - o P R L e l u t e d from the column was then assayed f o r b i n d i n g a c t i v i t y . A l s o p o r t i o n s of membranes were heated a t 100°/ f o r 30 seconds then cooled to room temperature. Receptor a c t i v -i t y was then determined as d e s c r i b e d i n the methods sec-t i o n . INHIBITOR CONCENTRATION SPECIFIC BINDING (mM) (% c o n t r o l ) 100°C/30 sec. - 0 DTT 1.0, 0.1, 0.01 34.1, 57.3, 94.3 pCMBA,+ membranes 1.0, 0.5, 0.1 1.0, 8.0, 98 pCMBA + 1 2 5 I - o P R L 1 mM 100 62 DISCUSSION There i s now abundant evidence that polypeptide hor-mones exert t h e i r primary actions on target c e l l s by binding to s p e c i f i c h i g h - a f f i n i t y receptor s i t e s i n the plasma mem-brane. The existence of such binding s i t e s for p r o l a c t i n i n mammary gland has been established (1) and t h e i r b i o l o g i -c a l significance has been demonstrated (2). Several recent studies have indicated the presence of p r o l a c t i n receptor s i t e s in various other tissues (1, 3). However t h e i r bind-ing c h a r a c t e r i s t i c s and b i o l o g i c a l significance has not yet been determined. The present study demonstrates that re-ceptors capable of binding p r o l a c t i n with high a f f i n i t y and s p e c i f i c i t y are present i n a plasma membrane containing 125 f r a c t i o n i s o l a t e d from rat l i v e r . The finding the I-oPRL i s accumulated by hepatocytes i n rat and mouse l i v e r i n vivo (68) suggests that the binding of p r o l a c t i n to membranes in v i t r o i s not an a r t i f a c t . Other peptide hormones such as LH, FSH and hCG which do not bind to l i v e r membranes i n v i t r o are accumulated by l i v e r macrophages i n vivo (69, 70). These results further suggest that the binding of p r o l a c t i n to l i v e r membranes i s b i o l o g i c a l l y s i g n i f i c a n t . As i n other studies on the binding of polypeptide hor-mones (74, 75), the subcellular f r a c t i o n that contains pro-l a c t i n binding a c t i v i t y also possesses 5 1-nucleotidase a c t i v i t y . Since 5 1-nucleotidase i s a v a l i d marker enzyme 63 for rat l i v e r plasma membranes (50), the enrichment of 5'-nucleotidase a c t i v i t y and the reduction of other marker assay a c t i v i t i e s i n the f i n a l tissue preparation, indicates that plasma membranes have been concentrated i n th i s f r a c t i o n . I t is therefore reasonable to assume that since p r o l a c t i n bind-ing capacity was also enriched i n th i s f r a c t i o n , the pr o l a c t i n receptors are located on c e l l surface membranes. The question now arises as to the extent to which the isolated plasma membranes represent the l i v e r c e l l surface i n s i t u . During preparation loosely bound surface components could be l o s t . Furthermore the surface of hepatocytes i s composed of heterogenous elements, for example, membranes l i n i n g Disse and b i l e canalicular spaces, membranes contact-ing the b i l i a r y c a p i l l a r i e s and membranes contacting neigh-bouring hepatocytes. Disruption by homogenization might create fragments of d i f f e r e n t size from the various areas. Thus small fragments could behave, during the d i f f e r e n t i a l centrifugations, as mitochondria and microsomes and subsequent-l y be l o s t . Studies have shown that only 13% of the t o t a l mem-brane area of rat l i v e r hepatocytes i s i n contact with the b i l i a r y c a p i l l a r y (71). It i s conceivable that i n rat l i v e r hepatocytes, as i n mammary alveolar c e l l s (75), p r o l a c t i n receptors are located only on that part of the membrane which i s adjacent to the vascular supply. The plasma membrane markers may also be r e s t r i c t e d to certai n regions of the mem-branes and thus would not be representative of the whole population of membrane fragments. This i s suggested by the 64 d i s c r e p a n c y observed f o r the y i e l d and p u r i t y as shown by 125 5'-nucleotidase and I-oPRL b i n d i n g c a p a c i t y (Table I ) . 125 Previous s t u d i e s have shown t h a t I-oPRL prepared by the LPO procedure bound much b e t t e r to membrane r e c e p t o r s and r e t a i n e d much more of i t s b i o l o g i c a l a c t i v i t y as compared to the l a b e l l e d hormone prepared by the chloramine-T procedure (1, 72). T h e r e f o r e the enzymatic procedure was used to prepare 125 125 the I-oPRL used i n the present study. The I-oPRL thus obtained demonstrated the b a s i c c h a r a c t e r i s t i c s needed f o r use i n RRA's; i t had good s t a b i l i t y , i t bound to r e c e p t o r s w i t h high a f f i n i t y and s p e c i f i c i t y , and n o n s p e c i f i c b i n d i n g was min-imal and remained constant throughout the i n c u b a t i o n p e r i o d . I t was f u r t h e r shown t h a t , a t p h y s i o l o g i c a l pH, i o d o p r o l a c t i n was as 125 potent as n a t i v e p r o l a c t i n i n d i s p l a c i n g I-oPRL from the r a t l i v e r r e c e p t o r s ( f i g . 12) suggesting t h a t the a f f i n i t i e s of the l a b e l l e d and n a t i v e hormones for. the r e c e p t o r s are equal. There-f o r e the a s s o c i a t i o n and d i s s o c i a t i o n constants determined f o r 125 I-oPRL would be the same f o r the n a t i v e hormone f u r t h e r i n d i c a t i n g t h a t the r e c e p t o r s are capable of b i n d i n g p r o l a c t i n a t p h y s i o l o g i c a l c o n c e n t r a t i o n s . However the serum p r o l a c t i n c o n c e n t r a t i o n i n normal female r a t s i s approximately 10 ng/ml and t h e r e f o r e with a half-maximal s a t u r a t i o n of 3 ng/ml, as determined here, the r e c e p t o r s would be s a t u r a t e d . T h i s would make the r e c e p t o r s b i o l o g i c a l l y i n s i g n i f i c a n t . Other h a l f -maximal s a t u r a t i o n c o n c e n t r a t i o n s r e p o r t e d f o r the b i n d i n g 125 of I-oPRL to female r a t l i v e r membranes range from 4 pg/ml 65 (42) to 22 ng/ml (40). The reason for such a large v a r i a t i o n i s d i f f i c u l t to assess, however there may be other.essential factors necessary for the hormone-receptor i n t e r a c t i o n which have not been recognized. 125 The e f f e c t of cations on I-oPRL binding to l i v e r recep-. . 125 tors was similar to t h e i r e f f e c t s on I-oPRL binding to mam-mary receptors (1). Binding could be activated either by b i -valent or monovalent cations - bivalent cations exerting a greater e f f e c t than monovalent cations at the same concentration. Also combinations of NaCl and CaCl 2 at concentrations that pro-duce maximal stimulation when used i n d i v i d u a l l y did not have an additive e f f e c t . Some noteable differences are (i) 10 mM CaCl 2 increased binding ten-fold i n the l i v e r but only three-f o l d i n the mammary gland, ( i i ) i n the l i v e r , MgCl 2 was not as e f f e c t i v e as CaCl 2 i n ac t i v a t i n g binding but i n mammary gland MgCl 2 was as e f f e c t i v e i f not more e f f e c t i v e than CaCl 2 i n a c t i v a t i n g binding. How cations influence binding i s not known. I t does not appear to depend on ioni c strength or os-molarity since 10 mM CaCl 2 and 150 mM NaCl, which activate bind-ing to the same l e v e l , have ion i c strengths of 0.03 and 0.15 respectively and osmolarities of 4 0 imosM and 300 imosM re-spectively. I t i s u n l i k e l y that the e f f e c t s of cations on bind-ing a c t i v i t y are mediated v i a an e f f e c t of the metal ions on the phase c h a r a c t e r i s t i c s of the membrane l i p i d s , since binding was activated i n the presence of monovalent cations which lack t h i s a b i l i t y (73). I t i s possible that these cations act by screening the surface charges on the membrane and p r o l a c t i n molecules, which would be negatively charged at physiological pH. 66 I t i s not known whether these c a t i o n s a c t d i r e c t l y w i t h the r e c e p t o r or not. A g r e a t d e a l of i n v e s t i g a t i o n must be done i n order to understand the e f f e c t s of c a t i o n s on b i n d i n g . S i m i l a r to the b i n d i n g of p r o l a c t i n to mammary gland 125 r e c e p t o r s (1), the b i n d i n g o f I-oPRL to r a t l i v e r r e c e p t o r s i s time and temperature dependent ( f i g . 8). In both mammary gland and r a t l i v e r b i n d i n g remained very low at 0° At 23° b i n d i n g increased,,' a t the same rate,' i n both r e a c h i n g a maxi-mum at 6 hours. However the b i n d i n g r a t e a t 37° was f a s t e r f o r p r o l a c t i n b i n d i n g to l i v e r r e c e p t o r s than mammary r e c e p t o r s . The b i n d i n g p a t t e r n observed a t 30° i s s i m i l a r to t h a t observ-ed f o r bGH b i n d i n g to i s o l a t e d mouse l i v e r c e l l s a l s o at 30° (9). However the cause of the decreased b i n d i n g a f t e r reach-ing a maximum was not i n v e s t i g a t e d . U n l i k e other hormone r e c e p t o r i n t e r a c t i o n s , i n c l u d i n g p r o l a c t i n b i n d i n g to mammary r e c e p t o r s , the i n t e r a c t i o n ob-served here does not appear to be f r e e l y r e v e r s i b l e e i t h e r i n the presence of 10 mM C a C ^ or 150 mM NaCI. However a t 37° using the p h y s i o l o g i c a l c o n c e n t r a t i o n of NaCI there i s a time dependent d i s s o c i a t i o n , but i t i s s t i l l extremely slow. T h i s slow d i s s o c i a t i o n r a t e i s i n disagreement wi t h Rojaniemi e t 125 a l (68) who found t h a t in v i v o I-oPRL i n the l i v e r o f fem-a l e r a t s r a p i d l y d e c l i n e d 15 minutes a f t e r hormone a d m i n i s t r a -t i o n . The la c k of d i s s o c i a t i o n i s a l s o - c o n f u s i n g s i n c e i t has been demonstrated t h a t the h a l f l i f e o f p r o l a c t i n r e c e p t o r s i n r a t l i v e r i s l e s s than 3 hours (45). I f the p r o l a c t i n mole-c u l e i s destroyed along with i t s r e c e p t o r , t h i s would be a unique mechanism not seen f o r other hormones. However i t may be p o s s i b l e t h a t d i s s o c i a t i o n occurs o n l y a f t e r the hormone i s i n a c t i v a t e d . I t has been demonstrated t h a t the b i n d i n g and i n a c t i v a t i o n of both glucagon and i n s u l i n by r a t l i v e r plasma membranes are two independent and u n r e l a t e d processes (77, 78). T h i s a l s o may be the case f o r p r o l a c t i n b i n d i n g , but the assay c o n d i t i o n s used to measure p r o l a c t i n b i n d i n g may not be optimal f o r the i n a c t i v a t i o n of the hormone. F u r t h e r i n -v e s t i g a t i o n s u s i n g other assay c o n d i t i o n s would have to be c a r r i e d out before any f i r m c o n c l u s i o n s were made. The pH optimum of b i n d i n g f o r a l a r g e v a r i e t y of p o l y -peptide hormones has been found to be between pH'7.0 and 7.4 125 i n c l u d i n g the mammary gland r e c e p t o r f o r I-oPRL, which has a pH optimum of 7.3 (1). The low pH optimum;' of 6.5 f o r the h e p a t i c p r o l a c t i n r e c e p t o r appears to be another unique c h a r a c t e r i s t i c of t h i s r e c e p t o r . As with many other p o l y p e p t i d e hormone r e c e p t o r s , the i n h i b i t i o n of b i n d i n g by pCMBA i n d i c a t e s t h a t c y s t e i n e r e s i d u e s are c r i t i c a l l y i n v o l v e d i n the b i n d i n g of p r o l a c t i n to i t s r e c e p t o r . D e s t r u c t i o n of r e c e p t o r a c t i v i t y by t r e a t -ment- ' wi t h pronase and phospholipase C i n d i c a t e s t h a t p r o t e i n and p h o s p h o l i p i d c o n s t i t u e n t s are s t r u c t u r a l components t h a t are e s s e n t i a l f o r r e c e p t o r a c t i v i t y . The l a c k of any e f f e c t on p r o l a c t i n b i n d i n g by RNase and DNase suggests t h a t n u c l e i c 63 a c i d s do not p l a y any s i g n i f i c a n t r o l e i n the formation of the hormone-receptor complex. The a c t i v a t i o n of b i n d i n g by treatment with neuraminidase i n d i c a t e s t h a t s i a l i c a c i d may not be e s s e n t i a l f o r b i n d i n g but i t may be a s t r u c t u r a l component of e i t h e r the r e c e p t o r or another p r o t e i n l o c a t e d i n c l o s e p r o x i m i t y to the r e c e p t o r s i t e . Recent s t u d i e s on the e f f e c t of l e c t i n s on p r o l a c t i n b i n d i n g to r a t l i v e r c e l l membranes have shown t h a t C o n c a n a v i l i n A, which binds to oi - m e t h y l g l u c o s y l or ex -methylmanosyl r e s i d u e s , i n h i b i t e d p r o l a c t i n b i n d i n g w h ile wheat germ a g g l u t i n i n which binds to s i a l i c a c i d d i d not i n h i b i t b i n d i n g (79). These r e s u l t s f u r t h e r suggest t h a t s i a l i c a c i d i s not i n v o l v e d i n the hor-mone r e c e p t o r i n t e r a c t i o n but r a t h e r °< - m e t h y l g l u c o s y l and oc -methylmanosyl r e s i d u e s may serve some f u n c t i o n i n the hormone r e c e p t o r i n t e r a c t i o n . Though s i a l i c a c i d does not appear to be necessary f o r p r o l a c t i n b i n d i n g i t may be r e q u i r e d f o r the f u n c t i o n of the r e c e p t o r . I t has been shown t h a t neuraminidase treatment of i s o l a t e d f a t c e l l s completely blocks the s t i m u l a t i o n of glucose uptake by i n s u l i n without i n f l u e n c i n g i n s u l i n b i n d i n g (80). I t may be p o s s i b l e t h a t s i a l i c a c i d i s necessary f o r a c t i v a t i o n of hepatocytes by p r o l a c t i n . 69 CONCLUSION Receptors for oPRL have been i d e n t i f i e d and characterized in a plasma membrane containing f r a c t i o n from r at l i v e r s . The finding that the Ka for the interaction varies widely and that the interaction i s not reversib l e casts some doubt on the b i o l o g i c a l significance of these receptors. However the demonstration that p r o l a c t i n binding i s in h i b i t e d only by other lactogenic hormones and not by unrelated hormones such as i n s u l i n and glucagon, which also bind to rat l i v e r , does in i t s e l f suggest some importance for these binding s i t e s . It would be very helpful to be able to correlate the binding of p r o l a c t i n with other important biochemical actions of pr o l a c t i n . 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