, . , ' - ‘ . . - , .t -O\ ' ~ . . -... Qt . a. - . - . S . ‘~ - . ‘ o"b" \ 'r.\-\\ . -‘ D . -0- I . . .' c .. - ‘_ — . . . - b . D 0- t A . ' - ' s . . .. . ' u. 7'. l .. . . ‘ ‘. . - - o , O y 'I I -. ‘~ x» n _ . ~ _ . . . \. ‘.| ‘ . -- - n ‘ I . “ Io.-§ '5‘ ~_ " . , -. a . -. - . ‘ - n v ‘ ..' . c ' D - . 0‘ V c ‘ ~ .3 . _. 4_. o - _ . l ‘ . ‘ v _ . ¢ .' n I “ . v t . . . . _ _ . ; . O _ I . ' . — . _ . . . |~ . . V . . - _ ~ . . ' , . ‘ _ -‘ . I .- .- a , . - ‘ . — . . . ‘ ' . ‘- - . . - . V v k- _ _ .. ‘ . . 'I ‘ ' . ' ‘ a -. . , ‘ _ |_ : ‘ - ‘ ' .~ ‘ a ‘ , . .. ., _ I . '0 .. . . _ - ‘ x . . . . v . _ o . \ _ ‘ . I . .- ~_ . FLUORESCENCE STUDIES or PROLACT-IN ,IODlNATI'ON AND ' 7. - _;; 7 ‘ . PROLAC‘TIN BINDING TOVPITT’ZU-I‘TARY TUMOR-CELLS ' ' ‘ ThesiSfortheDegree ofM,S.-- ‘ ‘ L S f} ‘ MICHIGAN IZIATEUNWERFS-ITY -- fl _ . SAMUEL :B.. RHODES. ' , . A . _ a5,- . I .. . -. Vv ‘ . ., ~5- . . . - -0 v . . — , , o - . ,- -. u - o . -. a- - - . .4 . . M- ¢ ‘ - - w. ' > -~'- '--A "f~" v-” 4‘“ °““".'- .---7-.-. “'9” . . - 4 ' ’ - - -o-,-.~.~‘-,.~oo..o.~ —-,.'..’v. l‘fiwo'V'.‘..if.'-N.ff~'~ M . I .4 4 av ’ - ’ v.\-r‘-w,n. .--~\- *'~ -— ‘ . M ‘J 33 'x . . MM n Q t ' ' 0.-a—-,-,wf,, -'—— ¢ rrr".‘ro.‘. .-- a*n!'vvfo.v’>*rr’.-~-k 000° Orr-'AOI’WPO‘I‘NO d~ ' 4" «mu 2 ”4%,. 0'. u to o l v. a in q 6 l V VI ‘ LIst - 'ZY um‘i J... at, ABSTRACT FLUORESCENCE STUDIES OF PROLACTIN IODINATION AND PROLACTIN BINDING T0 PITUITARY TUMOR CELLS By Samuel B. Rhodes The study of plasma membrane lactogenic hormone receptors 125I-labelled hromones has been done predominantly via the use of and partially purified membrane fractions. The kinetic and affinity constants derived from this radioreceptor assay (RRA) depend on the questionable assumption that the labelled and unlabelled hormones possess identical biochemical properties. A few investigators using unlabelled hormones and cell fractions have employed colorimetric analyses to describe hormone binding. In this study, the lactoperoxidase catalyzed iodination of ovine prolactin was monitored with a computer coupled spectrophotometric-spectrofluorometer. A 34.7% decrease in tryp- tophanyl fluorescence and a 35.1% decrease in partial quantum efficiency were shown to correlate with the enzyme-dependent iodina- tion reaction. The reaction mechanism appears to involve a tertiary complex of prolactin, lactoperoxidase and iodide. All fluorometric changes are complete within 5 minutes of the addition of hydrogen peroxide. The fluorescence response is clearly distinguishable from heavy ion collisional quenching and thus is believed to be the Samuel B. Rhodes result of stearic interference or conformational changes in the vicinity of the fluorophore. The binding of ovine prolactin to a clonal strain of rat anterior pituitary tumor cells was also investigated by fluorescence techniques. As a criterion for metabolic activity, at least 90% of the intact cells were required to exclude trypan blue before and after the experimental reactions. The reaction is complete within l2 minutes and is characterized by dose-dependent tryptophanyl fluor- escence enhancement. A sigmoid dose-response curve we interpret as showing potentiation of the binding site at concentrations between 18 and 30 ug/ml ovine-prolactin. There was no fluorescence enhance- ment in response to 25 ug/ml ovine growth hormone, but 45 ug/ml caused a small but significant degree of quenching. No hormone dependent changes in Tyndall or Rayleigh light scattering were detectable under the conditions of these experiments, however, methodological refinements are recommended for future investigations. FLUORESCENCE STUDIES OF PROLACTIN IODINATION AND PROLACTIN BINDING TO PITUITARY TUMOR CELLS By Samuel B. Rhodes A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1976 This thesis is dedicated to my mother whose strength and courage are a continuing inspiration. ACKNOWLEDGMENTS The author is indebted to the many friends and faculty whose encouragement and support during the past two years will long be remembered. Special thanks to Olan Dombroske for countless hours of valuable discussion and to Mark Halonen for help with cell cul- turing. I I would especially like to thank Drs. w. L. Frantz and John Holland for valuable counsel and expert technical assistance. Thanks are also due to Dr. C. Nelsch for the use of the laminar flow hood, to Dr. Jack Hoffert for help with the photography and to Nancy Turner for hours spent in preparation of the thesis. To Elaine, your patience, encouragement and understanding during the past months are deeply appreciated. I am also indebted to the National Science Foundation for support during part of the research period (NSF grant GBMS-7l-0l257). iii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES . INTRODUCTION . LITERATURE REVIEW 1. II. III. IV. Physical-Chemical Properties of Prolactin and Prolactin Iodination . . . . . . A. The Prolactin Molecule . B. Lactoperoxidase Catalyzed Iodination of. Prolactin . . . Prolactin Synthesis and Secretion . Radioreceptor Assays for Prolactin Binding Pituitary Cell Culture as a Model for Organ Function : Structure and Physical Properties of Cell Surface Membranes . . . . . . A. Overview 8. Infrared Spectroscopy. . C. Nuclear Magnetic Resonance (NMR) D. Electron Spin Resonance (ESR) E. Fluorescence Spectroscopy MATERIALS AND METHODS . I. II. III. IV. The Spectrophotometric-Spectrofluorometer Reactions with Prolactin . . A. Iodination . . B. Potassium Iodide Quenching Pituitary Cell Culture . A. Growth Medium . B. Harvesting and Preparation of Cells Fluorescence and Light Scattering Monitors of AP Cells: Prolactin Reactions . . A. Fluorescence . . B. Light Scattering Microphotography . iv Page vi vii EXPERIMENTAL . I. II. III. IV. Iodination and Iodide Quenching . A. Iodination . . . . l. Objectives 2. Procedures 3. Results a. Kinetics . . b. Analysis of fluorometric data B. Iodide Quenching l. Objectives 2. Procedures 3. Results C. Discussion . Photometric Characterization of CgllRAP Cells A. Objectives . B. Procedures C. Results D. Discussion . Fluorescence Monitor of Hormone- Cell Interactions . A. Objectives B. Procedures C. Results D. Discussion . Light Scattering of CgllRAP Cells A. Objectives - B. Procedures C. Results D. Discussion SUMMARY AND GENERAL DISCUSSION . APPENDICES A. Optics and Technical Specifications of the Spectro- fluorometer . . . B. PUCka Plus Glucose Balanced Salt Solution C. Dilution Corrected Photometric Data from Iodination Reactions . . D. Prolactin- Cell Interactions at Various Cell and Hormone Concentrations . . . E. Dose Response of Prolactin- Cell Reactions . F. Changes in Light Scattering of CgllRAP Cells Due to Dilution . . G. Statistics . REFERENCES 80 81 82 83 84 85 86 87 Table 1. 9A. 98. LIST OF TABLES Changes in fluorescence and partial quantum efficiency associated with iodination and iodide quenching Cell concentration and photometric data . Fluorescence of ovine prolactin and ovine growth hormone . . . . . . . . . . . Fluorescence response of CgllRAP cells to ovine growth hormone and ovine prolactin Pucksf plus glucose balanced salt solution . Dilution corrected photometric data from iodination reactions . . . . Prolactin-cell interactions at various cell and hormone concentrations Dose response of prolactin-cell reactions Changes in light scattering of C3llRAP cells due to dilution with buffer . Changes in light scattering due to simultaneous dilution and addition of 40 ug/ml PRL . vi Page 34 46 53 60 Bl 82 83 84 85 85 LIST OF FIGURES Absorbance, fluorescence and partial quantum effi- ciency changes during an iodination reaction plotted against time in minutes . Histogram of the percent change in fluorescence and partial quantum efficiency during iodination and iodide quenching . . . . . Excitation and emission scans of CallRAP cells Plot of C llRAP cell fluorescence as a function of cell co centration . . . . . . . . Fluorescence enhancement as a function of initial cell concentration Fluorescence enhancement as a function of prolactin concentration . . . . . . . . Changes in light scattering of C8llRAP cells due to dilution . Photmicrographs of C8llRAP cells Optics and technical specifications of the spectro- fluorometer . . . vii Page 31 35 44 47 54 57 69 72 8O INTRODUCTION The mammalian cell membrane is the structural and functional interface between the cell's environment and its cytoplasm. Electrical and chemical communication between the specialized tissues of the body are mediated and integrated by specific compo- nents of the plasma membrane of a cell. The exact compliment of proteins and lipids which compose the cell membrane is determined by the functional role that the cell plays and the physiological state of the animal. The plasmalemma also plays an important role as a transducer by converting electrical or bond energy into chemical signals which influence cytoplasmic processes such as protein synthe- sis, enzyme activation or nuclear transcription. Polypeptide hormone receptors comprise a subpopulation of membrane proteins which are capable of stimulating precise and genetically prescribed cytoplasmic processes when triggered by the binding of the appropriate hormone. At this time, little is known about the mechanism of hormone binding and the majority of the work 125I-labelled hormones. in this field has depended on the use of Using this radioreceptor assay, investigators have found that the binding is Specific and saturable and the association is assumed to follow first order equilibrium kinetics. Any attempts to derive affinity constants rely on indirect analyses such as the Scatchard plot. A serious deficiency of the radioreceptor assay is the l assumption that the labelled and unlabelled hormone possess the same biochemical properties. There is no means of evaluating the validity of this assumption within this system because the only measurable parameter is the radioactivity itself. In order to avoid this dilemma, a few investigators have turned to colorimetric methods and unlabelled hormones to monitor the binding of the hormone to its receptor. In recent years, prolactin and its receptor have been intensely investigated by a number of groups. The methods however have been limited to the use of I-labelled lactogens. Although the iodinated molecule is known to have a somewhat reduced biological potency, little effort has been spent in characterizing the molecular changes which account for the diminished activity. Because bio- chemical properties such as binding affinity and reaction rate con- stants have been derived for the iodinated species, it is essential to determine the applicability of these parameters to the native hormone. It would also be informative to develop a different assay method which could be compared to the radioreceptor assay. This research was designed to examine the kinetics, mechanism and effects of the lactoperoxidase iodination reaction and compare the iodinated and native molecule by fluorescence methods. We also wished to explore the possibility of deveIOping a fluorescence monitor of prolactin-receptor binding with intact cells. LITERATURE REVIEW I;_ Physical-Chemical Properties of Prolactin and Prolactin Iodination A. The Prolactin Molecule Prolactin is a polypeptide hormone secreted by the acidophil cells of the anterior pituitary in a variety of mammalian and sub- mammalian species. Purified monomeric prolactin, from the pitui- taries of sheep (o-PRL), was first characterized by a group headed by C.H. Li (Li et_al,, 1969; Li et_al,, 1970). They found the hormone to consist of a single chain of 198 amino acids with no sugar or lipid moieties and having a molecular weight of 22,550. Prolactin's secondary structure is conferred by three disulfide bonds between cystines 4 and 11, 58 and 173, and 190 and 198. The molecule's isoelectric point is at pH 5.73. More recently, a number of lactogenic hormones have been isolated and characterized, includ- ing human growth hormone (h-GH), human chorionic sommato-mammotropin (HCS) (Li, 1972), bovine prolactin (b-PRL) (Wallis, 1974) and porcine prolactine (p-PRL) (Bewley and Li, 1975). Each of these hormones have a number of regions of amino acid homology and Li (1972) has used circular dichroism and optical rotary dispersion to show that each lactogen consists of 45 to 55 percent a-helix, and less than 12 percent B-sheet conformation. The distribution of charged and uncharged residues appears to be random in all the lactogens, but ovine, bovine, and procine prolactins are distinguished by having 3 only two tryptophan residues (Bewley and Li, 1975). Recently, Dombroske and Frantz (1976) have shown 1n_vjtrg that rat prolactin may exist in monomeric, dimeric, or trimeric forms and the relative abundance of each species may be related to the rate at which they are secreted from the pituitary. The structural-functional similarity of the various lactogens may be evaluated by comparing each hormone's capacity to stimulate a specific biological response. Accordingly, Niall (1972) finds that human prolactin (h-PRL), h-GH, and o-PRL all stimulate comparable epithelial growth of the pigeon crop sac. These findings were con- firmed by Posner (1974), whose radioreceptor work shows that a number of lactogens, including rat prolactin (r-PRL) are all competitive for the same membrane receptor, but not all to the same degree. The bio- »logically active region of the prolactin molecule has not yet been identified. However, Kawauchi (1973) has modified the biological activity of ovine prolactin by forming o-nitrophenylsulfenyl (NPS) derivatives of the native molecule. If a single NPS molecule is covalently bound to tryptophan-149, 80% of prolactin's biological activity is retained. In contrast, if NPS is bound to both tryptophan-9O and tryptophan-149, the resulting prolactin derivative is devoid of biological potency. These experiments suggest that the region surrounding trp-90 is essential to the biological activity of the hormone. Unfortunately, the tertiary folding of the prolactin amino acid sequence is not known,1 and little information concerning prolactin's active site can be obtained from Kawauchi's experiment. 1Alexander Tulinski is presently attempting X-ray crystallography of o-PRL. B. Lactoperoxidase CataLyzed Iodination of Prolactin Radioiodination of polypeptide hormones, to high specific activity, can be accomplished by using chloramine T to oxidize the iodide (Greenwood §t_al,, 1963). However, this reaction appears to destroy a large percentage of the hormone's biological activity. To alleviate this problem, Frantz and Turkington (1972) developed an enzymatic method of prolactin iodination using lactoperoxidase from cow's milk to catalyze the reaction. Low concentrations of peroxide are provided to energize the reaction. Similar methods have also been used by others for other polypeptide hormones (Thorell and Johansson, 1971; Posner, 1974) and all report high specific activity (about 130 uCi/ug) and about 60% biological activity. The pH maxima of lactoperoxidase are at 5.6 and 7.4; the former being nearly twice as great as the latter (Rogol and Chrombach, 1975). In Spite of the widespread use of the lactoperoxidase iodina- tion method, little is known about the mechanism of the reaction for large polypeptides. The reaction involves three substrates: peroxide, iodide and the phenolic compound which is iodinated. Morrison and Bayse (1970) have studied the kinetics of tyrosine iodination and report that a quaternary complex is not formed, but that a “ping-pong- type" mechanism is involved. They caution however, that their pro- posed mechanism may not be applicable to protein substrates. In fact, there is no final evidence which implicated tyrosine as opposed to phenyalanine or tryptophan as a site of iodide fixation in polypep- tides. However, lactoperoxidase has been used to iodinate the tyrosine side chains of thyroglobulin (Monoco gt_gl,, 1975), but because these tyrosines are not in peptide linkage, we cannot con- clude that tyrosine is a preferred substrate for enzymatic reaction with proteins. Moreover, there is some evidence that lactoperoxidase may also catalyze the oxidation of indole compounds such as trypto- phan. This indole oxidation (Alexander, 1974) may partially account for the reduced biological activity of radioiodinated prolactin. II. Prolactin Synthesis and Secretion Both prolactin and growth hormone are synthesized and secreted by the acidophil cells of the anterior pituitary (Li, 1969). A specific messenger RNA has been isolated and used to synthesize prolactin in cell free systems (Maurer, 1976; Evans and Rosenfeld, 1976). The regulation of prolactin release is under the control of a number of inhibiting and stimulating factors. Meites (1959) found that prolactin secretion was stimulated by surgically separating the pituitary from the hypothalmus, and Talwalker (1963) was able to inhibit prolactin secretion jg_vitro by exposing pituitary explants to hypothalamic extracts. Both of these phenomena illustrate the effects of a prolactin inhibiting factor (PIF). A prolactin releasing factor was first proposed by Meites (1961), who was able to stimulate lactation by injecting a crude hypothalamic extract into estrogen primed rats. It soon became evident however, that other humoral factors were effecting the secretion of prolactin. For example, 14C-leucine into male and female newborn Yamamoto (1970) injected rats and followed the secretion of prolactin during the course of the rat's development. Between days 37 and 45, the female rats showed a rapid increase in the concentration of labelled serum pro- lactin. The males also had increased serum prolactin levels by day 45, but in all cases the radioactivity per hundred grams body weight was higher for females than males. It was also found that the pituitary content and plasma concentrations of prolactin in female rats, are higher during estrus and proestrus as compared to diestrus (Ieiri, 1971). More recently, Meites (1972) has reported that estrogen can stimulate prolactin secretion not only via the hypo- thalamus, but directly at the pituitary. The action of progesterone as an effector of prolactin secretion is not clear, but Blake (1972) concludes that the roles of estrogen and progesterone in prolactin secretion are probably dose and system dependent. Thyrotropin releasing factor, the catecholamines and the biogenic amines also appear to moderate the rate of prolactin secre- tion. Shaar and Clemens (1974) report that 2.5 to 5 ug/ml of dopamine are sufficient to significantly inhibit prolactin secretion jg_yjt§g, while equivalent doses of catecholamines have no apparent effect. On the contrary, Chen and Meites (1975) found that both catecholamines and dopamine precursors are able to stimulate the release of prolactin inhibiting factor, and thus effect a decrease in serum prolactin titers. However, it may be that pharmacological doses of catecholamines are necessary to be effective. Serotonin and serotonergic precursors by contrast tend to stimulate prolactin secretion (Kamberi et_al,, 1971). These findings have led to the suggestion that the biogenic amines control diurnal fluctuations in prolactin serum levels (Meites et a1., 1972). Tashjian (1971) has shown that the tripeptide, thyrotropin releasing hormone (TRF), is capable of stimulating the secretion of prolactin from cultured pituitary tumor cells when administered at 0.1-10 pg/m1.concentra- tions. He also notes that TRH increases the rate of prolactin synthesis while the rate of degradation remains constant. In addi- tion, 5 x 10‘6 M hydrocortisone was sufficient to inhibit the release of prolactin (Dannies and Tashjian, 1973). In_vjyg_stimula- tion of prolactin secretion in the rat by TRF was demonstrated by Rivier and Vale (1974) who were also able to quantitate the various effects of TRF analogs. The rye ergot derivatives such as CB-154 are also able to reduce serum prolactin levels in rats as was reported by Brooks and Welsch (1974). The mechanism of their action is probably via increased PIF from the hypothalamus. There are conflicting reports concerning the effect of pro- lactin on its own secretion. Voogt and Meites (1971) have shown that a 250 ug implant of prolactin in the median eminence of pseudo- pregnant rats inhibits the secretion of prolactin probably by stimulating dopaminergic neurons in the hypothalamus (Fuxe and Hokfelt, 1970). In addition, the prolactin implant caused leutein- izing hormone and follicle stimulating hormone titers to increase, and the rats resumed their estrus cycles. Macleod and Abad (1968) on the other hand, observed direct inhibition of prolactin secretion in rats with multiple pituitary tumor explants. It is interesting to note that Frantz's group (1975) has found specific radioreceptor 125 binding sites for I-prolactin in cultured anterior pituitary tumor and normal cells; and Payne (1975) correlated the binding capacity of these cells with the rate at which they secrete prolactin. There is no final conclusion nontheless, and the mechanism of the short-loop feedback system awaits more detailed investigation. III. Radioreceptor Assays for Prolactin Binding 125I-labelled ovine Birkinshaw and Falconer (1972) injected prolactin into female rabbits and followed the tissue distribution of radioactivity by autoradiographic techniques. Their report showed that the radioactivity appeared to be bound to the plasma membrane of the alveolar secretory cells of the mammary gland. This study prompted the development of a radioreceptor assay which proved to be a sensitive and specific method of detecting prolactin binding to target tissues. The pr0perties of the lactogenic receptor were first described by Frantz (1972) who showed that the binding sites are protease sensitive, responsive to nanogram concentrations of hormone, and distributed to a number of tissues including mammary, adrenals, liver, prostate and seminal vesicles. The kinetics of the binding reaction are still not clearly understood. Frantz reports saturation times of about thirty minutes and Shiu and Friesen (1974) report saturation times of up to three hours. The functional significance of prolactin binding has been established by a number of physiological events including casein synthesis and a-aminoisobutyric acid (AIB) transport in mammary tissue (Frantz et_al,, 1973; Shiu and Friesen, 1976). Thus, the prolactin receptor is consistent with the structural-functional criteria set forth by Cuatrecasas (1974). More recently, prolactin receptors have been found in the pituitary (Frantz et a1., 1975), 10 and in estrogen receptor-deficient mammary carcinomas (Costlow et_gl,, 1973). Moreover, Kelly's group (1974) has shown that specific bind- ing of prolactin to mammary tumors correlates strongly with the dependence of the tumor on prolactin. The relative abundance of prolactin receptors in rat liver correlates to sex, pregnancy, and state of lactation. This observa- tion led several investigators to explore the effects of sex steroids, hypophysectomy and prolactin itself on the regulation of liver receptors. Kelly et_al, (1975) noticed that in female rats, receptor induction occurs during estrus and diestrus I, while receptor reduction occurs during proestrus and diestrus II. Similarly, estrogen injections can augment prolactin binding in males. These effects however, take up to six days to be manifested, while hypOphysectomy has a comparable effect within forty-eight hours (Posner et;al,, 1974). When pituitary glands are implanted in the renal capsule of hypophysectomized female rats, a rapid rise in serum prolactin titers ensues which is paralleled by receptor induc- tion in the liver (Posner et_al,, 1975). These effects can be duplicated by a single 2 mg injection of ovine prolactin, and the results are independent of serum steroid levels (Costlow gt_gl,, 1975). Thus, prolactin itself is at least partially responsible for the induction of its own receptor. Shiu and Friesen (1974) have solubilized and purified the membrane receptors from rabbit mammary tissue and found that it is proteinaceous in nature. More- over, a guinea pig antiserum to prolactin receptors inhibits not 11 only prolactin binding, but the ability of the hormone to stimulate casein synthesis and A18 transport in mammary explants (Shiu and Friesen, 1976). In spite of the apparent utility of the radioreceptor assay, a number of discrepancies are seen when it is compared to biological assays. The ultimate reliability of the system has not yet been proven, and a number of criticisms have been advanced by Nicoll (1975). In particular he finds that prolactin binding does not always correlate with the initiation of a biological response. IV. Pituitary Cell Culture as a Model for Organ Function Long term culture of mammalian pituicytes is limited to the use of tumor cells because normal cells have a tendency to de- differentiate or die after several weeks in culture. Several anterior pituitary cell lines were isolated and characterized from X-ray irradiated Nister-Furth rats by Tashjian (1968). He found that three of these clonal strains secreted both growth hormone and prolactin and were stable in culture over extended periods of time (Tashjian et_al,, 1970). The karyotype, specific function, and appearance of the cells were also documented (Sonnenschein gt_al,, 1970). Following this extensive characterization of the cell lines, a specific functional criterion was examined: the effect of thyroid releasing factor (TRF) on the secretion of prolactin. They found that the rate of prolactin secretion by one strain of cells was quantitatively related to the amount of TRF provided in the growth medium, while a second strain of cells was relatively unresponsive 12 to TRF (Tashjian, 1971; Dannies and Tashjian, 1973). These biological responses were subsequently correlated with specific binding of radio- labelled TRF to a membrane receptor (Tashjian, 1973). The receptor affinities and biological potency of TRF analogs were also studied and the active site of the hormone molecule was postulated (Hinkle, 1974). Finally, differential fluorometry was used to monitor the conformational changes of membrane proteins which accompany the bind- ing of TRF to cell homogenate particles (Imae et_al,, 1975). A second group of cell lines cloned from estrogen induced rat anterior pituitary tumors was characterized by Sonnenschein (1973). The morphology, growth pr0perties, and secretory products of the cells have also been described (Sonnenschein gt_al,, 1974). The authors caution however, that morphology does not correlate with function, and both histologically different cell types secrete pro- lactin and growth hormone. Three of Sonnenschein's clonal cell strains were assayed for prolactin binding. One was found to have slightly greater binding capacity than normal pituitary cells, while the other two strains were devoid of prolactin receptors (Frantz gt_al,,l975). The physiological significance of these binding sites is still undetermined. Although pituitary tumor cells appear to be excellent models for endocrinological investigation, there are important physiological properties which distinguish these cells from normal cells. First of all, tumor cells generally have lost their density dependent regulatory apparatus (no contact inhibition) and thus will continue to multiply indefinitely (Abercrombie, 1970). Franks (1968) has l3 determined that tumor cells often possess surface antigens which distinguish them from normal cells. In addition, the carbohydrate content of the surface proteins of tumor cells appears to be altered. The latter property permits tumor cells to be selectively agglutinated by plant lectins such as Concanavalin A and wheat germ agglutinin (Cook and Stoddart, 1973). All of these characteristics suggest that tumor cells have unusual and distinctive membrane properties which may be significantly different from normal cells. Therefore, any conclusions drawn from in vitro experiments must be cautiously and prudently generalized to jg_vivo conditions. V. Structure and Physical Properties of Cell Surface Membranes A. Overview The plasma membrane of the eukaryotic cell is the only structural and functional barrier between cytoplasmic processes and the cell's environment. Thus, all cellular nutrients and waste products must either diffuse or be carried across this lipo-protein partition; and electrical or chemical stimuli must be communicated to the cytoplasm by membrane-mediated phenomena. In the present study, the hormone-membrane interactions of prolactin and its membrane binding site have been investigated, and a bried review of the litera- ture concerning the structural-functional properties of membranes is appropriate. The classical article by Danelli and Davson (1935) gave us the first insights into the conformational arrangements of lipids and proteins into a bimolecular sheet which was believed to be essentially rigid and inelastic. The concept of a rigid membrane 14 was also supported by electron microscopic and X-ray diffraction studies which presented the membrane as an orderly array of lipids and imbedded proteins (Hendler, 1971). However, during the past fifteen years, a number of biochemical and biophysical techniques have led us to believe that the components of the plasma membrane are not static, but undergo numerous conformational and configura- tional changes. A few of the pertinent techniques and results which have furthered this dynamic concept of the plasma membrane are presented below. B. Infrared Spectroscopy Interatomic distances within molecules fluctuate about average values through one or more vibrational motions. Such motions can change the dipole moment of a given bond and the resulting electric field will oscillate at the same frequency as the bond vibrations. If the bond is irradiated with electro- magnetic waves of the same frequency, it absorbs some of the radiant energy in a quantal fashion. The absorption spectra of various chemical bonds have been catalogued and serve as a reference for the interpretation of macromolecular microenvironments. Thus, infrared (IR) absorbance spectra are useful for determining the interactions of macromolecular components of membranes. For example, Maddy and Malcolm (1965) have determined that the phosphate ester stretching (P=O) frequency in erythrocyte ghosts is the same as that observed in phospholipids in water. This suggests that the polar heads of membrane lipids are oriented toward an aqueous 15 environment. Wallach and Zahler (1968) compared the IR spectra of Ehrlich ascite tumor cell membranes with the IR spectra of free lipids and concluded that 25% of the lipid must be hydrophobically bound to membrane proteins. Moreover, the IR spectra of erythro- cyte membranes indicate that the imbedded proteins are capable of conformational changes which correlate with metabolic processes such as ATPase activity. There is also evidence that membrane proteins may possess a substantial amount of B structured H-bonding (Chapman, 1973). C. Nuclear Magnetic Resonance (NMR) The nuclei of many atoms are like spinning charged spheres. These oscillating electric fields induce localized magnetic moments which can be oriented in an applied field. If electromagnetic radiation is applied, the nucleus may be forced to realign itself. NMR essentially measures the energy required for the realignment, and thus supplies information about neighboring molecules and their configurations (Wallach and Ninzler, 1974). Of particular interest in the study of biological membranes is proton magnetic resonance (PMR), which in general is thought to arise primarily from the membrane lipids. There is a distinct difference in NMR and PMR spectra of erythrocyte ghosts and sonicated membranes. The sonicates tend to have sharper, well defined peaks and longer relaxation times (Chapman et_al,, 1968). The broad and indistinct peaks found in the membrane ghosts implies a more structured and thermodynamically stable arrangement of molecules. Conversely, excitable membranes 16 such as the sciatic nerve of the rabbit, appear to have regions of relative fluidity (Dea et_al,, 1973). Glaser (1970) has studied the effects of temperature on PMR and reports that an increase from 18 to 40°C will greatly enhance the liquid content of the membrane lipids. Only at temperatures above 60°C will the methyl groups from proteins contribute to the spectra. In addition, they found that phOSpholipase C can increase the fluid nature of erythrocyte mem- branes by 75%, without changing the tertiary structure of the mem- brane proteins. They concluded that the proteins and lipids inter- act in a "fluid mosaic“ medium. 0. Electron Spin Resonance (ESR) The Spinning charge of an electron will induce a magnetic field. Generally, electrons are paired in chemical bonds and their magnetic moments cancel. In contrast, molecules such as nitroxides contain linkages with unpaired electrons. When placed in a magnetic field, the unpaired electrons orient themselves either with or against the field. If electromagnetic radiation, of appropriate frequency is applied, energy may be absorbed or emitted and the electron can "flip" its alignment. A difference in the relative populations of the two alignment states is the basis of electron spin resonance spectroscopy. Because most biological molecules do not possess unpaired electrons, covalently bound nitroxide labels must be affixed to the molecule being studied. Kornberg and McConnell (1971) used spin labelled ESR to monitor the inside-outside exchange of lipids between the bimolecular layers of phosphatidylcholine 17 micelles. They estimated the "flip—flop" half times to be about 6.5 hours at 30°C. On the other hand, lateral diffusion of lipids in the plane of the membrane is relatively rapid. Thus, neighboring lipids can exchange places with a frequency on the order of 107 per second (Devaux and McConnell, 1972). However, this lateral diffusion rate is apparently proportional to the lipid/protein ratio in the Fe: membrane. Jost (1973) found that as the concentration of cytochrome I oxidase in a model membrane was increased, the fluidity of the lipids decreased. They determined that about 0.2 mg of phOSpholipid is hydrophobically bound to each mg of protein. It should be noted i that some researchers believe that the spin-label itself may con- ‘ tribute to the fluidity of the membrane. Recently, this criticism has been avoided by Stanacev and Stuhne-Sekalec (1974) who have perfected an enzymatic incorporation of radio-labelled and spin- labelled phosphatidic acid. The radiolabel (3H) permits easy quantitation and the spin-label is biologically active stearic acid. E. Fluorescence Spectroscopy Electrons revolving in their respective orbits are capable of absorbing electromagnetic energy in a quantal fashion. The fate of the absorbed energy is a function of the molecular environment of the chromophore. Ideally, an electron can emit radiation one of two ways: emit a photon from the same vibrational level to which it was excited, or undergo changes in vibrational levels prior to emission of radiation. In solution, only the latter phenomenon is observed and the emitted radiation (fluorescence) is always of longer wave- length (Hercules, 1966). Proteins are complex molecules and usually 18 contain more than one chromophore and fluorophore. Under favorable conditions an excited residue may transfer its energy to an absorbing residue which in turn will emit photons at its characteristic wave- length. The efficiency of the energy transfer varies with the inverse sixth power of the separation of residues so that generally a donor-receptor pair must be within 20-100 A of each other for ::.. appreciable energy transfer to be detected (Van Holde, 1971). 1 X-ray diffraction analyses have shown that such distances are common between the aromatic residues of biological proteins (Tulinsky et_al,, 1973; Liljan et_al,, 1972). Because of this energy transfer and the fact that the tryptophan absorption band is at the longest wavelength, it is not surprising that when phenylala- nine, tyrosine and tryptophan are all present in a protein, tryptophan fluorescence usually constitutes the major emission peak. Fluorescence spectroscopy may be used as a sensitive monitor of many substrate-ligand binding reactions. The association of a protein with a substrate is often accompanied by fluorescence perturbation which can be stoichiometrically related to the concen- trations of reactants. Examples of such reactions are the tubulin- colchicine system (Bhattacharya and Wolff, 1974) and lysozyme- saccharide binding (Halford, 1975). In addition, selective quenching of protein tryptophan fluorescence can be effected by exposing the protein to various concentrations of heavy ions such as iodide (Lehrer, 1967). If a mathematical analysis of tryptophan quenching is subsequently made, valuable information about protein tertiary structure can be resolved (Lehrer, 1971). 19 The conformational and structural arrangement of membrane proteins have been studied by a number of fluorescence techniques. In particular, l-anilino-B-naphthalene sulfonate (ANS) and 2-p- toluedinylnaphthaline-6-sulfonate (TNS) have been employed exten- sively as membrane fluorescent probes. The major factors effecting ANS and TNS fluorescence are viscosity, polarity, and polarizability IF‘ of their microenvironment (Oster and Mishyma, 1956). For example, I the transfer Of a naphthaline sulfonate derivative from an aqueous ; to an organic solvent will cause a large enhancement of fluorescence I and an accompanying blue shift, i.e., fluorescence at a lower wave- 5 length. There is also energy transfer from membrane tryptophans to membrane bound ANS. This is apparent both from the excitation spectra of free and membrane bound ANS, and the quenching of tryptophan fluorescence by binding of ANS (Wallach, 1970). Gulik- Krzywichi (1970) has used X-ray diffraction and fluorescence to show that ANS is usually bound in the hydrophobic regions of protein- lipid contact. The utility of ANS as a membrane probe was admirably demonstrated by Tasaki (1968) and Teisse (1975) who have monitored fluorescent changes which parallel depolarization of isolated nerve axons. Feinstein (1970) has also reported fluorescence enhancement of membrane bound ANS during treatment of erythrocytes with butacaine and calcium. Each of these experiments suggest that membrane pro- teins undergo conformational changes which facilitate or inhibit the transfer of electromagnetic energy from tryptophan to ANS. The fluorescence of membrane protein itself can be monitored when appropriate excitation and emission wavelengths are selected. 20 Membrane fragments from Erhlich ascite tumor cells emit light at 335 nm when excited at 275 nm which implies that membrane tryptophans are located in a somewhat non-polar environment (Wallach and Zahler, 1966). These membrane fragments also lacked an emission peak at 303 nm which suggests that energy transfer from tyrosine occurs. Similarly, Sonenberg gt_al, (1971) reports that human erythrocytes reacted with human growth hormone emit 20% less fluorescence than membranes alone. Concurrent with this apparent tryptophan quenching is an observed decrease in fluorescence polarization. The effect was restricted to pH 7.4 and physiological temperatures. When growth hormone is reacted with rat liver membranes a corresponding fluorescence quenching is observed and a greater negative ellipticity (monitored by circular dichroism) is found to correlate with 5'-nucleotidase activity (Postel-Vinay, 1974; Rubin, 1973). Imae §t_al, (1975) could also observe fluorescence quenching associated with treatment of cultured pituitary cell fractions with TRF. The quenching effect could be titrated with increasing quantities of hormone. One of the most revealing experiments con- cerning membrane structure and functions was conducted by Taylor (1971). His group reacted fluorescence-labelled anti-immunoglobulin with intact mouse spleen cells. They found that immediately following the reaction, the labelled antigen was scattered randomly over the surface of the cell. However, within 30 minutes, the fluorescing particles had migrated to one of the poles of the cell: a phenomenon called capping. These experiments suggest that proteins as well as lipids can be highly mobile in the plane of the membrane. 21 In light of the experiments outlined above, it is not surprising that models of cell membranes have undergone considerable revision during the past decade (Bretscher, 1973; Siekevitz, 1972). It is also quite reasonable to suspect that membrane reactions may have direct effects on cytoplasmic phenomena (Edleman, 1976). MATERIALS AND METHODS I. The Spectrophotometric-Spectrofluorometer The instrument used for the majority of the present study is a spectrOphotometric-spectrofluorometer (Holland et_al,, 1973) coupled to an on-line computer which applies corrections to many of the instrumental and photophysical variables of fluroescence mea- surements. The unique feature of this instrument is its capacity to measure absorbance and fluorescence simultaneously from paired quartz cuvettes. The contents of one cuvette serve as a reference, while the contents of the second cuvette are varied through one or several experimental parameters. The excitation radiation to each cuvette is derived from a single monochromatic light source which is chopped by an oscillating mirror, and passed intermittently (25.0 milli- seconds) to each cell. The transmitted photons are then fed to a photodetector which in turn feeds the signal to the computer. The 90° emission from the experimental (or sample) cuvette is passed to a second monochrometer and photodetector and ultimately to the com- puter. Thus, a single excitation scan will provide the data needed to compute simultaneous absorbance and fluorescence spectra. More- over, when the number of quanta fluoresced is divided by the number of quanta absorbed, a unique datum is derived. Since at any point along the scan axis, the emission detector will see only a fixed part of the total quanta fluoresced, this datum has been called 22 23 partial quantum efficiency (PQ). Ideally, changes in PQ linearly exhibit the changes in total quantum efficiency. Thus, chemical reactions in the sample cuvette which produce subtle changes in solvation, conformation, or bonding in the vicinity of the fluoro- phore may also produce changes in PQ. Therefore, like fluorescence, PQ presents an intrinsic quantity which can be used to detect struc- tural changes affecting the relaxation processes of the photoexcited molecule. Because PQ is the quotient of the number of quanta fluoresced and the number of quanta absorbed, it is unitless, and thus, in the case of a pure fluorophore, independent of excitation wavelength and concentration. The term Relative Fluorescence Effi- ciency has also been used to define this quantity. The computer which is coupled to the fluorometer is connected to a teletype which is equipped with a phosphorescent screen. The data collected during an excitation or emission scan can be displayed visually in tabular or graphic form. There are three programs which are employed extensively during the present study. (1) The gxgjtay tion scan is simply the collection of absorbance, fluorescence and PQ values while the emission monochrometer is held at a constant setting and the excitation monochrometer is driven over a pre- determined range. (2) Similarly, the emission scan is the collection of data while the excitation wavelength is held constant and the emission monochrometer is varied. (3) In the time scan, both mono- chrometers are held at pre-determined settings and data is collected over discrete periods of time. In the present study, all time scans were performed during five second intervals when 12-15 data points 24 were collected for absorbance, fluorescence, and PQ. These data were averaged by the computer and displayed on the fluorescent screen of the teletype. Henceforth, the averaged time scan data shall be referred to as AB (absorbance), CO (corrected fluorescence), and PQ (partial quantum efficiency). With this instrument, the units of absorbance and ffiuorescence are relative and can be varied by u. xvcxrj adjusting the photomultiplier amplifier voltage and the monochrometer slit width settings. Throughout this study the instrument was 5 calibrated with the same 10' M quinine sulfate solution in 0.1 M H2504. A diagram and brief discussion of the spectrofluorometer is provided in Appendix A. II. Reactions with Prolactin A. Iodination Preparations of ovine prolactin (NIH-P-S-ll), unlabelled KI (Baker Chemical Co., Phillipsburg, N.J.), H202 (Mallinckrodt Chemical Works, St. Louis, Mo.) and lactoperoxidase (Calbiochem, San Diego, Ca.) are made to equal ten times the concentrations used by Frantz (1972). The prolactin and lactoperoxidase are weighed on a Cahn model 4100 electrobalance (Cahn Corp., Paramount, Ca.) and stored at -27°C until two hours before use. Experiments in our lab (unpub- lished) have shown that prior dissolution of prolactin in 0.01 M NH3HCO3 (pH 8.3) will facilitate the solvation of hormone in the buffer which provides the optimal pH for lactoperoxidase activity: 0.4 M sodium acetate, pH 5.3. Accordingly, 25 pg of KI, and 100 25 nanomoles of H202, directly to the prolactin solution. Each solute in 0.25 ml of sodium acetate (Baker Chemical Co.) buffer is added via a Hamilton microliter syringe. The reaction is monitored by the spectrophotometric-spectrofluorometer described in the previous section. 8. Potassium Iodide Quenching The selective quenching of tryptOphanyl fluorescence by K1 has been demonstrated by Lehrer (1971) for a number of model poly- peptides. In the present study, 25 ug/ml solutions of ovine pro- lactin (NIH-P-S-ll) were prepared in 0.4 M Na Ac (pH 5.3) containing 0.00, 0.04, 0.12 or 0.2 M KI. To inhibit 13 formation 10-4 M Na2503 was also provided. (All chemicals are analytical grade.) The rela- tive fluorescence of each solution was determined with an Aminco- Bowman spectrofluorometer. III. Pituitary Cell Culture A. Growth Medium C811RAP rat anterior pituitary tumor cells are raised in 3 liter roller bottles at 37°C essentially according to the method of Payne (1975). The growth medium is prepared using a modification of Sonnenschein's medium (1974), consisting of 13.47 g per L Dulbesco's Modified Eagle Medium Powder (GIBCO, Grand Island, N.Y.), 15% Horse Serum (Difco, Detroit, Mi.), 2.5% Fetal Calf Serum (Difco), 812 mls triple distilled water, and 0.5 m M N'—2-hydroxyethylpipera- zine-N'-ethanesulfonic Acid (Hepes) buffer (GIBCO). An antibiotic- antimycotic mixture (GIBCO) and 72 pg of Anti-PPLO-agent (GIBCO) 26 are also added (a Tylocine preparation). The medium is pH adjusted to 7.2 with sodium bicarbonate (GIBCO). Before use, the medium is filtered through a 0.45 0 pore filter (Gelman Instrument Co., Ann Arbor, Mi.) utilizing a sterile pyrex Millipore-filtering apparatus (Millipore Corp., Bedford, Ma.). The medium is then frozen (-20°C) until needed. 8. Harvesting~and Preparation of Cells Cells are harvested and fed under a laminar flow hood (Type W S series 300, Westinghouse, Grand Rapids, Mi.). Within six hours of an experiment, cells are harvested and centrifuged at 1800 rpm's for 15 minutes in a Servall mOdel 554 centrifuge (Sorvall Corp., Newton, Conn.). The culture medium is decanted and the pellet resuspended, washed three times, and_concentrated in Pucksf plus glucose (a protein-free balanced salt solution, pH 7.4; see Appendix B). Aliquots of the resulting cell suspension are counted with a Neubaurer hemocytometer and tested for viability by excluding 0.01% trypan blue in 0.02 M citric acid (or 0.01% Erythrocin B). Tennant (1964) found 85% of the cells counted by this method were capable of replication under optimal conditions. In this study, 90% of the cells were required to exclude the dye both before and after the experimental reactions. The cells were viable in Pucksf plus glu- cose for at least six hours. 27 IV. Fluorescence and Light Scattering Monitors of AP Cells: Prolactin Reactions A. Fluorescence The reaction of a polypeptide hormone with erythrocyte mem- brane homogenates was first performed by Sonenberg (1969). He found that picomolar quantities of human growth hormone were sufficient to cause a twenty percent reduction in the intrinsic fluorescence of membrane tryptophans. Similarly, Imae (1975) found that TRF will effect comparable quenching of membrane fluorescence of cultured AP cell particles. In the present study, 87.5, 125, 150, 225, 250, and 500 pg/ml solutions of ovine prolactin (NIH-P-S-ll) were prepared in Pucksf saline plus glucose. With a microliter syringe, 250 pl ali- quots of those solutions were then injected into 2 mls of various concentrations of CBllRAP pituicytes in the sample cuvette of the fluorometer. Thus, the resulting suspensions contained 17.5, 25, 30, 45, 50, and 100 pg/ml of hormone, respectively; the reference cuvette contained only Pucksf. A8, C0, and PO readings of the preinjection and postinjection suspensions were taken at various time intervals. The pituitary cells were prepared as described above, and su5pended in Pucksf plus glucose. As a control, 87.5, 125, and 225 ‘pg/ml solutions of ovine growth hormone (NIH-GH-SlO) were also pre- pared and reacted with the cells as above. All reactions were con- ducted at 4°C in the water cooled fluorescence chamber of the spectrofluorometer. 28 8. Light Scattering Individual CallRAP cells have a diameter on the order of 7 microns (Sonnenschien, 1973). Thus, they are capable of scattering a substantial portion of the light which is incident on them (Van Holde, 1971). In order to distinguish the light scattering effects from fluorescence effects it is appropriate to conduct light scat- tering experiments which parallel the fluorescence analysis experi- ments. We prepared various concentrations of 0811RAP cells which were reacted with o-PRL at a final concentration of 40 pg/ml of hormone. The conditions of the experiments were identical to those used in fluorescence experiments except the excitation and emission monochrometers were both set at 330 nm wavelengths. As a control, comparable suspensions were injected with 250 pl aliquots of Pucksf plus glucose. V. Microphotography Photomicrographs of C811RAP suspensions were taken with a 35 mm Yashika TTL reflex camera (Yashika Co., Tokyo, Japan) using a Honeywell Pentax microscope adapter (Honeywell Corp., Denver, Colo.) and a Unitron model Mic 2312 inverted microscope. The film used was Kodak Tri-X (Eastman Kodak Co., Rochester, N.Y.). EXPERIMENTAL I. Iodination and Iodide Quenching A. Iodination 1. Objectives The radioiodination procedure of Frantz (1972) is known to yield some prolactin species which are deficient in biological activity as measured by the radioreceptor assay method. In this experiment, the iodination reaction is monitored by fluorescence instrumentation in order to derive information concerning the mechanism, kinetics, and site of iodide fixation. 2. Procedures Solutions of 25 pg/ml of o-PRL were prepared two hours prior to iodination. Quadruplicate 2 ml aliquots of PRL in sodium acetate buffer were pipetted into the sample cuvette of the fluorometer. Five time scans were performed at two minute intervals on each sam- ple. Then 100 pg of lactoperoxidase was injected into the cuvette in 0.25 ml of acetate buffer, followed by syringe agitation. The contents of the cuvette were allowedto equilibrate for about two minutes and then 3-5 time scans at 2 minute intervals were performed. Similarly, 0.26 pg K1 was injected and followed by time scans. When 0.25 ml of the peroxide was added an immediate time scan was taken and 5-10 subsequent time scans at one minute intervals were per- formed. A final time scan was taken approximately 30 minutes 29 30 following the initial injection of peroxide. The protocol for the control experiments was identical to the above procedure except lactoperoxidase was not added to the cuvette. For all reported experiments, the excitation monochrometer was set at 288 nm and the emission monochrometer at 380 nm. AB, C0, and PQ values for each time scan were recorded and cataloged according to the time course of the reaction and the addi- tion of reactants, respectively. All other possible combinations of PRL, lactoperoxidase, KI, and H202 were also assayed for fluoro- metric responses according to the protocol outlined above. Of all the control experiments, only the PRL plus H202 and the lactoperoxi- dase deficient control elicited measurable responses; and both of these were significantly less than the complete reaction mixture response. 3. Results a. Kinetics.--AB, CO, and PQ values obtained from the com- puter printout are expressed in arbitrary units which are a func- tion of the excitation slit width and the voltage applied to the photomultiplier amplifier; both of which were set at constant levels. The raw data obtained from various stages of the reaction are also proportional to the concentration of reactants in the cuvette. Since each reactant is added in a 0.25 ml sodium acetate vehicle, a proportional dilution correction must be applied to the respective raw data. No dilution correction is necessary for PQ. Figure l is a representative graph of dilution corrected AB, CO, and PQ versus time. The addition of each reactant is marked 31 Figure 1.--Absorbance, fluorescence and partial quantum efficiency changes during an iodination reaction plotted against time in minutes. 0 = absorbance x 10. o = fluorescence. A = partial quantum efficiency. Letters beneath the abscissa designate the addition of PRL(P) lactoperoxidase (L), KI(K), and H202(H). A] = change in CO or PQ six minutes following the addition of K1. At = change in C0 or PO eight minutes following the addition of H202. 32 FIGURE 1 25‘ 204. - 5 0 1 1.. mHHZD wm 0.85), but only the 25.0 pg/ml curve has a slope which is significantly different from 0 (p < 0.1). The curvi- linear appearance of the 45 pg/ml plot led us to believe that a log- dose rather than a linear relationship existed between fluoresence enhancement and cell concentration. It is also apparent that the greatest changes occur at higher cell concentrations. Figure 6 is a dose-response curve of fluorescence versus prolactin concentration for a given concentration of cells. The ordinate values were obtained from equation (1), and each point represents the results of a single experiment. It was extremely difficult to pipette the 20 identical aliquots for the dose-response experiments, and a wider experimental variance was tolerated. The initial CO values of the cell suspensions varied from 290 to 330 6 cells/ml units yielding a mean concentration of 3.65 1 0.26 x 10 (mean 1 standard deviation; see Appendix E). The resulting graph has a distinct sigmoid shape with an exponential rise between the concentrations of 18 and 30 pg/ml O-PRL. The slope decreases 57 Figure 6.--F1uorescence enhancement as a function of prolactin con- centration. Each point is the result of a single experiment. Vertical bars indicate experimental variance of 3 time scans. Concentration of cells for each experiment = 3.65 1 0.26 x 106 cells/ml. Fluorescence enhancement = COexp - cocontrol‘ Units are arbitrary. o and A = two different series of experiments. 58 L r8 0 D O Q 11 O N 1 l r I I I ‘ G O G O D ID Q (0 N P lNSWBONVI-INS SONSOSBHOH'H ing/ml o- PRL 59 rapidly above 50 pg/ml but continues to rise until 100 pg/ml. Similarly, there is a gentle decrease in slope between 17.5 and 3 ug/ml. Such a graph would be consistent with what one expects in a positively cOOperative system. Table 4 presents the pooled results of two or more experi- 6 cells/ml. ments with various concentrations of hormone and 3.65 x 10 The mean and standard deviation for each hormone treatment were cal- culated and compared statistically. The data compiled for PRL treat- ments in Table 4 correspond to the exponential phase of Figure 6. In spite of the small sample number, all of the means are signifi- cantly different from each other at the 99% confidence level. The statistics employed in this analysis assume GausSian distribution of the individual trials which is a questionable assumption for data derived from a dose response curve; especially in the case of the pooled 45-50 pg/ml datum. However, to a first approximation the Student's t test provides a reasonable description of the results. Note that no significant response is elicited by cells treated with 25 pg/ml o-GH while a 45 pg/ml treatment causes fluor- escence quenching. 0. Discussion The fluorescence of photoexcited cell membranes represents the emissions from a heterogenous population of surface proteins. Although these proteins cannot be distinguished solely on the basis of their colorimetric properties, they can be distinguished func- tionally. Thus, only a given proportion of the proteins will be 60 TABLE 4.--Fluorescence response of C311RAP cells to ovine growth hormone and ovine prolactin (288ff348).aab l 2 3 4 5 25 ug/ml 45 ug/ml 17.5 pg/ml 25 pg/ml 45-50 pg/ml o-GH o-GH o-PRL o-PRL o-PRL 1.3 -8.5 5.0 18.6 31.0 0.5 -6.0 4.5 19.0 29.4 -l.0 22.2 02711.03 -7.2511.77c 4.751035C 19.931202“d 30.211.1°*"ie n = 3 n = 2 n = 2 n = 3 n = 2 a _ All values - mean 15.0. (COexp - cocont). bEach datum rgpresents the result of a single experiment with 3.65 1 0.26 x 10 cells/ml, and was derived from equation (1). cDifferent from 4 (p < 0.01). dDifferent from 3 (p < 0.01). eDifferent from 4 (p < 0.01). involved in any given physiological activity. If the biological activity necessitates a conformational or structural change in the protein, then a concomitant change in fluorescence may also occur (i.e., quenching, enhancement, peak shigt, etc.). Polypeptide hor- mones are known to have membrane bound protein receptors in a variety of target tissues (Frantz et_al,, 1974; Shiu and Freisen, 1975; Cuatrecasas, 1974) which doubtlessly contribute to the total fluor- escence emitted from photoexcited membranes. The interaction of prolactin with its receptor is believed to be analagous to enzyme- substrate binding (Shiu and Freisen, 1974) and thus probably involves tertiary changes in either the hormone or the receptor or both. 61 Consequently, we would anticipate fluoremetric changes in membrane emissions during PRL binding. The changes, however, are not directly proportional to the total membrane fluorescence but to the propor- tion of the total fluorescence which is contributed by the binding site alone. It is therefore misleading to express the fluorometric changes as a percent of the total. In the present study, an additional complicating factor is involved. The concentrations of hormone used also possess a signifi- cant intrinsic tryptOphanyl fluorescence (Table 3). We are inter- ested in the response of the membranes alone, yet are unable to distinguish hormone from membrane fluorescence. The recorded fluor- escence enhancements (Figures 5 and 6) in some cases equals more than twice the fluorescence of the hormone alone. If the enhance- ment arose from changes in the hormone, then PRL would be required to double its quantum efficiency. 0n the other hand, if the enhance- ments reflect changes at the membrane, a lesser, more plausible augmentation of membrane quantum efficiency would explain the fluor- escence enhancement. Both explanations are reasonable; unfortunately, we cannot yet resolve which (if not both) mechanisms are operative. Throughout these experiments, periodic emission scans were taken both before and after the addition of hormone. Although the intensity of fluorescence varied accOrding to the experimental pro- cedure, there was no evidence of an emission peak shift. Since the peak emission was not shifted, the environment surrounding the fluor- escent tryptophan residues probably is not greatly changed in polarity. Thus, fluorescence enhancement would be caused only by 62 (a) a conformational change in the membrane whereby a tryptophan group is brought near to a tyrosine (energy transfer), (6) non- radiative competitive quenching processes were altered, or (c) the hormone binding reaction causes an increased conformational organi- zation of the hormone or the receptor. We see in Figure 5 that high concentrations of cells (about 3.6 x 106/ml) are necessary before dose dependent changes in fluor- escence are resolvable. This may be due to the existence of rela- tively few PRL specific binding sites per cell. There are, however, certain trends which seem to be present which merit some discussion. For PRL doses of 25 and 17.5 pg/ml a plateau region on the graph is distinguishable between cell concentrations of 3.5 and 5.5 x 106 cells/m1. Note that the graph of 25 pg/ml of PRL appears to be shifted upward and to the right with respect to the 17.5 pg/ml graph. In contrast, the graph of 45 pg/ml of PRL undergoes a rapid rise over the same concentration ranges. The significance of these phenomena was not clear, so we decided to try dose-response experiments which are illustrated in Figure 6. At a constant concentration of 3.65 x 106 cells/ml the fluorescence enhancement undergoes the most dramatic changes between 18 and 30 pg/ml o-PRL. At higher concentrations (30-100 pg/ml), a less pronounced augmentation occurs which may con- tinue indefinitely. When experiments with comparable cell and hor- mone concentrations were pooled (Table 4), we found that the mean fluorescence enhancement values in the 17.5, 25 and 45 pg/ml treat- ments were all significantly different from each other. Thus, the protocol and instruments employed in these experiments were sufficient 63 to resolve fluoremetric changes resulting from small changes in hor- mone concentration. We also see in Table 4 that the response of CallRAP cells to O-GH is opposite to that of o-PRL. That is, o-GH causes a quenching of fluorescence at 45 pg/ml doses while o-PRL causes enhancement. If both of these responses are due to conforma- tional changes in specific membrane receptors, then two distinct populations of binding sites can be identified by these techniques. Quenching is associated with o-GH binding and enhancement is corre- lated with o-PRL binding. At this time, no O-GH binding sites have been identified on C811RAP cells by radioreceptor methods, but tryp- tophanyl quenching does occur in rat liver membranes exposed to b-GH (Postel-Vinay e_t_a_1_., 1974). L We are very pleased with the ability of the spectrofluorome- ter to resolve changes in membrane conformation associated with small changes in hormone concentration. However, it is unknown whether this system will be applicable to other target tissues and lower concentrations of hormone. Optimall y, we would like to detect changes in membrane components in response to picomolar concentra- tions of PRL. Although Frantz gt_gl, (1975) have demonstrated specific binding to C811RAP as well as normal pituitary cells, no physi- ological response has been identified. It was postulated that PRL binding may be the stimulus for a short loop feedback mechanism. In this study, fluorescence enhancement has been correlated to o-PRL cell interactions, which suggest that the hormone is capable of stimulating conformational changes in the membrane or hormone 64 protein structure. Concentrations of 18-30 ug/ml of o-PRL appear to be a critical range through which the largest changes in fluor- escence occur. Quite possibly, the changes occurring at the membrane at these specific hormone concentrations could trigger intracellular mechanisms which function to regulate such cytoplasmic processes as transcription or secretion. Although microgram concentrations would be considered extremely high as compared with normal physiological levels (2-200 ng/ml), microgram concentrations might be expected in the capillaries bathing the anterior pituitary. Furthermore, since C811RAP cells secrete prolactin (Sonnenschein gt_gl,, 1974), they are constantly exposed to relatively high levels of hormone. It is not surprising that even higher levels are necessary to initiate the binding process. This hypothesis was indirectly supported by experi- ments conducted in our lab with o-PRL and partially purified membrane particles (unpublished data). We found that comparable fluorescence changes could be effected by reacting the liver membranes with 100 ng/ml of hormone. We are presently planning to obtain cultures of liver and mammary cell lines to be studied in the future. The fluorescence response of the PRL-cell association appears to be very rapid; being complete within 10 minutes. If fluorescence is a monitor of receptor binding, then the kinetics are very rapid. This evidence lends support to the work of Frantz gt_al, (1974) and appears to contradict the studies by Shiu and Friesen (1974). Alternatively, the fluorescence response may only monitor the ini— tial phases of PRL-receptor association, giving a misleading estimate of the reaction time. However, because the PRL used in this study 65 was not altered by iodination, we feel that the system provides a more accurate reflection of physiological conditions. In addition, the cells are intact and metabolically active, whereas the bulk of the radioreceptor work was done with cell homogenates or purified membrane preparations whose structure may no longer be like that of the intact cell. The 4°C temperature used throughout these experiments may have had a significant effect on the results. Sonenberg (1969) found that erythrocyte membranes were only responsive to h-GH in the range of 21-40°C, being maximal at 37°C. Similarly, Shiu and Friesen (1974) found that radioreceptor binding to rabbit mammary homogenates was greatly decreased by low temperatures. It would be very informative to evaluate the effects of temperature on fluorescence-monitored binding reactions. The tendency of intact cells to settle out of solution was a serious problem throughout the study. In order to obtain consistent results, the fluorescence cuvette had to be agitated every 8-12 min- utes. An important step in the refinement of this system would be the develOpment of a matrix which would immobilize the cells, be light transparent and penmeable to hormone solutions. Possibly a polymeric gel such as FicolTM should be investigated. IV. Light Scattering of CallRAP Cells A. Objectives The surfaces of individual or aggregates of cells are large relative to the wavelengths of the incident radiation used in the 66 fluorescence experiments and therefore will reflect a substantial portion of the excitation radiation. If ignored, these surface reflections (Tyndall light scattering) may produce inappropriate absorbance and fluorescence values which can lead to incorrect interpretation of photometric phenomena (Willard gt_g1,, 1970). In addition, the proteins on the surface act as conglomerates of oscil- lating charges which can disperse some of the incident photoenergy in directions other than the direction of the incident radiation (Rayleight light scattering; Van Holde, 1971). Both of these effects were presumed to be operative during the experiments described in the previous section. Since Tyndall light scattering is purely a reflec- tive process, the intensity of the scattered light is directly pro- portional to the surface area and density of the scattering centers. Unfortunately, cell suSpensions are a heterogenous mixture of indi- vidual and aggregates of cells and we suspected that the relative abundance of disaggregated cells increases as the suspensions were diluted. In the fluorescence experiments, 0.25 ml injections of hor- mone and control solutions were made directly into the fluorescence cuvette which meant that the cell suspensions were diluted by a fac- tor of 0.888. This dilution may have disrupted cell aggregates and therefore decreased both the density and the surface area of the scattering particles. Furthermore, because we postulated that mem- brane proteins undergo conformational changes during the hormone binding process, the Rayleigh light scattering properties of the 0811 cells may have also changed. Thus, we felt it was essential to 67 evaluate the light scattering properties of the cells under the conditions of the earlier fluorescence experiments. B. Procedures 'The light absorbance of C811RAP ce11s falls off rapidly at wavelengths above 325 nm and thus by monitoring light scattering of monochromatic light at 330 nm, an absorbance artifact can be avoided. In these experiments individual 2.0 ml aliquots of various concen; trations of cells were pipetted into the sample cuvette of the spec- trofluorometer and allowed to equilibrate for ten minutes at 4°C. The collection of data and protocol was identical to that used in the fluorescence experiments except that both monochrometers were set at 330 nm. The 90° emission data were Collected both before and after the addition of hormone or Pucksf at the intervals specified above. In the light scattering experiments, duplicate suspensions were not compared, but a series of cell concentrations were injected with either o-PRL (final concentration of 40 pg/ml) or Pucksf. C. Results The intensity of scattered light (330ff330) at the cell con- centrations used in the fluorescence experiments tended to be higher than the intensity of fluoresced light (288ff348). Thus, after determining the cell concentration for the standard curve (Figure 4), the voltage applied to the emission photomultiplier amplifier was reduced from 8.0 to 6.5 Kvolts. This had the unfortunate effect of changing the arbitrary value of the light scattering units. However, the relative effects of the dilution phenomenon are still evident, 68 though the absolute value of the units is different from those used in the fluorescence experiments. A 40 pg/ml solution Of o-PRL in Pucksf plus glucose also produces a small but detectable amount of light scattering. In three consecutive trials, the solution pro— duced a light scattering of 2.6 1 0.4 units (mean 1 experimental variance). Thus, in order to evaluate only the changes in membrane light scattering, 2.6 units were subtracted from the final light scattering values for C811 cell suspensions. Figure 7 is a plot of the decrease in light scattering which occurs following the addition to various initial concentrations of cells of 250 pl of PRL or Pucksf plus glucose. The ordinate values were derived by subtracting the diluted cell suspension light scat- tering value from the initial light scattering value. Thus, the graph 1§_flot_analogous to Figure 5. The solid line denotes the decrease which is anticipated for the dilution of homogenous suspen- sion of light scattering particles. (Note that at low cell concen- trations, there is very good agreement with the theoretical line, but at concentrations above 1.3 x 106 cells/ml, a dilution affect gives rise to a disproportionate decrease in light scattering. Within experimental variance (vertical bars) there was no detectable difference between the light scattering changes in the hormone (o) and the Pucksf (o) injected treatments. Thus, if the Pucksf—injected light scattering values were subtracted from duplicate o-PRL-injected values, the result would be 0 for all concentrations of cells. It should be remembered that only the 90° emissions can be monitored with this instrument. It is assumed that unobserved changes in 69 Figure 7.--Changes in light scattering of C811RAP cells due to dilution. Decrease in light scattering = Initial - Final. 0 = dilution with Pucksf. o = dilution with Pucksf + O-PRL: final concentration 40 pg/ml. Light scattering units are arbitrary. INITIAL LIGHT SCATTERING 150 1 120 1 1 80 1 7O i-ID in but On 10- o c‘. A A N I N g» in SNIHBLLVOS 1H9” NI 381738030 60~ 06/1111 1 X CELL NUMBER 71 Rayleigh light scattering occur at other angles, but are propor- tionate and randomly distributed. Photographs were taken of cells suspended in culture medium at two different concentrations. In Figure 8A the cell concentration was approximately 2.0 x 106 cells/ml. We see that most of the cells exist as aggregates of five or more cells and very few free cells are present. Figure 88 is a photograph of the same solution after diluting by a factor of four and agitating with a glass pipette. In this case, most of the cells are in groups of 2 or 3 and more indi- vidual cells are present. Presumably, the disaggregation was a direct result of dilution. 0. Discussion The light scattering data obtained in these experiments pro- vide us with valuable information concerning the treatment of intact cell suspensions in fluorescence experiments. We see that the dilu- tion of cell suspensions at concentrations greater than 1.3 x 106 cells/m1 will cause a disproportionate decrease in light scattering. We also found in the control fluorescence experiments that a dis- proportionate decrease in light emitted at 348 nm accompanied the addition of 250 pl of Pucksf. Because the light scattering and the apparent fluorescence decreases were proportionately equivalent, we believe that both phemonema were due to changes in Tyndall light scattering alone. We furthermore demonstrated (Figure 8) that CellRAP ce11s exist both as aggregates and free cells and the rela- tive abundance of each appears to be related to the cell concentration. 6 A. 2.0 x 10 cells/ml. 6 B. 0.5 x 10 cells/ml. Figure 8.--Photomicrographs of 0811RAP cells. 73 Therefore, we feel that it is reasonable to postulate that the light scattering phenomena is a result of the disaggregation of cells. That is, when a cell SUSpenSion is diluted, not only are the scat- tering centers dispersed but the reflective area of each center is also reduced. Although the Tyndall perturbations are real and measurable, there is no distinguishable difference in light scattering with the PRL or control vehicle that is injected in the cell treatments. It should be noted, however, that the experimental variance was rather large in the light scattering experiments such that small differ- ences may have been obscured. If any trend does exist, it would be that PRL seems to augment the decrease in light scattering as com- pared to vehicle-injected suspensions. Possibly this is the result of Rayleigh effects which accompany conformational changes of the surface proteins. This would be opposite to the fluorescence effect described in the previous section. But as a first approximation, it is sufficient to assume equivalent light scattering decreases. We must remember that only the 90° emission was monitored and, thus, larger and undetected light scattering changes may have occurred at smaller angles with respect to the incident light beam. These, how- ever, would have no effect on the fluorescence measurements as reported. In conclusion, we see that Tyndall light scattering changes constitute a serious experimental error in fluorescence studies of C811RAP cells. However, by expressing fluorescence enhancement as 74 the difference between duplicate samples of hormone and control injections, an internal correction is made (equation 1). In retro- spect, it would be preferable to establish an experimental design which did not depend on the dilution of the cell suspensions. SUMMARY AND GENERAL DISCUSSION Lactogenic hormones and their receptors have been the sub- ject of intense investigation during the past five years. We now know that prolactin binds specifically to a variety of tissues including mammary, liver, adrenals, prostate, seminal vesicles and the pigeon crop sac. The receptor itself is located on the membrane surface; is protease sensitive, neuraminidase indifferent and has been partially purified. Most of the work with the radioreceptor assay has relied on the lactoperoxidase catalyzed iodination reac- 125I fixation to the native hormone. tion for In this study, we have demonstrated specific quenching of tryptophanyl fluorescence of O-PRL which is associated with the lactoperoxidase catalyzed iodination. The fluorescence quenching (34.7%) is rapid, irreversible and clearly distinguishable from collisional quenching. We believe the fluorometric effect to arise from (1) iodide fixation on or near tryptophan, or (2) con- formational changes in o-PRL tertiary structure. In addition to their fluorescent behavior, one of the two tryptophans (Try-90) is believed to be located on or near the biologically active region Of the hormone (Kauwachi gt_al,, 1973). Thus, stearic or conformational changes at this locus could have serious consequences for the bio- chemical potency of the molecule. Unfortunately, the radio receptor and radioimmunoassays (RRA and RIA) operate under the assumption 75 76 that the ‘25 I-labelled hormone is functionally identical to the native hormone. Similarly, the Scatchard analysis relies on equiva- lent competition of the labelled and unlabelled hormone for the same population Of receptors. In light of the present findings, we are compelled to question these fundamental assumptions. It is there- fore reasonable to question the physiological significance of the 10 to 10'9 M dissociation constants which have been estimated for 10' the various PRL-receptor complexes. Using fluorometric methods we have also monitored the inter- action of o-PRL with intact rat anterior pituitary tumor cells which are known to possess specific and saturable binding sites (Frantz gt_gl., 1975). The system which we developed is capable of detecting tryptophanyl fluorescence enhancement from membrane proteins of C811RAP cells in response to micromolar concentrations of o-prl. The effect is specific to o-PRL as compared to o-GH. The exponential rise of a sigmoid dose-response curve occurs between 18 and 30 pg/ml of hormone. It appears that a threshold is reached at a concentration of approximately 18 pg/ml which potentiates the binding of additional molecules of hormone. It is difficult to compare these results to those obtained from RRA methods, because the experimental designs differ. In the RRA, specific binding is defined by the capacity of native hormone to displace or compete with labelled PRL. In con- trast, the fluorescence assay uses only native hormone and the speci- ficity of the response is defined by comparison to the zero control and the effects of o-GH. Although the former method permits easier quantitation under ideal conditions, the latter method does not 77 depend on a chemically altered hormone. In spite of these differ- ences, there are several observations which should be discussed. With the fluorescence method we have estimated a saturation time of approximately 10 minutes. This datum was derived from a system utilizing intact, metabolically active cells and native hormone. Furthermore,'Uwaphotophysical response is the direct result of a macromolecular conformational change. The reaction time is also consistent with those obtained by Sonenberg (1969), Postel-Vinay gt_al, (1974) and Imae et_al, (1975). In contrast, the RRA gener- ally depends on experimental methods using cell homogenates, indirect calculations of specific binding and a particular batch of labelled hormone. By using disrupted ce11s, one risks exposing the hormone and receptor to intracellular enzymes which may destroy a significant portion of the tissue's receptor activity. Moreover, the response is usually defined as the difference of total and nonspecific bind- ing and the 90% saturation times are equivocal (Frantz gt_al,, 1974; Shiu and Friesen, 1974; Nicoll, 1975). We have also found in our lab that the fluorescence method produces more consistent results than the RRA (personal observation). Although we have not exploited its potential, it appears that the fluorescence method may be capable of distinguishing at least two populations of binding sites simultane- ously. That is, the sequential addition of o-PRL and o-GH to a sus- pension of CBllRAP cells should elicit fluorescence enhancement and fluorescence quenching, respectively. It is perhaps unfortunate that anterior pituitary cells were used for these initial studies because masking of binding sites by 78 endogenous r-PRL may have given misleading results. At present the sensitivity and general applicability of the fluorescence method is unknown. However, the success of the experiments of the Sonenberg and Tashjian groups with b-GH, TRH and various membrane fractions is a promising incentive for the development of intact cell systems. The ultimate success of the fluorescence assay will depend on more extensive investigations with liver and mammary cell lines. Method- ological refinements should also be explored because light scatter- ing changes associated with cell suspension dilution are a possible source of error. Apart from the endocrinological aspects of this study, we have obtained supporting evidence for the theory of a dynamic mem- brane. Although there is some ambiguity in the interpretation of the data, it is likely that PRL and GH both CONfer their biological activity by causing conformational changes in specific membrane proteins. At this time we haverunzascertained what cytoplasmic processes are linked to the membrane phenomena, but a feedback mechanism is certainly operational and should be further studied. APPENDICES 79 APPENDIX A ”Gm _____, MONO 1 sounce ' LIGHT SOURCE--150-watt Xenon arc MONOCHROM. l--EXCITATION MONOCHROMETER R --REFERENCE CUVETTE s --SAMPLE CUVETTE MONOCH ROM. 2--EMISSION MONOCHROMETER ‘ PUT COMPUTER --PDP--8/I (DIGITAL CORP.) Figure 9.--Optics and technical Specifications of the spectro- fluorometer. Four analog signals, two photomultipler signals and two wavelength encoder outputs are connected to a multiplexer-A to O converter combination (DEC model AFOl). Under program control, the computer can switch the multiplexer to admit any one of these volt- ages to the A to D converter where it is digitalized and from which it can be read into the computer. The computer applies instrumental and photophysical corrections to the data and stores it in the memory buffer until called up for display on the screen of the con- trol terminal. A more complete description is available in the paper by Holland et a1. (1973). 80 TABLE 5.--Pucksf plus glucose balanced salt solution. 81 APPENDIX B Inorganic Salts mg/liter CaCl2 - ( 2 H20 ) 16.0 KCl 285.0 MgSO4 ° ( 7 H20 ) 154.0 NaCl 7,400.0 NaHC03 1,200.0 NazHPO4 - ( 7 H20 ) 290.0 Other Components Glucose 1,100.0 ( GIBCO ) 82 118888888 80 888888888 one 88888 .8888888888 5288888 8888888 u 08 8888888888888 888888188 u M88888 88888ea8 8888c88e 8:8 .1888 888888 888888888 .18888 8888888 88 88:88 Fmowugm> ”cowuummg we mmuscwe n mesapou Pmuwpgm> ou mmucmngomn8 n 8.8 8.8 8.8 8.8 8.88 8.88 8.8_ 8.8_ 88 8.8 8.8 8.8 8.8 8.8 8.8 8.8 8.8 88 8 888. 888. 888. 888. 888. 888. 888. 888. 88 8.8 8.8 8.8 8.8 8.88 8.8_ 8.88 8.8_ 88 8.8 8.8 8.8 8.8 8.8 8.8 8.8 8.8 88 8 88_. 888. 88.. 88,. 88.. 888. 88_. 888. 88 8.88 8.88 8.88 8.88 _.88 8.88 8.88 8.88 8.88 8.88 8._8 8.88 8.88 8.88 8.88 88 8.88 8.8_ 8.8— 8.88 8.88 8.8. 8.88 8.88 8.88 8.88 8.88 8.8_ _.P_ 8.88 8.1_ 88 8 888. 888. 888. 888. 888. 888. 888. 888. _88. 888. 888. 888. 888. 8.8. 888. 88 8.8. 8.88 8.8. 8.88 8.88 8.88 8.88 8.88 8.88 8.88 8.88 8.88 8.88 8.88 8.88 88 8.8 8.8 8.8 8.8 8.88 8.88 8.8_ 8.__ 8.8_ 8.__ 8.__ _.8 8.8 8.8 8.8 88 _ 888. 888. _88. 888. 888. 888. 888. 888. 888. 888. 888. 888. 88_. 888. 888. 88 88 88 88 88 88 88 88 8F 88 88 8_ 8 8 8 8 18128 888: 88 888 888 8888 .888888881 cowomcwuow 5888 8888 u xHszmm< 88888888888 888888288 =888=_88--.8 88888 83 APPENDIX D TABLE 7.--Prolactin-cell interactions at various cell and hormone concentrations (288ff348). Initial CO PRL C0 Final CO - PRL CO Exp. - Control (X) (Exp) (Y) 17.5 ug/ml 64 1 1 16.1 43.9 1 l 0.0 1 2 141 1 1 16.1 114.0 1 l -2.0 1 2 225 1 1 16.1 163.9 1 l 2.9 1 2 311 1 1 16.1 227.9 1 l 4.9 1 2 424 1 2 16.1 .310.9 1 2 5.0 1 4 25 ug/ml 48.4 1 1 23.4 34.1 1 1 2.1 1 2 65.4 1 1 23.4 50.4 1 1 6.4 1 2 89.8 1 1 23.4 70.3 1 1 7.3 1 2 169.5 1 1 23.4 127.6 1 1 7.6 1 2 183 1 1 23.4 139.9 1 l 9.9 1 2 250 1 1 23.4 194.6 1 1 15.6 1 2 302 1 l 5 23.4 235.6 1 1.5 18.6 1 3 338 1 l 5 24.0 263.0 1 1.5 20.0 1 3 430 1 2 24.0 326.0 1 2 20.0 1 4 45 pg/ml 92 1 1 42.0 73 1 l 9.0 1 2 187 1 1 42.0 145 1 1 12.0 1 2 248 1 1 42.0 196 1 1 18.0 1 2 375 1 l 5 42.0 309 1 1.5 39.0 1 3 434 1 2 42.0 377 1 2 65.0 1 4 diff. 1 experi- diff. 1 experi- mental variance* mental variance** All units are arbitrarily standardized against 10"5 M quinine sulfate in 0.1 M H2504. Initial CO can be converted to cell concentration by use of the standard curve in the text. *Experimental variance = variance of three sequential scans. **Exp. var. = sum of variances from control and exp. data. Control = C0 of 2 ml of C811RAP cells + 0.25 mlinyucka + glucose. Initial CO = C0 of 2 ml of C311 RAP cells alone. PRL C0 = C0 of PRL in 2.25 ml of Pucka + glucose. Exp. CO C0 of PRL - Cell mixture (2.25 ml - PRL CO. 84 APPENDIX E TABLE 8.--Dose response of prolactin-cell reactions. Initial PRL C0 [PRL] Final CO - PRL CO Exp. - Control co (x) (Exp) (Y) 330 4.0 5.6 290 1 1 2.0 1 2 310 16.1 17.5 281 1 1 5.0 1 2 312 15.8 17.5 283.5 1 l 4.5 1 2 332 21.0 22.0 300.5 1 1.5 12.5 1 3 302 23.4 25.0 282.0 1 1.5 18.6 1 3 321 24.0 25.0 307.0 1 1.5 19.0 1 3 326 24.0 25.0 314.1 1 1.5 22.0 1 3 290 28.1 30.0 278.0 1 1.5 32.0 1 3 290 48.0 50.0 286.0 1 2.5 38.2 1 5 309 91.4 100.0 311.2 1 2.5 46.6 1 5 312 1 15.2 diff. 1 experi- diff. 1 experi- (Mean 1 S.D.) mental variance mental variance Symbols as in Appendix D. All units are arbitrary but standardized against 10"5 M quinine sulfate in 0.1 M H2504 (350ff450). 85 APPENDIX F TABLE 9A.--Changes in light scattering of C811RAP cells due to dilution with buffer. C.C. 1.4 1.9 2.9 5.6 I 8.9 10.9 19.1 25.5 30.1 37.0 50.0 52.1 75.5 107 131 169 F 8.5 9.6 17.6 24.3 26.5 33.0 37.5 36.2 51.5 75.2 96 119 A 0.4 1.3 1.5 1.2 3.6 4.0 12.5 15.9 24.0 31.8 35 50 I.V. 11 11 11 11 11 11 12 12 13 14 15 15 initial light scattering in arbitrary units. light scattering following addition of 0.25 ml PUCka + glucose. I - F. instrumental variance of three sequential time scans. - D'nH II II II II I.V TABLE 9B.--Changes in light scattering due to simultaneous dilution and addition of 40 ug/ml PRL. C.C. 2.1 3.1 4.7 I 41 60 77 109 133 4 167 IPRL 2.6 2.6 2.6 2.6 2.5 2.6 F 32.2 35.2 58 75 92.7 108.4 A 8.8 24.8 29 34 38.3 58.6 I.V. 12 12 :3 :4 :5 :5 initial light scattering of cells. o—c II II light scattering of PRL in 2.25 and Pucksf. PRL F = light scattering of cells following addition of 0.25 ml of buffer and 80 g PRL - IPRL‘ A = I - F. I.V. = instrumental variation of three sequential scans. C.C. = cell concentration x 106/m1. I Rum: II. 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